The reported incidence of sexually transmitted infections (STIs) in China has been increasing over the last decades, especially among drug users, which has become one of the main burdens of public health in China. This study was conducted to estimate the prevalence and associated factors of STIs among non-injecting methamphetamine (MA) users in Eastern China.A cross-sectional survey was conducted among 632 MA users in Eastern China in 2017. Demographic characteristics, sexual behaviors, behaviors of MA use and sexual health knowledge were collected through questionnaire. First pass urine specimens were collected and detected for deoxyribonucleic acid (DNA) of Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) with Nucleic Acid Amplification Technology (NAAT), while blood specimens were collected and detected for antibodies of Human immunodeficiency virus (HIV), Herpes simplex virus type-2 (HSV-2), and syphilis with enzyme-linked immune sorbent assay (ELISA).Among the 632 MA users, 464 (73.42%) were males, 60.92% were < 35 years of age, 546 (86.39%) were Shandong residents. 317  participants were tested positive for at least one kind of STIs, including 242  for HSV-2, 107  for active syphilis, 46  for treated syphilis, 40  for CT, 6  for HIV, and 3  for NG infection. 99  participants were co-infected with two kinds of STIs, including 91  participants were co-infected with HSV-2 and syphilis. 14  participants were co-infected with three kinds of STIs, and 4 HIV positive participants were co-infected with both syphilis and HSV-2. In the multiple logistic regression analysis, the results showed that females  and individuals ≥ 35 years of age  were more likely to test positive for STIs among MA users, whereas participants who acquired sexual health knowledge primarily from the Internet  and those whose regular partners did not use drugs  were less likely.This study found that the prevalence of HSV-2 and syphilis are alarming high among non-injecting MA users in Shandong Province in Eastern China. The prevention and control intervention of STIs among MA users in Shandong were needed, especially on females and MA users ≥ 35 years of age. According to the World Drug Report 2020, there are about 27 million amphetamines users  worldwide . In receThe burden of sexually transmitted infections (STIs) in China has been increasing within the last decades. The incidence of syphilis, Neisseria gonorrhoeae (NG), Human immunodeficiency virus (HIV), the three notifiable STIs, has increased from 33.09 cases per 100,000 residents in 2009 to 51.91 cases per 100,000 residents in 2019, with an average annual increase of 4.61% . And theAccording to findings of published studies, frequent use of methamphetamine has neurotoxic effects on the dopaminergic and serotonergic systems, leading to potentially irreversible loss of nerve terminals and/or neuron cell bodies –14. In aThe official data showed that there were 30 thousand registered drug users in Shandong province as at 2012 . HoweverFinding the epidemiological characteristics of STIs among MA users are essential for designing control program. To our knowledge, previous studies in China have been conducted with more focus on injecting drug users and other key population, only a few studies have focused on non-injecting MA users and estimated its impact on STIs in China –28. HoweThis study was conducted to estimate the prevalence and associated factors of STIs among non-injecting MA users in Eastern China to provide information for the formulation of targeted intervention to control the spread of STIs.α2 * p * q/d2 = 1.962 * 0.14*(1 − 0.14)/0.0282 = 590. Following this, cluster sampling was adopted to select the sample. We randomly selected two prison areas in each site, and all individuals in the selected area that met the inclusion criteria were included in our study. A total of 642 MA users were initially included in the study. However, 10 MA users were excluded because of ineligible blood (6 cases) and urine samