Neonates with intrauterine growth restriction (IUGR) have a high lipid profile that predisposes them to cardiovascular disease later in life. We aimed to evaluate the effect of omega 3 supplementation on serum leptin level, lipid profile, and growth in neonates with IUGR.This clinical trial was conducted on 70 full-term neonates with IUGR. Neonates were randomly divided into two equal groups; the treatment group: received omega 3 supplement (40 mg/kg/day) for 2 weeks after the establishment of full feeding, and the control group, who were followed up to full feeding without any supplementation. Serum leptin level, total cholesterol (TC), high-density lipoprotein (HDL), triglycerides (TG), low-density lipoprotein (LDL), and anthropometric measurement were evaluated at admission and after 2 weeks of omega 3 supplementation in both groups.After treatment, HDL significantly increased, unlike TC, TG, LDL, LDL, and serum leptin levels, which significantly decreased in the treatment group compared to the control group after treatment. Interestingly, weight, length, and ponderal index greatly increased in omega 3-treated neonates compared to the control group.Omega 3 supplementations lowered serum leptin level, TG, TC, LDL, and VLDL but increased HDL and growth in neonates with IUGR.The study was registered at clinicaltrials.gov (NCT05242107).Neonates with intrauterine growth retardation (IUGR) were reported to have a high lipid profile that predisposes them to cardiovascular disease later in life. Leptin is a hormone that adjusts dietary intake and body mass and has a significant role in fetal development. Omega 3 is known to be essential for neonatal growth and brain development.We aimed to evaluate the effect of omega 3 supplementation on serum leptin level, lipid profile, and growth in neonates with IUGR.We found that omega 3 supplementations lowered serum leptin level and serum lipid profile but increased high density lipoprotein and growth in neonates with IUGR. This early misprogramming increases the susceptibility to cardiovascular diseases, coronary heart disease, hypertension, and type 2 diabetes mellitus.2Intrauterine growth restriction (IUGR) is a significant public health problem with significant perinatal mortality and morbidity.3Neonates with IUGR were reported to have abnormal lipid profiles. High triglycerides (TG) and low high density lipoprotein (HDL) levels in neonates with IUGR may be due to enhanced cholesteryl ester transfer, which could explain why neonates with IUGR have a higher risk of coronary heart disease later in life.3 While excess leptin provides energy expenditure by suppressing food intake and increasing thermogenesis, decreased leptin increases the storage in adipocytes. Several studies reported that plasma leptin levels were positively correlated with body mass index (BMI) values and total body fat.6 Also, Marchini et al.5 found that the plasma leptin level decreases in connection with the initial physiological weight loss in newborn infants. Following this discovery,7 leptin and infant development have gained momentum.10Leptin adjusts dietary intake, body mass, and has a significant role in the fetal development, angiogenesis, and lipolysis. Trials reported that the level of serum leptin is reduced during fasting or energy restriction and increased during re-feeding, overfeeding, and surgical stress.2Understanding the leptin and cholesterol metabolism in the fetus and neonates is very critical, not only for the nourishment of neonates but also for the strategies to decrease the future recognized risk of cardiovascular diseases (CVD) in fetuses with IUGR.11 Omega 3 supplementation has multiple proposed mechanisms that could improve the metabolic adipocyte and lipid profile.12 These involve changes in the gene expression of adipose tissue, adipokine-mediated or related pathways, changes in adipokine release, alterations in the carbohydrate metabolism, appetite suppression, the increases in energy expenditure (possibly through thermogenesis), the increases in fat oxidation, activating mechanisms involved in muscle anabolism, and its effect on epigenetics.13 Omega 3 supplementation effect on leptin levels is still controversial; some studies on omega 3 supplementation reported that it decreases circulatory leptin levels17; however, other investigations do not support this beneficial effect of omega 3 intake impact on leptin concentrations.20Higher intake of anti-inflammatory omega 3 polyunsaturated fatty acids (PUFA) in pregnancy has been associated with lower adiposity, higher birth weight and longer gestation.There is a paucity of trials that focus mainly on the omega 3 effect on leptin serum level in neonates with IUGR. Therefore, our study aimed to assess the effect of omega 3 supplementation on serum leptin level and lipid profile in neonates with IUGR.This randomized open-label clinical trial was carried out on 70 full-term neonates with IUGR who were referred to the neonatal intensive care unit (NICU), Pediatric Department, Tanta University Hospitals from June 2021 to January 2022. The trial was done after approval from the Ethical Committee of the Faculty of Medicine at Tanta University (code:35199/1/22). The trial was registered at clinicaltrials.gov (ID: NCT05242107). The guardians of included neonates provided informed consent.Exclusion criteria were preterm neonates, neonates who died before feeding was established, neonates with congenital infection, neonates with respiratory distress, neonates with cholestasis, and neonates who had multiple congenital anomalies.19 After the establishment of full-feeding and for 2 weeks duration, mothers learned how to use the capsule after calculating the suitable dose for their baby after recovery. The contents of the capsule were taken by a syringe, and the mother dissolved it in 10 ml of expressed breast milk or freshly prepared formula and then shook it very well; then the calculated dose was given to the baby once daily for 2 weeks.We used omega 300  capsule, which contains 1000 mg Omega 3 fatty acids 3 in a dose of 40 mg/kg/day in the study group.Neonates who met the inclusion criteria were randomly assigned to one of two study groups in a 1:1 ratio using a random block size of six utilizing computer-generated random numbers by an independent statistician. Allocation concealment was accomplished using sequentially numbered sealed opaque envelopes. The sealed opaque envelope was opened once the patient’s guardians signed the written agreement and the neonates were enrolled into the appropriate group.The treatment group: include 35 neonates who received omega 3 supplements (40 mg/kg/ day) after the establishment of full feeding for 2 weeks.The control group: included 35 neonates who were followed up to full feeding without receiving any supplementation.All patients were subjected to complete history taking such as maternal age, mode of delivery, and maternal dietary intake of omega 3 during pregnancy. Full clinical examinations were done for all included neonates e.g., gestational age assessment, vital signs, and anthropometric measurements such as weight, length, and head circumference (HC).21The Ponderal index (PI) can be used to detect newborns with soft tissue mass that is below average for their skeletal development stage. PI has been used to assess the IUGR because low birth weight and IUGR tends to reoccur in siblings, and clustering of PI in sibling even persists after controlling for factors such as race, sex, maternal age, gravidity, year of birth, gestational age, pregnancy complications and poor maternal illnesses.3 X 100. IUGR is diagnosed when PI is below 2. All measurements were taken at admission and after 2 weeks of omega 3 supplementation.The formula for calculating the PI is as follows: PI = birth weight (g)/(length in cm)Routine laboratory investigations such as complete blood count, serum urea and creatinine, blood urea nitrogen, C- reactive protein, and serum electrolytes such as serum sodium (Na), potassium (K), and calcium (Ca) were also performed at admission and after 2 weeks from omega 3 supplementation.Serum leptin level was measured using commercial ELISA assay kits .Serum lipid profile included total cholesterol (TC), high density lipoproteins (HDL), triglycerides (TG), low density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) were measured by enzymatic colorimetric method using commercial kits .All laboratory investigations were measured at admission and 2 weeks after omega 3 supplementation. Blood sampling was obtained before breastfeeding and at least 2 h after the previous feed between 8 and 10 am.All cases were followed up as regards the establishment of full feeding, weight gain, length, and HC catch up of growth 2 weeks after completion of omega 3 supplementation. Development of signs and symptoms of sepsis, feeding intolerance, or necrotizing enterocolitis were observed. Moreover, any side effects of the drug were also recorded.The primary outcome was to assess the effect of omega 3 supplementation on serum leptin levels. The secondary outcomes were to assess the effect of the supplementation of omega 3 on serum lipid profile and on the anthropometric measurement.The sample size calculation was done by G*Power 3.1.9.2 . We performed a pilot study on five cases in each group. The mean ± SD of leptin level after 2 weeks (the primary outcome) was 1.42 ± 0.148 ng/ml in the control group and 1.74 ± 0.52 in the study group. Four cases were added to each group to overcome dropout. We needed 35 cases in each group to achieve 90% power of the study with a 0.83 effect size and 0.05 α error.The CONSORT 2010 checklist is included in the supplementary files (supplementary file 1).t-test, while non-parametric quantitative data were compared using the Mann–Whitney test. Categorical data were compared between the two groups using Chi-square or Fisher’s exact tests. Paired T-test was performed to compare variable quantitative data within the same group. A two-tailed P ≤ 0.05 was considered statistically significant.SPSS v27  was used for the statistical analysis. Shapiro–Wilks test and histograms were used to determine the normality of the data distribution. The quantitative data were presented as mean and standard deviation (SD) if normally distributed, while non-parametric quantitative data were presented as the median and interquartile range (IQR). Qualitative variables were expressed as frequency and percentages (%). Normally distributed quantitative data were compared between the two groups using the unpaired Student The flow chart of the present study was presented as Fig. P = 0.007) , or maternal use of omega 3. Antenatal risk factors were significantly higher in the study group compared to the control group (7) Table .Table 1HAt admission, weight, length, head circumference, and ponderal index were insignificantly different between the two groups. Moreover, leptin, TC, TG, HDL, LDL, and VLDL were comparable between the two groups. No significant difference was found between both groups as regards CBC, renal function tests, serum electrolytes, or CRP Table .Table 2AP < 0.001). However, leptin significantly increased in the control group after treatment compared to their levels at admission (P < 0.001).Leptin level significantly decreased after 2 weeks of omega 3 treatment in the study group compared to their levels at admission (P ˂ 0.05), unlike HDL, which significantly increased after treatment in the study group compared to the control group  and/or docosahexaenoic acid (DHA) supplementation decreased leptin levels that may be due to decreased inflammation through altering gene expression, reducing adipocyte size, decreasing weight and total adiposity, and improving insulin sensitivity.In the current study, omega 3 significantly decreased leptin levels in neonates with IUGR compared to the control group in which leptin levels significantly increased after 2 weeks of treatment compared to their levels at admission. In line with our study, Hariri et al.27 It has also been shown that n-3 FAs are ligands for peroxysomal proliferator-activated receptor α (PPARα) and PPARγ and can heterodimerize with retinoid X receptor to increase the expression of FA oxidizing enzymes and reduce the expression of sterol regulatory element-binding protein (SREBP)-1, promoting reduced lipogenesis.28 Hence, because omega 3 fatty acids seem to influence fatty acid metabolism markedly and leptin is important for adipose tissue size, omega 3 fatty acid supplementation exhibit a promising role in managing abnormal high leptin levels in IUGR.29Omega 3 fatty acids reduce the synthesis and increase the oxidation of fatty acid (FA).30 Metabolomic analysis of cord blood from early and late-onset IUGR revealed an abnormal lipid metabolism in both early and late-onset IUGR.31 Higher circulating triglycerides and cholesterol lipoproteins in growth-restricted fetuses were noted.32High lipid profile was reported in neonates with IUGR that indicate intrauterine malnutrition and lipolysis, and this is a key biochemical indicator of IUGR and contributes to a higher risk of metabolic and coronary heart diseases in the adult life.33 who investigated the effect of omega 3 on serum lipid profile level in 25 full-term neonates with IUGR compared to controls. They noted an increase in the level of TC and f TG initially in IUGR neonates, and this level was decreased after supplementation of Omega 3 for 2 weeks.In our study, we found that TC, as well as TG levels, significantly decreased in omega 3 treated neonates compared to the control group, which came in agreement with EL-alkamy et al.25 We noted that LDL and VLDL significantly decreased after 2 weeks in the study group compared to the control group. In line with our results, EL-alkamy et al.33 found a significant decrease in LDL after 1 week and in VLDL after 2 weeks of Omega 3 supplementation in the cases group more than in the control group.VLDL and IDL were found to be the most deregulated lipoproteins in fetuses with IUGR, suggesting that VLDL rich in triglycerides may be an alternative fuel mobilized by the growth-restricted fetus.34 reported in their study, which was done on 25 participants with a mean age of 16 years, that there was a significant decrease in levels of LDL after a daily supplementation of fish oil for 16 weeks (p < 0.05). Also, this came in line with Dallongeville et al.35 who reported a significant decrease in levels of VLDL in adult patients suffering from type 3 dys-beta-lipoproteinemia on receiving 6 grams of Omega 3 for 12 weeks.Also, Garaiova et al.36Mechanistically, most evidence suggests that Omega 3 fatty acids reduce the synthesis and secretion of VLDL particles and increase TG removal from VLDL and chylomicron particles through the upregulation of enzymes, such as lipoprotein lipase.37 In the present study, HDL significantly increased in omega 3 treated neonates compared to the control group, which agreed with the results of El-alkamy et al.33 and Mohamed et al.38 studies. This witnessed effect might be attributed to that Omega 3 PUFAs, especially DHA, increase HDL-C.39 The formation of HDL is related to the catabolism of TG-rich lipoproteins such as VLDL or intermediate-density lipoprotein (IDL) by LPL.40 Therefore, increased LPL activity reduces IDL and VLDL and increases HDL. Increased pre-heparin LPL due to omega 3 PUFA was observed.41On the other hand, a low HDL level was noted in fetuese with IUGR when compared to appropriately grown fetuses. Although the cause of the low fetal HDL concentration in neonates with IUGR is still a matter of debate, it was suggested that HDL is the result of diminished fetal de novo synthesis.42 who supplemented DHA in two different doses to premature infants with less than 1250 g and more than 1250 g, and they found that infants with a birth weight of more than 1250 g had a significant increase in weight and length at 12 months of age. As regards head circumference in our study, there was no significant difference in the head circumference between both groups after treatment. In contrast to the current study, Collins et al.42 reported that the supplemented group with high DHA had greater head circumference than the standard control group at 12 weeks of age.Interestingly, the weight and length of the omega 3 supplemented group were significantly higher than that of the control group after treatment. This was in agreement with the results of Collins et al.43 Additionally, based on our findings, we can theorize that omega 3 could be safe in IUGR neonates for controlling the serum leptin level and lipid profile; therefore, screening them is important to be done in all IUGR neonates at birth and during follow-up.Adequate consumption of omega 3 fatty acids is vitally important during pregnancy as they are critical building blocks of the fetal brain and retina. Omega 3 fatty acids may also play a role in determining the length of gestation and in preventing perinatal depression.44; in addition, neonatal cord leptin concentrations correlate well with birth weight and BMI.45 As a result, the maternal serum leptin is an important covariate in the changes in serum leptin levels in neonates that should be considered in future studies. Second, the intervention period (2 weeks) was relatively short and could not be enough to reflect precisely the outcomes of measure. Third, we did not use a nationally representative sample in our research. As a result, before generalizing to different demographics or geographic regions, results must be carefully understood. Longer follow-up periods with a larger sample size along with long-term surveillance of all IUGR newborns for CVD detection and addressing the influence of Omega 3 on these groups are needed to better understand our findings.Nonetheless, there were certain limitations to our research. First, we did not assess important maternal information; it was reported that the mean maternal serum leptin concentration in pregnancies with IUGR was significantly higher than in normal pregnanciesOmega 3 supplementations lowered serum leptin level, TG, TC, LDL, and VLDL but increased HDL and growth in neonates with IUGR.


Colorectal cancer (CRC) is often diagnosed in advanced stages. Circulating tumour DNA (ctDNA) has been proposed as an early diagnostic biomarker. However, as a screening tool, ctDNA has mainly been studied in selected populations at the time of clinical diagnosis. The aim of this study was to detect CRC by known ctDNA markers up to 2 years prior to clinical diagnosis.In this case–control study, methylated ctDNA markers were detected in plasma samples from 106 healthy controls and 106 individuals diagnosed with CRC within 24 months following participation in The Trøndelag Health Study.BMP3, FLI1, IKZF1, SFRP1, SFRP2, NPTX2, SLC8A1 and VIM (specificity >70%). When combining these into a panel, the CRC sensitivity was 43% (95% CI 42.7–43.4) and the CRC specificity was 86% (95% CI 85.7–86.2). The findings were reproduced in an independent validation set of samples.The most specific single markers were Detection of known methylated ctDNA markers of CRC is possible up to 2 years prior to the clinical diagnosis in an unselected population resembling the screening setting. This study supports the hypothesis that some patients could be diagnosed earlier, if ctDNA detection was part of the CRC screening programme. Colorectal cancer (CRC) is one of the most common malignancies worldwide. The incidence of CRC in Norway is one of the world’s highest with 83.8/100.000 new cases in 2021 and still increasing [Established tools for CRC screening are FIT  , HSgFOBT  , colonoscopy  and CTC (Computed tomographic colonography)  [n = 4) and focused on the detection of several cancers simultaneously [Circulating tumour DNA (ctDNA) has shown promising results as liquid biopsy for diagnosis, treatment and follow-up of colorectal cancer , 7. ctDNaneously . Luo et aneously . The metaneously . Methylaaneously . ctDNA cAPC, MGMT, RASSF2A and WIF1) have reached sensitivities and specificities above 90% [Approximately 70 methylated promoter regions have been identified as possible blood or stool-based biomarkers for CRC . Some bibove 90% .To date, studies investigating ctDNA and its diagnostic ability as a screening tool have mainly been performed in selected populations, including cases at the time of diagnosis  and matching healthy controls either being colonoscopy negative or self-reported healthy. To our knowledge, no studies have so far systematically searched for methylated ctDNA in blood plasma prior to the clinical diagnosis of CRC in unselected populations, such as in cross-sectional health surveys, which resemble the actual screening setting. In the present study, we aimed to detect known colorectal ctDNA markers in plasma samples from participants of The Trøndelag Health Study (HUNT), up to 2 years prior to the clinical diagnosis of colorectal cancer.This is a nested case–control study based on the Trøndelag Health study (HUNT). HUNT is one of the largest longitudinal population health studies ever performed. The HUNT Research Centre has collected data in four cross-sectional surveys: HUNT1 (1984–1986), HUNT2 (1995–1997), HUNT3 (2006–2008), and HUNT4 (2018–2019). The entire adult population ≥ 20 years old in Nord-Trøndelag county was invited to give a blood sample in the third wave of the Trøndelag Health Study (HUNT3), where of ~60,000 participated . ThroughWe defined the outcome as being diagnosed with CRC or not, within the 24 months following participation in HUNT3. A panel of promising methylated regions within 20 different genes detected in early diagnosis of CRC were selected based on systematic reviews , 12, 13 g and aliquoted within 24 h after venepuncture. The EDTA plasma aliquots were stored at minus 80 °C for future use. For the present study, all frozen samples were given a unique ID-number (phenotypes blinded to assay operators) before being couriered to Aalborg University Hospital for the methylation analyses, where plasma specimens were randomly processed.All blood samples were obtained by a skilled technician at the time of participation in HUNT, transported to HUNT Research Centre at 4 °C, centrifuged at 6 °C for 10 min at 2500 × Two targeted markers were designed within each gene of interest. All primer and probe sequences along with amplicon sizes and detailed PCR descriptions are presented in Supplementary Methods and Supplementary Tables MEST1 was used as a quality control parameter (reference gene for methylation), to ensure the correct assessment of the effect of methylation changes in the other methylation markers. The corrected Ct-value was used for calculations, where a correction factor was used to account for differences in PCR effectivity in different promoter sequences. The correction factor was calculated as mean Ct-value of eight replicates of global methylated DNA (EpiTect methylated human control DNA) minus mean Ct of MEST1.The method was not quantitative and hence there were no reference intervals, ranges or CVs (coefficient of variation). Units of measurement was cycle threshold (Ct) values. The naturally hemimethylated 2). Comparisons of clinical variables and methylation status between cases and controls were made by Χ2 test for categorical variables and t test for continuous variables (two-sided).The outcome variable (CRC/no CRC) and the following potential predictor variables were handled as binary: methylated/not methylated for each of the biomarkers, sex , diabetes/no diabetes and other cancer/no other cancer. The following potential predictor variables were handled as continuous: age, smoking (pack-years), BMI, Hb- and CEA-level. The number of pack-years was missing for 16 of the 212 subjects in the study. These missing values were replaced by the median pack-years of the entire population (7.0 pack-years). Similarly, one of the 212 subjects had no BMI calculated and the missing value was replaced by the median of the entire population  in the AROC analyses were considered possible candidates for a diagnostic methylation panel and used as a panel of markers in the test set. Ultimately, the resulting panel was tested in the validation set. All statistical analyses were done with the statistical software packages SPSS® version 28.0.1.0.SPSS split the samples into a test set (70% of samples) and a validation set (30% of samples), by simple randomisation based on the personal identification number. The test set was used for analyses leading to the selection of a marker panel, whereas the validation set was used exclusively to determine if the results of the selected marker panel could be reproduced in an independent sample. In the test set, the cut-off corrected Ct-value to define a sample as methylated/not methylated for each marker was defined as the value that maximised the Youden index . All samfounders . Sensitifounders . The putn = 143) and one validation set containing 30% of the samples (n = 69). The test set and the validation set did not differ regarding sex, age, BMI, diabetes, Hb, CEA, TNM-stage or AJCC stage (P > 0.05). In the validation group, cases had smoked more than controls   was sent for ctDNA analysis  , FLI1  and SFRP2  were all independent predictors (P < 0.05) after adjusting for age, sex, BMI and smoking (Table The number and percentage of cases and controls deemed positive for each of the putative diagnostic markers in the test set are presented in Supplementary Table NDRG4 (62.5%) and WNT5A (51.4%). The most specific single markers were AGBL4, BMP3, FLI1, IKZF1, NPTX2, SFRP1, SFRP2, SDC2, SLC8A1 and VIM (specificity >70%). As expected, based on previous studies showing that no single ctDNA methylation marker is considered adequately sensitive and specific for cancer screening, we found it important to investigate the combined effect of the most specific and significant markers in a panel. BMP3, FLI1, IKZF1, NPTX2, SFRP1, SFRP2, SLC8A1 and VIM were combined into the HUNT-CRC diagnostic panel (HUNT-CRCd), considered positive if two or more of the eight markers were present  of being clinically diagnosed with colorectal cancer within the subsequent 24 months with AUC of 0.669 , makes it attractive to use in clinical settings where the sensitivity is adequate. Possible areas of use could be family members of colorectal cancer patients , and as a supplement among those refusing colonoscopy or iFOBT/FIT . In linel cancer . This feMany studies have used an arbitrary cut-off Ct-value  to deem a sample as methylated or not, while some have classified all samples with Ct-values as methylated. This study use the Youden index to define a cut-off Ct-value for each biomarker. The Youden index maximises the sensitivity and specificity of the test, and could therefore be beneficial in a diagnostic setting. The number of cycles required for detectable amplification of colorectal ctDNA markers is dependent on a long list of factors beyond the amount of tumour DNA present in the specimen. Ct-values are influenced by pre-analytic variables such as efficiency of the blood sampling, storage and transport, analytic variables including DNA extraction efficiency, analysis platform used, nature of the target marker, design of the primers/probes and finally by the clinical variables such as stage of disease and presence of other diseases including other cancer. If researchers control their experiments, Ct-values can be used in one experiment as guide to compare one result to another, but the comparison of Ct-values between labs or experiments is not meaningful because of the abovementioned variations. This is a hindrance to commercialisation of methylation-based tests for CRC screening, but not prohibitive, as demonstrated by tests already available in the market  were largely ruled out. In addition, decay of ctDNA due to lengthy storage is well-known and the plasma samples in this study were prepared 11–13 years prior to ctDNA analyses . HoweverA strength of this study is that the controls are very likely true controls, as the observation time after blood sampling was long (9–11 years), all Norwegian inhabitants have the same access to health care and the quality of the CRN is high. The quality of plasma samples in HUNT Biobank is high . CompareIn conclusion, detection of known methylated ctDNA markers for CRC is possible up to 2 years prior to the clinical diagnosis. As suggested by others, this study supports the hypothesis that some patients could be diagnosed earlier, in a possible curable setting, if ctDNA detection was implemented as part of the CRC screening programme. To evaluate this hypothesis further, prospective studies and cost analyses addressing the overall benefit of ctDNA in CRC screening are needed.Supplementary materialsSTARD


Drosophila subobscura is considered a good model species for investigation of a population’s ability to adapt and cope with climate changes. Decade long research has shown that inversion frequencies change in response to environmental factors indicating their role in adaptation to novel environments. The mechanisms behind organisms’ responses to temperature are complex, involving changes in physiology, behavior, gene expression and regulation. On the other hand, a population’s ability to respond to suboptimal conditions depends on standing genetic variation and population history. In order to elucidate the role of local adaptation in population response to the changing temperature, we investigated the response to temperature in D. subobscura individuals originating from two different altitudes by combining traditional cytogenetic techniques with assessing the levels of Hsp70 protein expression. Inversion polymorphism was assessed in the flies sampled from natural populations and in flies reared in laboratory conditions at three different temperatures after five and sixteen generations and Hsp70 protein expression profile in 12th generation flies at the basal level and after heat shock induction. Our results indicate that local adaptation and population history influence population response to the changing temperature. Drosophila species, most of them having in common that due to the absence of recombination in heterokaryotypes, each inversion captures specific alleles in a beneficial combination, and as such, their frequency changes in response to the environment.Chromosomal inversions are traditionally described as adaptive structural mutations that become advantageous in populations by capturing favorable allele combinations . Since DDrosophila subobscura is particularly well documented, with numerous evidences of long-, mid- and short-term changes in their frequencies correlating with environmental variables. Parallel latitudinal distribution in inversion clines [D. subobscura natural populations [Regardless of mechanism, environmental variables found to influence changes in inversion polymorphism are many. Climatic factors such as temperature, humidity, precipitation and isolation are the most common factors correlated with shifts in inversion distribution in natural populations ,11,12, bn clines  and incrn clines ,15,16, in clines ,18. Despn clines ,20, withDrosophila subobscura is a Palearctic, temperate region species, with a thermal range between 6 and 26 °C, but it is generally considered a cold-adapted species with a thermal optimum of 18 °C [Drosophila genus [D. subobscura the Hsp70 family consists of two intronless and closely spaced Hsp70 genes arranged as a head-to-head inverted repeat (Hsp70IR), and that this gene family has identical genomic organization in Ost and O3+4+7 karyotypes [D. subobscura strains revealed that O3+4 flies have higher basal expression levels of Hsp70 proteins not boosted after heat shock and that Ost individuals show a different pattern [3+4, O3+4+7 and Ost files from different geographical regions [Drosophila species adaptation to colder climates strongly influences the magnitude of that response, indicating that changes in regulatory regions may be a target of selective pressure. Other experiments have demonstrated genome-wide changes in inversion polymorphism in response to temperature [D. subobscura associated with thermotolerance dispersed across all chromosomes has revealed that breakpoints do not disrupt any candidate gene directly associated with temperature adaptations, thus further corroborating the genetic complexity of thermal traits [By origin, of 18 °C . With mola genus ,23,24. Sla genus ,18,25. Tla genus , is consla genus ,26,27 asla genus , but allla genus ,30. The la genus . Sequencryotypes ,32. Expe pattern ; however regions . Howeverperature  and diffl traits ,39,40,41Although molecular genetic analysis has provided deeper insights into genes and mechanisms involved in temperature adaptation in this species, many evolutionary issues still remain unresolved. Despite the power of sequencing technology for the characterization of inversion polymorphisms, the cytogenetic technique, although with lower resolution, still remains a valuable tool for the analysis of the adaptive significance of this structural chromosomal polymorphism.D. subobscura individuals originating from two different altitudes by combining traditional cytogenetic techniques with assessing the levels of Hsp70 protein expression. Inversion polymorphisms were assessed in the flies sampled from natural populations and after 5 and 16 generations in laboratory conditions at three different temperatures. The Hsp70 protein expression profile was performed on the 12th generation at the basal level and after heat shock induction. Laboratory temperature regimes were chosen as stressful but not lethal for D. subobscura, with 16 °C prolonging development time, 19 °C considered as optimal, and 25 °C as a temperature which may induce male sterility [Our previous work on the effect of population history and different laboratory thermal conditions, performed with populations from different altitudes, indicated that both factors shape responses to stressful conditions . In the terility ,25. The Wild caught females were sampled in mid-August on Stara planina mountain, in Serbia, from two different altitudes: 1080 m and 1580 m . After establishing isofemale lines (IF) in the laboratory, their progeny  was mixed to make two mass laboratory populations that differ in sampling location: high altitude (marked as H) and lower altitude (marked as L). The first generation progeny of these two laboratory mass populations was used to establish three experimental groups per population which differed in their maintenance temperature: 16 °C, 19 °C and 25 °C. There were six experimental groups in total, reared on standard Drosophila medium , at approx. 60% relative humidity, light of 300 lux and 12/12 h light/dark cycles.D. subobscura males or F1 males of individual females from natural populations and with those from F5 and F16 generations, in all experimental groups from different temperature regimes. Each unique cross between 4 and 5 virgin females from the Küsnacht laboratory strain and between 25 and 35 males from each sample was performed. The Küsnacht strain was used to identify chromosomes with inversions in the progeny since it is homokaryotypic for the standard chromosomal configurations on all five chromosomes . The sample size of the studied populations is given in Inversion polymorphism analysis was performed with wild Experimental groups established twelve generations earlier were maintained at the above-mentioned temperature regimes and conditions. Their female and male freshly emerged progeny was collected and separated in order to prevent mating. For each assay, approximately 50 ♀ and 50 ♂ virgin flies were used per experimental group. Five flies were placed into empty falcon tubes (50 mL volume) with moistened plugs to prevent desiccation and placed in an incubator; set to 37 °C for 90 min. Flies were allowed to recover at room temperature for 30 min and then frozen at −80 °C for further analysis. To measure basal levels of Hsp70 expression, the same number of flies from each experimental group were collected and frozen in the same way.A total of six biological replicates per group were analyzed. All protocols were performed according to the manufacturer’s instructions. Homogenizations of five flies per experimental group and per sex  were performed in 1.0 mL of a suitable mix  following the protocol in . The totO) and expected (HE) heterozygosity, were calculated using Arlequin ver. 3.5.2.2 software under the Hardy–Weinberg equilibrium model [STF values and Nei’s distances. The statistical significance of all performed tests was assessed with 10,000 permutations. The matrix of pairwise population STF values was visualized using a multi-dimensional scaling method (nonmetric MDS) implemented in the PAST 4.03 software [Population genetic analysis was based on the frequencies and distribution of different inversions. For the analysis of differences in individual chromosome arrangements we used Z statistics. Observed  we used karyotypes of each individual. This method consists of performing the linear discriminant analysis (LDA) on the principal components analysis (PCA)-transformed matrix. LDA was performed on the first 14 PCs, cumulatively conserving 98.9% of the total variance. Additionally, the PCA-transformed matrix was used to find the optimal number of clusters using Ward’s method. We tested 2–50 clusters, and the optimal number of clusters was chosen using BIC statistics using the “diffNgroup” method. The relative expression of Hsp proteins was analyzed using the full factorial general linear model (GLM) procedure and Fisher’s post hoc test in the STATISTICA ver. 12 with fixed factors: Population, Rearing temperature, Treatment and Sex.O) was detected in the H natural population, with a general trend of higher HO in natural compared to laboratory populations, and a weaker trend of higher HO in laboratory populations derived from H then its L counterparts. Inversion polymorphism frequencies are shown in 3+4 is the most frequent arrangement for the O chromosome, but with significantly lower frequency in the L population. Complex arrangements in the O and E chromosomes are mostly absent from the L lab populations but retain small frequencies in the H lab populations regardless of temperature conditions. It is interesting to note that arrangement frequencies in the J chromosome behave differently in the H and L populations. While Jst in the H natural population has a much lower frequency than J1, its frequency rises in laboratory conditions, but the opposite trend is observed for the L population where J1 is dominant in the natural population and preserves its frequency in laboratory conditions. In the H population, after five generations at different temperatures, the Ost is more frequent in warm conditions, but after 16 generations of standard arrangements in A, J and O chromosomes have significantly higher frequencies in both cold and optimal conditions compared to the warm. In the L population after five generations, there are no differences in Ost frequencies at different temperatures, but it is more frequent in warm conditions after 16 generations.Standard parameters of genetic diversity per group are shown in STF statistics  with an inferred number of clusters and multi-dimensional scaling (MDS) showed a similar pattern of population differentiation . The natWhen the H and L populations were analyzed separately, strong differentiation could be observed between the natural populations and corresponding groups reared in laboratory conditions, and such differentiation was more prominent in populations from the higher altitude .p = 0.000668). Besides this, individuals in the H group tended to have higher levels of expression, rising with temperature with no difference between sexes.The results of the relative Hsp expression in each experimental group are presented in Although no significant interaction was observed between the combinations of factors, it is interesting to note the results of Fisher’s post hoc analysis of population/rearing temperature/treatment . IndividD. subobscura has been in the focus of scientific research for over seven decades, as one of the first genetic variability markers linked to a species adaptation to climate change [D. subobscura is considered to be adaptive [Abundant inversion polymorphism e change . Althouge change ,48, recee change . Naturaladaptive ,13,18,47D. subobscura from two altitudes in constant laboratory conditions under three thermal regimes, and assessing inversion polymorphism change over generations. Furthermore, we assessed whether adaptation to different thermal regimes in laboratory conditions influences the expression of the Hsp70 protein, both on a basal level and after heat shock.We attempted to experimentally validate the decade-old assumption that temperature is the main force shaping the distribution of chromosomal inversion frequencies across latitudes and climates by maintaining two distinct populations of 3+4 increasing in warm conditions. In laboratory groups derived from the L population, no such pattern could be detected, and Ost frequency was elevated in warm conditions. For other chromosome arrangements associated with thermal response [D. subobscura [Under the assumption that temperature was the main selective factor forming latitudinal clines ,25,47, cresponse , no cleabobscura ,20, suggbobscura ,25,52,53bobscura ,55,56,57bobscura ,59,60. STF values. A similar pattern was observed at the chromosomal level by [1 in L groups, irrespective of the thermal regime. Higher heterozygosity may be associated with the distribution of differentially expressed genes dispersed across all chromosomes [Further evidence that population history may have an effect on how the population responds to temperature changes is observed in differences in population structure. Our chosen natural populations from different altitudes across the mountain slope of Stara planina showed initial differences in inversion polymorphism, and that differences are preserved across generations and laboratory conditions with high level by , where tlevel by . In addiomosomes , providiDrosophila species is not ubiquitous [D. subobscura there is clear evidence of a plastic response to thermal variation [The observed changes in the Hsp protein expression patterns are in line with the observed differences between the H and L populations in laboratory conditions based on inversion polymorphism. Although not significant, the observed increase in Hsp70 levels with rising temperature as well as different patterns of expression in the H and L population further confirm the influence of population history. Expression levels are lower in H groups without temperature stress, but after heat exposure, all H groups have higher expression levels compared to their L counterparts, indicating a better ability to alleviate the influence of the extreme rise in temperature. Moreover, thermal shock induces higher protein expression, but significant difference between the basal- and temperature-induced levels could be observed only in H groups reared in cold and optimal conditions. Hsp protein expression is a balance between their protective role in various stressful conditions ,63 and tiquitous ,76,77,78ariation .D. subobscura showed the presence of population variation in evolutionary potential for long-term thermal adaptation suggesting that environments with higher thermal variation and pronounced thermal extremes may enhance reproductive success at higher temperatures [Recent studies with eratures . Our resD. subobscura populations, pushing them to their upper thermal limits, but the contribution of some other factors, such as population history, has to be considered. The results obtained in our study reveal that local adaptations significantly shape the populations’ response to the changing temperature as one of the climate parameters. Combined with phenotypic assays and functional expression of relevant proteins, chromosomal inversions and frequency changes of gene arrangement seem to be a relevant marker for inferring population structure, differentiation, and ability to withstand environmental challenges, such as temperature stress.Climate change and rising temperatures strongly influence changes in the genetic structure of natural


High physical work demands can cause musculoskeletal disorders and sick leave in petroleum workers. However, our knowledge of their physical work demands is scarce and based on self-report. The objective of our study is to work towards closing this knowledge gap by assessing the physical work demands of onshore petroleum maintenance workers using body-worn sensors.A total of 46 of 69 eligible maintenance workers (37 mechanics and 9 process technicians) from three onshore petroleum facilities in Norway filled in a questionnaire and diary and wore five accelerometers and a heart rate sensor for up to six consecutive workdays. Work-related physical activity and postures were classified using rule-based modelling in a modified version of the validated Acti4 software.The onshore maintenance petroleum workers were working an average of 10 h a day and spent on average this time with 48% (SD = 16.5) sitting, 1% (SD = 2.8) lying down, 39% (SD = 16.2) in light physical activity, and 9% (SD = 3.8) in moderate to vigorous physical activity. During work hours while at feet, we found arm elevation ≥60° to be 11% (SD = 7.1) (68 min), and forward bending of the trunk ≥60° to be 2% (SD = 2.2) (14 min). The workers spent 2% (SD = 2.5) (12 minu) of the workhours kneeling. We observed a high inter-individual variation for all these work exposures. Moreover, 26% (12) of the workers conducted static standing for >30% of the workday, and 17% (8) spent more than half of the work hours >33% of their estimated maximal cardiovascular capacity.While onshore maintenance petroleum workers on average spend about half of the workday sitting or lying down, the remaining worktime is spent with a rather high duration of arm elevation and forward bending. Quite high fraction of the workers spends much of the workhours in static standing and kneeling. We see a substantial variation in these work exposures between the workers. The findings indicate a need for preventive measures in how work is organized and performed. This study is important because it comprehensively measured the physical work demands among petroleum maintenance workers. The workdays involved rather long durations of arm elevation, forward bending and kneeling, and static standing; with high variation observed among workers. These findings indicate a need to modify how work is organized and performed to prevent musculoskeletal disorders and pain, injuries, disability pension, adverse cardiovascular events and sickness absence.Workers in the petroleum industry are often exposed to challenging working environments. From 1992 to 2003, 47% of the total illness cases reported in the offshore petroleum industry in Norway were due to work-related musculoskeletal disorders . AccordiMechanics and process technicians perform primarily maintenance tasks, such as troubleshooting, disassembly, installing, repairing machines and piping systems, design and construction of process facilities, changing, and adjusting various components on gas tanks, pumps, and valves  were recruited from three of eight onshore petroleum facilities in Norway. All workers were provided written and oral information about the research project and gave written consent prior to the study. Inclusion criteria included that the primary work tasks had to be manual material handling. Exclusion criteria were: (i) office work, (ii) night work, (iii) physical disability not allowing normal behaviour, (iv) pregnancy, and (v) bandage, band aid, or adhesive allergies. The study was conducted according to the Declaration of Helsinki and approved by the Regional Committees for Medical Research Ethics, Central Norway (No.: 190964). We have previously investigated the physical work demands of home care workers and the description of the methods and results are partly overlapping  were used for baseline measurements of height and weight.Technical devices can pose a risk of ignition under certain operating conditions in petroleum facilities. Thus, risk assessment of the devices utilized in this research project was carried out, and a temporary approval for use of equipment at the given locations was given.Five triaxial AX3 accelerometers  were attached to the skin of participants, using double-sided adhesive tape  and sealed with waterproof medical tape . The sensors were worn 24 h each day for a period of up to six consecutive workdays with a sampling frequency of 25 Hz and a range of ± 8G. The accelerometers were mounted: (i) below the head of the fibula, on the proximal and lateral position of the calf for classification of kneeling, (ii) on the distal, anterior, and medial position of the femur (approximately 10 cm above the superior crest of the patella), (iii) approximately 10 cm below the iliac crest of the hip for classification of lying, sitting, moving, walking, running, stair climbing, and running, (iv) on the upper back at the level of Th1–Th2 vertebrae for classification of forward bending of the trunk, and (v) on the dominant upper arm, approximately at the insertion (tuberositas deltoidea) of the deltoid muscle for classification of arm elevation    was used for configuration of the AX3 accelerometers, and a modified version of the custom-made MATLAB software Acti4 , running, cycling, stair climbing, arm elevation, forward bending of the trunk and kneeling with high sensitivity and specificity (≥95%)  sensor calibration, (ii) working hours, (iii) after working hours, and (iv) sleep. After processing all data, a batch analysis was conducted in the Acti4 software and imported to a CSV file. Working hours from the dataset were derived using Python . At least two workdays with ≥4 working hours each day were required from each worker to be eligible for further analysis  of the worktime. The fraction of workhours spent statically standing and kneeling, and with arm elevation and forward bending of the trunk was rather high. The cardiorespiratory aerobic workload was measured to be within the recommended ‘safe limits’. Notably, there was an extensive uneven distribution of physical work demands among workers, meaning that some workers were exposed to much higher physical work demands than others.In contrast to the well-documented benefits of leisure-time physical activity, large prospective cohort studies report that high amounts of work-related walking and lifting are risk factors for adverse cardiovascular events (The average time spent static standing was 26% (about 2.6 h), with a considerable inter-individual variability . As muchOn average, onshore maintenance petroleum workers were well within the proposed safe threshold for cardiovascular workload. A proposed upper safe limit by the International Labor Organization for whole-body work is that workers on average, should be within one third of their maximal aerobic capacity during an 8-h workday , and morRegarding demanding postures, we found that on average, onshore maintenance petroleum workers were conducting arm elevation while on feet ≥30° for 43% (254 min), ≥60° for 11.3% (68 min), and ≥90° for 1.8% (11 min) during working hours. Moreover, a high inter-individual variation was found. The Danish Dphacto cohort used the same methods to assess physical work demands in various blue-collar professions, where manufacturing was perhaps the most comparable to the workers in the present study. Danish manufacturers were exposed to ≥60° of 6.3% and ≥90° of 1.3% arm elevation while on feet ,B, showeWe found that the average levels of exposure to work-related forward bending of the trunk while on feet ≥30° was 7.5% (47 min) and ≥60° was 2.2% (14 min) with a high inter-individual variation. Onshore maintenance petroleum workers were conducting less work-related forward bending of the trunk than manufacturers (≥60° of 3%) in the Danish Dphacto cohort . AlthougOnshore maintenance petroleum workers were on average conducting 1.8% (12 min) of work-related kneeling. Additionally, we found a high inter-individual variation with some workers exposed to durations of kneeling for up to 40 min during working hours. There were only comparable data of device-worn measurements of kneeling from Danish childcare workers (2.5%) (Predominant preventive strategies to reduce musculoskeletal pain has been by implementing ergonomic aids to reduce loads, or by organizing work so that it is varied and do not cause harm . DespiteA limitation of the current observational study was that only 67% of the eligible onshore maintenance petroleum workers completed the study. Thus, we cannot eliminate the possibility of selection bias, as workers not willing to participate could have different demographics, for example, higher age and poorer health. Due to a limited time window of one week of data collection in each onshore petroleum facility, we only had access to process technicians assigned to these dayshifts; therefore, the number of available process technicians was lower than for mechanics as fewer process technicians were working day shifts. In addition, we had only access to three of eight onshore petroleum facilities in Norway. As sensors were only mounted on one side of the body, it is possible that the amount of kneeling was underestimated (This study helps to fill a knowledge gap on the physical work demands of onshore maintenance petroleum workers by using device-worn measurements. While onshore maintenance petroleum workers on average spend about half of the workday sitting or lying down, the remaining worktime is spent with a rather high duration of arm elevation, forward bending and kneeling. Quite high fraction of the workers spend much of the workhours in static standing and kneeling. We see a substantial variation in these work exposures between the workers. The findings indicate a need for preventive measures in how work is organized and performed. We encourage future research to use this knowledge to investigate health-promoting measures for onshore maintenance petroleum workers.


DNA metabarcoding is rapidly emerging as a cost-effective approach for large-scale biodiversity assessment and pest monitoring. The current study employed metabarcoding to assess insect diversity in citrus orchards in Ganzhou City, Jiangxi, China in both 2018 and 2019. Insects were sampled using Malaise traps deployed in three citrus orchards producing a total of 43 pooled monthly samples.The Malaise trap samples were sequenced following DNA metabarcoding workflow. Generated sequences were curated and analyzed using two cloud databases and analytical platforms, the barcode of life data system (BOLD) and multiplex barcode research and visualization environment (mBRAVE).H′) showed a similar pattern of BIN assortment at the three sampling sites. Beta diversity analysis by Jaccard similarity coefficient (J) and Bray-Curtis distance matrix (BC) revealed a high level of BIN similarity among the three sites . Comparison of BIN records against all those on BOLD made it possible to identify 40% of the BINs to a species, 57% to a genus, 97% to a family and 99% to an order. BINs which received a species match on BOLD were placed in one of four categories based on this assignment: pest, parasitoid, predator, or pollinator. As this study provides the first baseline data on insect biodiversity in Chinese citrus plantations, it is a valuable resource for research in a broad range of areas such as pest management and monitoring beneficial insects in citrus gardens.These platforms assigned the sequences to 2,141 barcode index numbers (BINs), a species proxy. Most (63%) of the BINs were shared among the three sampling sites while BIN sharing between any two sites did not exceed 71%. Shannon diversity index ( China ranks among the top three countries globally for citrus cultivation and production . In factcytochrome c oxidase I gene (COI), has gained global acceptance for specimen identification and species discovery (www.boldsystems.org). The BOLD system assigns eligible barcode sequences  to barcode index numbers (BINs) which serve as proxy for species  based on HTS of a specific DNA marker . It diffcpis.hzau.edu.cn) and the literature to allow their classification into pest, parasitoid, predator, or pollinator  and large . Large specimens were subsampled to obtain a tissue block  of a size similar to the small ones while small specimens were used in entirety. This was done to achieve comparable tissue-mass representation for all specimens in the DNA extracts. The specimens/tissue samples from each bottle were mixed, frozen in liquid nitrogen and then ground to a fine powder using a disposable mortar and pestle. DNA was extracted with the TIANamp Genomic DNA kit  following manufacturer’s protocols. Briefly, 500 mg of the powder was lyzed in 2 ml Buffer GA overnight at 56 °C in the presence of 20 µl proteinase K. The lysate was centrifuged at 5,000 g for 10 min and the supernatant was then aliquoted 40 µl into ten equal volumes for DNA extraction. The quality and purity of the DNA was assessed using a Nanodrop 2000 and 1% agarose gel electrophoresis. Three of the 10 DNA extracts from each sample were randomly selected for PCR, creating a total of 129 DNA extracts for analysis.A single Malaise trap was deployed in three citrus orchards  about one kilometer apart in Ganzhou City, Jiangxi Province, China . SamplesA first PCR was used to amplify the target region of COI while the second PCR added adapters to allow discrimination of the sequences derived from each DNA extract . The firTaPurified PCR2 products (126 reactions) were sequenced separately on an Illumina Miseq PE300 platform following standard protocols. Each reaction had two sequencing replicates to generate 252 products for sequencing. The amplicon libraries were prepared using MiSeq Reagent Kit v3 . Briefly, the pooled library was thawed on ice along with HT1 (hybridization buffer), and then diluted to 2 nM in EB buffer. A total of 5 µl of the library were mixed with 5 µl of NaOH at 0.2 N in a microcentrifuge tube with brief vortexing and 1 min of centrifugation at 280 g. After 5 min incubation at room temperature, 990 µl pre-chilled HT1 was added to the tube containing denatured library providing 1 ml of a 10 pM denatured library. The denatured library was diluted to 8 pM  by inverted mixing and then pulse centrifugation. Subsequently, the libraries were loaded onto the reagent cartridge to set up the sequencing run. After cleaning the flow cell, the reagents were loaded into the flow cell to initiate the sequencing.www.mbrave.net). mBRAVE integrates common analytical methods and links to the BOLD system for access to reference datasets (www.mbrave.net) under the project “MBR-MTCHN1” where they were analyzed using a standard pipeline  statistical environment . To reduDiversity and species richness analyses were performed in R (package “vegan”) using BINs (as species proxies) recovered from the HTS sequence data. Alpha diversity at individual sites was analyzed by Shannon-Wiener index  while thH′) value  for the three Malaise sites. Biodiversity overlap determined by Jaccard similarity coefficient (J) suggested a high level of BIN sharing among the three sites in pairwise comparisons . Beta diversity assessed by Bray-Curtis index showed a low level of BIN dissimilarity in the pairwise site comparisons . All samples combined yielded 2,141 BINs, but estimates range between 2,233 (Chao), 2,260 (Bootstrap) and 2,373 (Jack1). At all three sampling sites, most BINs  were encountered only once in the 14/15 collection events and just a few  were detected in all samples. However, the variance (R2) of BIN occurrence in the collection events at the three sampling sites remained low  of the BINs showed a match to a known species while 59%  were placed to a genus, 97%  to a family, and 100%  to an order. However, a check of the correspondence between the BINs and their associated taxonomy revealed 235 discordances at the species level, 98 at the genus level and 20 at the family level. At GAN site, 220 discordant BINs were found at the species, 85 at the genus and 15 at the family level. At QIU, 218 discordant BINs were found at the species, 87 at the genus and 18 at the family level. At SHI, 216 discordant BINs were found at the species, 89 at the genus and 19 at the family level .Most (96%) of the 2,141 BINs were linked to 14 insect orders, with a 49% to Diptera, 22% to Hymenoptera, 14% to Lepidoptera, 5% to Coleoptera, and 4.5% to Hemiptera. 875 of the 2,141 BINs were linked to a known insect species which included 443 pests, 223 pest/pollinators, 140 parasitoids, 52 predators, two predator/pollinators and 15 pollinators .Challenges of morphology and lack of taxonomic experts have limited the understanding of insect pest and natural enemy complexes in citrus orchards, compromising the efficacy of pest management tactics . The preBINs were used as a proxy to count species and to link the generated sequences to species represented on BOLD. This approach not only revealed the presence of potentially more than 2,000 insect species, but it also linked 866 (40%) of the BINs to known insects. This result supports the utility of BINs as an effective approach for counting species  and asseS = 0.67–0.68) and low beta diversity values (BC = 0.19–0.20). This means that there was little variation in the insect species at these locations. There are several possible reasons for the low insect beta diversity. One reason could be that due to a similar habitat of citrus plantations and small distance (1 km) between the three traps the physical characteristics of the habitat were relatively similar across a wide area, leading to a homogenization of insect species. For example, if the vegetation and climate are very similar across a region, or the geographic range is small, it may be more likely that the same insect species will be found throughout that region  of the BINs were common among sites, and this was reflected in the high alpha diversity  that are potential biological control agents for Lepidoptera (Biological control is the most environmentally safe and cost-effective pest management strategy. The present study revealed the presence of BINs linked to known species of Diptera and Hymenoptera which may have potential as biological control agents. For example, BINs linked to 32 species of Braconidae were encountered which belong to three genera (nine idoptera . In addiidoptera .In summary, metabarcoding of bulk insect collections provides a cost- and time-effective way to assess insect communities in citrus orchards. However, the present study only examined one region. Constructing a comprehensive insect inventory for citrus orchards across China will require surveys at the representative sites in other citrus areas.Our study analyzed the composition of insect communities in citrus orchards using DNA metabarcoding followed by data analysis on BOLD and mBRAVE. The species revealed by BIN matches on BOLD were then searched in citrus pest database and the literature to allow their classification into pest, parasitoid, predator, or pollinator. Our results provide a valuable resource for research on citrus pest management and beneficial insect exploration.10.7717/peerj.15338/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.15338/supp-2Supplemental Information 2Click here for additional data file.


Carica papaya L.) is one of the few fruit crops still propagated by seeds. However, its trioecious condition and the heterozygosity of the seedlings make urgent the development of reliable vegetative propagation procedures. In this experiment, we compared, in a greenhouse sited in Almería (Southeast Spain), the performance of plantlets of ‘Alicia’ papaya originated by seed, grafting, and micropropagation. Our results show that grafted papayas were more productive than seedlings papayas , while in vitro micropropagated papayas were the least productive . Root density and dry weight were both higher in grafted papayas, while the seasonal production of good quality, well-formed, flowers was also enhanced in grafted papayas. On the contrary, micropropagated ‘Alicia’ plants yielded less and lighter fruit despite these in vitro plants blooming earlier and setting fruit at desirable lower trunk height. Less tall and less thick plants and reduced production of good quality flowers might explain these negative results. In addition, the root system of micropropagated papaya was more superficial, while in grafted papayas, the root system was larger and had more fine roots. Our results suggest that the cost-benefit ratio does not favor the choice of micropropagated plants unless elite genotypes are used. On the contrary, our results encourage more research on grafting, including the search for suitable rootstocks for papaya.Papaya ( Carica papaya L.) is a tropical fruit crop of growing interest due to its enormous productivity and rapid entry into production. Agricultural statistics indicate an average yield of 30 t ha−1, but high variability exists depending on the country and cultivation system. According to FAOSTAT [−1 per year. Unfortunately, papaya lifespan in a greenhouse is limited to around 2 years because its rapid growth makes the plants reach the roof of the greenhouses at that time. Very tall papayas also lead to higher costs in harvest and in flower and fruit thinning operations. Papaya  and gynoecious plants (producing only pistillate flowers). However, the appearance of hermaphrodite plants by mutation has allowed their commercial production by directed crosses . GrowersThis system of seed multiplication presents many drawbacks. The more obvious are, the higher expenses of buying more plantlets than finally necessary; also, the additional costs of the initial cultivation of the surplus seedlings and the negative effects of the competition among them on plant growth . Plant sDespite the very remarkable interest in developing multiplication by grafting , the resThe objective of this work is to compare in a greenhouse the growth, rooting, yield, and fruit quality in ‘Alicia’ papaya cultivar obtained by seeds, grafting, and in vitro micropropagation in order to recommend a propagation system for protected cultivation.The monthly average temperature inside the greenhouse ranged from 14.6 °C in January 2021 to 28.1 °C in July 2020. The average monthly minimum temperatures ranged from 10.3 °C in January and February 2021 to 23.4 °C in July 2020, while the monthly maximum temperatures ranged from 21.6 °C in January 2021 to 35.6 °C in September 2019. On the other hand, the monthly mean relative humidity (RH) fluctuated between 59% recorded in June 2019 and 83% in December 2019, being the mean RH of the minima between 34% in June 2019, and 69% in December 2019, while the mean RH of the maxima ranged between 82% measured in March 2021 and 88% in December 2019.‘Alicia’ papayas propagated by seed, grafting, and in vitro followed a similar growth pattern during the first year . HoweverAll plants grew rapidly during the initial period (June–December 2019), exceeding PHT 200 cm in all treatments B. Reduce−2, respectively) than seedlings (106 roots cm−2). Heavier root dry matter (3.51 and 3.80 kg per plant) was obtained for the formers compared to 2.80 kg of roots per plant in seedlings. Similar root distribution pattern in depth was observed in all treatments. The highest roots density (205 roots cm−2) was found near the surface (10–15 cm), followed by medium depth (25–30 cm) with 114 roots cm−2 and finally by the greatest depth analyzed (40–45 cm) (32 roots cm−2) with smaller differences among soil depths at the furthest distance from the trunk analyzed (50 cm apart) (−2), evidence of a more horizontal roots distribution  . Despiteribution , explainribution . In thisThe lowest insertion points to the trunk for the first flower and first fruit were measured for in vitro papayas, as previously observed by Fitch et al. , indicatThe seasonal frequency of different floral types was evaluated by inspecting all open flowers on each sampling date. The highest percentage of elongata flowers, hermaphrodite flowers that later will produce commercial fruits, were obtained in all treatments in autumn and spring . Spring Harvest started in February or March 2020, depending on the treatment; it was carried out on 10 and 24 February for in vitro and seedlings plants, respectively, and later, on 10 March, in grafted papayas, likely an expression of the higher vigor of the latter. The total and commercial yield was higher  in grafted than in vitro plants. The plants that originated directly from seeds showed intermediate values . DiscardVegetative propagation provides clear advantages, such as higher yield, if selected elite plant material is selected ,6,18,19.In the evaluation of fruit quality carried out in June, fruits from in vitro plants were again smaller and significantly lighter than those harvested from seedlings but significantly sweeter (an average > 9 °Brix) than those from the other two propagation treatments . No signIn addition to the fruit quality evaluation in June, the total soluble solids content (TSS) of fruits of the propagation procedures was compared on different dates. In these measurements, we observed that the TSS values were again higher for in vitro plants in April and May of 2020 but lower one year later, although at that time, the differences were slight . In earl2 area).This study was carried out at the Cajamar Experimental Station ‘Las Palmerillas’, located in El Ejido  . The papaya crop grew in a multi-tunnel type greenhouse made of galvanized steel, with a 200 μm thick asymmetrical low-density polyethylene cover. The greenhouse consists of eight spans E-W oriented, 7.5 m wide each, with 3.4 m height to the eaves and 5.4 m to the ridge. The trial took place in a southern-oriented individual module composed of four spans , obtained by different propagation procedures, were compared. Seed-propagated plants, selected in a sexing process based on the use of molecular markers, were compared to plants obtained by vegetative propagation procedures, specifically grafting and in vitro multiplication . For gra−1 double emitter spaced 0.1 nozzles m−2 and 1 L m−2 h−1 flow, activated when the relative humidity was below 60%. A heating system consisting of a hot flow air Ermaf RGA95 (Elster), with diesel as fuel, was activated during cold periods (from November to March) when the temperature was ≤12 °C. Temperatures and relative humidity inside the greenhouse were recorded during the experiment.Natural ventilation through a zenithal window per span and a lateral panel improved climate conditions inside the greenhouse. The temperature to open greenhouse windows was managed through a Priva weather controller and set at 24 °C. In addition, roof whitening was performed to improve the climate conditions inside the greenhouse in summer. The greenhouse climate was regulated with a cooling system consisting of a low-pressure nebulization system CoolNet Pro (Netafim), with 5.5 L h2 subareas at three different soil depths  and later similarly at three distances from the trunk , obtaining thus the root density at each soil depth and trunk distances on both cardinal sides (East and West) (Plant height from the ground to the head (PHH) and from the ground to the top of the canopy (PHT), that is, including erect leaves, were measured monthly using a graded bar. The trunk perimeter (TP) at 15 cm from the ground was measured with the same periodicity using a tailor tape ruler. After pulling up the plants in November 2021, the power and extension of their root system were studied. The root analyses proceeded as follows: a trench (40 cm wide × 100 cm long) was made in the east and west positions of the plants, 50 cm away from the trunk. Then, the roots in the trench were counted with the help of a mesh with 10 cmnd West) . Then, tnd West) A at one nd West) B to estiThe distance from the ground to the first flower and to the first fruit was measured using a tailor tape ruler. All flowers in anthesis were inspected from the beginning to the end of the blooming period (August 2019 to June 2020) to determine the frequency of elongata flowers  and also of non-elongata, pentandric, and carpeloid misshapen flowers, and of functionally male flowers . The seaFruit quality was evaluated on 5 June 2020, just before the end of the trial. For this, one fruit per plant  was collected at 50% of yellow skin and analyzed four days later, when they showed their skin 100% yellow. Fruits were weighed on a balance (d = 0.1 g) , and their longitudinal and equatorial perimeter were measured using a tailor tape ruler, as it was the cavity width, with a digital caliper . Pulp firmness was determined on each fruit in two opposite equatorial points using a digital penetrometer  after removing the skin of the fruit. Skin and pulp color were determined in three positions of each sampled fruit, with the help of a colorimeter . These results were expressed considering hue angle (hue°), which indicates the color tone of the fruit so that a hue angle of 0° corresponds to red color, 45° orange color, 60° to yellow-orange color, 90° to yellow color and 120° to yellowish green color. Total soluble solids content (TSS) and titratable acidity (TA) of the juice of the fruits were also evaluated. TSS was measured using a digital refractometer , while TA was assessed by titration with 0.1N NaOH, using phenolphthalein as an indicator, and expressed as grams of citric acid per liter of juice. In addition, TSS has compared during the experiment. A randomized complete block with three treatments  and three replicates was designed. A tree row per treatment constituted each replicate, in which the four central trees per row (12 plants per treatment) were selected for measurements, thus avoiding border plants. The data were subjected to analysis of variance (ANOVA), and the means were separated by Tukey’s Test using R software version 4.2.1 .Our results show that micropropagated ‘Alicia’ plants produced less and lighter fruit, despite these plants bloomed earlier and set fruit into a lower trunk height. Less tall and less thick plants with a reduced production of good-quality hermaphrodite flowers might explain the negative results. More superficial root distribution in micropropagated plants has penalized too this treatment in terms of total and commercial yield. On the contrary, grafted papayas performed better than micropropagated plants and than seedlings, without differences with the latter above ground, because no selected (elite) material was used as scion. The difference between them is that in seedlings, the root system is from hermaphrodite plants, while in grafted plants, the root system comes from female seedlings, and the latter had more root density and a larger root system. Vasconcellea and the species Carica pentagona.Further confirmation of our results might open a new vegetative procedure for producing more productive papayas. Knowing that obtaining propagules as rootstocks from mother plants is costly and that rooting plants in vitro are not easy nor cheap, our results encourage more research on grafting, including the search for suitable compatible rootstocks for papaya. We initiate this search within the genus


Our results confirm that chelonians bioaccumulate uranium radionuclides and do so sequentially over time. This technique provides both a time series approach for reconstructing nuclear histories from significant past and present contexts throughout the world and the ability to use chelonians for long-term environmental monitoring programs .Chelonians  grow scute keratin in sequential layers over time. Once formed, scute keratin acts as an inert reservoir of environmental information. For chelonians inhabiting areas with legacy or modern nuclear activities, their scute has the potential to act as a time-stamped record of radionuclide contamination in the environment. Here, we measure bulk (i.e. homogenized scute) and sequential samples of chelonian scute from the Republic of the Marshall Islands and throughout the United States of America, including at the Barry M. Goldwater Air Force Range, southwestern Utah, the Savannah River Site, and the Oak Ridge Reservation. We identify legacy uranium ( Since chelonian scute grows sequentially over time, this approach provides the ability to reconstruct nuclear fallout or contamination histories and conduct environmental monitoring, in localized settings. We identify uranium signatures relating to historic nuclear testing or accidental release in a green sea turtle from the Republic of the Marshall Islands, a desert tortoise from southwestern Utah (United States of America), a river cooter from the Savannah River Site, and a box turtle from the Oak Ridge Reservation.Chelonians  bioaccumulate anthropogenic radionuclides (e.g. 235U and 236U) in a variety of chelonian scute tissue from areas with a known history of contamination from nuclear testing  and/or nuclear processing (waste). Our research highlights that Bert's fictional experience was in some ways true—turtles, tortoises, and sea turtles live in areas with historic nuclear activity and may have survived the immediate effects of nuclear events—but our study also confirms that the record of this nuclear activity is present and measurable within these same animals long after first exposure.In a 1952 United States Civil Defense Administration educational film titled “Duck and Cover”, an animated turtle named “Bert” showed the American public what to do in case of a nuclear attack—the words were simple, you should “duck” and “cover”, and for millions of schoolchildren, this meant seeking shelter under their desks. Shelter for the anthropomorphic Bert involved a functional characteristic present in some chelonian taxa , the ability to retract his head and feet into his bony shell. Although a fictional depiction, Bert provides a useful analogy for understanding the effects of nuclear activities in the environment today. Effects from nuclear activities leave a legacy of specific elemental and isotopic signals in environments near and far from nuclear sites or detonation locations . In this3 of contaminated soil and 1.8–4.7 billion m3 of contaminated water from legacy nuclear activities .Globally, there are numerous potential areas to investigate legacy radionuclide bioaccumulation in chelonians. Throughout the 20th-century Cold War, the United States developed a large nuclear weapons complex, involving multiple sites across the country devoted to producing fissile and other nuclear weapons materials from natural uranium. These early activities produced contamination in the environment, before our understanding of its impact on the environment led to better management practices. In the United States alone, there is an estimated 30–80 million m235U) involved enrichment to produce highly enriched uranium  for direct use in nuclear weapons or for use in nuclear reactors to produce fissile plutonium. Depleted uranium , a by-product of both enrichment and reactor processes, was produced in large quantities and initially found limited use, so was stored or disposed of as waste . Significant amounts of 236U were also produced anthropogenically by thermal neutron activation of 235U during nuclear fission, and a lesser amount by the 238U(n/3n)236U nuclear reaction. When input into the environment, this anthropogenic 236U easily overrides the natural 236U/238U signature , and provides a clear indicator of contamination through anthropogenic uranium  scute collected on 1978 May 1 from the stomach of a tiger shark caught by line fishing off a pier at Enewetak Island, Enewetak Atoll, Republic of the Marshall Islands (RMI). Enewetak Atoll, and neighboring Bikini Atoll, were the site of 67 nuclear weapons tests from 1946–58  collected on 1959 April 23 from Washington County, southwestern Utah. This scute is from an old, mature, individual tortoise with homogenized layers (i.e. no layer rings present due to its old age). Washington County is approximately 150 miles due east of the (formerly) Nevada Test Site  where the Atomic Energy Commission (now Department of Energy) tested 100 atmospheric nuclear weapons between 1951 and 1962. Radionuclide data indicate that regions of Washington County, southwestern Utah, such as St. George and Cedar City, received low levels of fallout from weapons tests approximately 5–6 times through 1959  collected in 1999 at the Barry M. Goldwater Air Force Range (BMGAFR) in southwestern Arizona. Unlike our other samples, this region experienced no direct input of anthropogenic uranium (e.g. no known use of DU munitions) and therefore represents a natural control from an arid environment.The third bulk specimen scute is from a Sonoran desert tortoise  collected from the Savannah River Site (SRS), South Carolina in 1985. From the 1950s to the late-1980s, the SRS was an important location of uranium fuel fabrication, target fabrication, and spent fuel reprocessing. A historical inventory of uranium waste disposed at the SRS indicated a total of 198.15 metrics tons present during this period from at least one location at the site, primarily comprised of fuel fabrication and spent fuel processing, including DU (The fourth bulk specimen scute is from a river cooter (2) in size. River cooters are aquatic, move easily within aquatic habitats, and may travel short distances by land if necessary  collected in 1962 from the Oak Ridge Reservation (ORR). Like the SRS, ORR was the site of significant uranium production and processing beginning in 1943 , we identified a large degree of variability in the uranium isotopic composition across the bulk scutes analyzed, including significant variations in both n nature .235U/238U deviated from that observed in natural terrestrial materials  and the green sea turtle from Enewetak Atoll, RMI (0.00981 ± 0.0002).In four out of the five samples analyzed, the aterials , and incaterials . Only th235U/238U results, an anthropogenic 236U signature was observed in four out of the five scute samples analyzed  × 10−5, 5.15(±0.20) × 10−5, and 1.42(±0.08) × 10−5, respectively, all clearly above analytical detection limits.Similar to the zed Fig. ; Table 1Macroscopic, scanning electron microscopy and energy dispersive X-ray spectroscopy (SEM-EDS) identified seven distinct yearly growth layers in the eastern box turtle collected in 1962 from ORR Fig. . The pos235U/238U isotopic signature in the box turtle from ORR. However, the degree of depletion varies over time (1955–62) with the most depleted signature in the neonatal portion (corresponding to 1955) with a 235U/238U = 0.0032  radionuclides, even when the total amount of uranium bioaccumulated is a minute fraction of the sample. In addition, the unique age and sampling locations of the turtles in this study present the opportunity to determine whether chelonian taxa provide historical information on the characteristics of uranium contamination from individual sites and nuclear events, and for environmental monitoring programs.Our data support previous studies on chelonian taxa—and numerous other organisms —determinWe recognize the limitations present in our study, specifically that our analysis only includes individual turtles from each location and time. Furthermore, due to fragmented and mature (homogenized) scutes present in our other samples, only at the ORR was there potential to sample sequentially grown scute to obtain a diachronic record. As elaborated below, this limitation does not minimize the significance of our results as our evidence clearly associates each turtle with nuclear activities at each site during the 20th century.Here, we discuss each individual sample in context to the environment of anthropogenic contamination that it was likely exposed to, based on historical knowledge of nuclear activities in each area. We conclude with a discussion of the wide potential for future research based on our findings.235U/238U and a 236U/238U isotope signature. While the measured uranium concentration in the scute is low (20.17 ng/g), the measured 235U/238U of 0.00981 ± 0.0002 is approximately a factor of 1.3 times higher than natural uranium (235U/238U = 0.007253–0.007256). We note that the RMI received a high degree of fallout from the testing of 67 nuclear weapons between 1946 June 30 and 1958 August 18. Fallout products at Enewetak Atoll contaminated the immediate environment, flora, and fauna, following each detonation  in Hiroshima, Japan . Our rea, Japan , 32. BlaThese results are significant when placing this green sea turtle in context of its date of collection. In May 1977, one year prior to the collection of this sea turtle in 1978, cleanup activities began at Enewetak Atoll resulting in the creation of the Runit Dome containment structure. Green sea turtles are migratory, but live, forage, and nest, at Enewetak Atoll , 34. The235U/238U ratio coupled with a minor 236U/238U signal. These results are notable, however, given the distance of this location from the (formerly) Nevada Test Site (∼150 miles due east). We note that southwestern Utah did receive nuclear fallout from testing at the NNSS during the 1950s and early 1960s —and previous research indicates that contamination and pollutants are passed through eggshells to offspring , the possible habitat of our box turtle. Variation in the isotope signature recorded in these successive layers indicates that the turtle continued to record environmental uranium contamination over time. Indeed, recorded data from the East Fork Creek highlight a consistent concentration of uranium (measured in mg/L) within this waterway during our study years  specifically in 1958.Although we do not have access to the mother of this turtle, the presence of anthropogenic uranium within this turtle's neonatal scute suggests that nuclear contamination passed between at least two generations of turtles at ORR during the mid-20th century. In addition, we note that the highly depleted uranium isotope signature matches historical records that show pre-1955 uranium waste streams at ORR were of a heavily depleted nature. For example, documented releases of wastewater from ORR's Y-12 Complex—originally constructed during the Manhattan Project to enrich uranium during the 1940s–1960s—resulted in depleted uranium contamination throughout the environment , 41, 42.ars Fig. ; 1955–62th-century nuclear deployment, testing, and production, we must now also consider chelonians and their lived experience in areas where these activities occurred. Our results confirm modeled expectations (When we consider the legacies of 20ctations  showing Mauremys japonica]), nuclear test histories (e.g. Kazakhstan steppe tortoise [Testudo (Agrionemys) horsfieldii]), or nuclear processing, production, and accident histories (e.g. Ukrainian pond turtles [Emys orbicularis]) will undoubtedly clarify the extent to which these reptiles bioaccumulate and reflect anthropogenic contamination in the environment. We anticipate that our green sea turtle results may influence future sea turtle-based environmental monitoring and tracking of contaminates at Runit Dome in the RMI, and potentially long-term releases of radioactive water from the Fukushima Daiichi reactors, into the Pacific Ocean.For example, given our limited sample size, we anticipate that future studies will focus on expanding the types of chelonians, and chelonian scute sequences, analyzed for anthropogenic radionuclides (including other nuclear elements). Turtles inhabiting locations with significant nuclear deployment histories , coralsWe obtained chelonian scute samples from natural history collections for this analysis. Prior to obtaining each sample, we received Los Alamos National Laboratory Institutional Biosafety Committee approval.To age the eastern box turtle specimen, we used macro and microscopic techniques. Four of our five chelonian samples only included a single bulk tissue specimen for this analysis, therefore, we accept the date at death (or collection) as the calendrical date for that sample . Turtles grow scute layers in a variety of forms, including sequentially in overlapping concentric rings, or by growth of a new scute layer while retaining former layers in sequence . River cSamples were mounted on aluminum stubs with double-sided carbon tape, and carbon coated using a Cressington 108carbon/A system. Images and analyses were collected with an FEI Quanta 200F field emission SEM coupled with an EDAX Octane Silicon Drift Detector for EDS analysis. Images were collected using both secondary electron and backscatter electron detection for textural and Z-contrast information. Images and EDS point analysis spectra were collected with a 20-kV accelerating voltage and an FEI spot size setting of 4 (moderate spot size optimized for both imaging and EDS).EDS point analyses were collected using a working distance of 10 mm, a duration of 2 min, and an amp time of 7.68 μs. EDS element composition data were processed using EDAX Team software and employed the PeBaZAF (Peak to Background ZAF—chosen due to sample surface roughness) Smart Quant method, using an assumed 50-nm carbon coat. Elemental compositions are standardless and normalized, and are therefore semi-quantitative. EDS element maps were collected with a 20-kV accelerating voltage with a larger FEI spot size setting of 5. EDAX Team software parameters were as follows: an amp time of 0.12 μs, a dwell time of 200 μs, a resolution of 1280 × 1024 pixels, and 256 frames, for a resulting map run time of 13.5 h.2O. The high organic content of the samples required a two-step dissolution procedure. Samples were first dissolved at ∼120°C for 12 h in 5-mL concentrated HNO3. The samples were then evaporated to dryness, reconstituted with a 5-mL mixture of concentrated HNO3, and 30% H2O2 (4:1 v/v) and placed on the hotplate for a further 24 h. This second dissolution step was found to be critical in eliminating any remaining organics from the sample, which have the potential to interfere with mass spectrometry. The samples were then evaporated to dryness and reconstituted in 3N HNO3 prior to chemical separation of uranium. Uranium separation was completed using a TRU spec  method from Reinhard et al. , Optima grade reagents  and 18.2-MΩ Hd et al. , which h3 solution and introduced into the MC-ICP-MS using a Cetac Aridus 3 desolvating nebulizer system and a 100-μL/min Savillex nebulizer. The samples were measured using a combination of ion counters  and a Faraday detector (for 238U). The baseline and gain for the Faraday detector and dark noise for the ion counters were determined at the start of the analytical session. The instrument mass bias and ion counter yields were determined by measuring a solution of U010 throughout the analytical session bracketing the samples and quality control (QC) standards. We used two QC standards to verify the accuracy of the mass bias correction and ion counter yields as follows: CRM U005A (a DU standard containing 234U and 236U) and NBS SRM 960 . The QC standards were run at concentrations to match the signal intensities measured in the samples.We measured the isotopic composition of the scute samples using a Thermo Neptune Plus MC-ICP-MS at LANL. The dissolved and processed samples were redissolved in a 2% HNOUranium concentration measurements were made using an Agilent 8900 triple quadrupole ICP-MS (QQQ-ICP-MS) at LANL. The first quadrupole was arranged as an ion guide, and no gas was introduced to the collision/reaction cell. The samples were introduced into the QQQ-ICP-MS using a PFA Microflow (200) nebulizer and a double pass quartz spray chamber. An acid blank and NBS SRM 960 uranium standards were measured throughout the analytical session to evaluate sample washout and analytical drift. Uranium concentrations were determined by building an eight-point calibration curve, including an acid matrix blank, and increasing concentrations of SRM 960 solutions of 0.01, 0.12, 0.26, 0.50, 0.72, 1.07, and 2.96 ng/g. The concentration of the samples was calculated by creating a linear calibration curve and applying to the measured intensities of the samples.All our data and supplemental coding script are available through an open-access repository at osf.io .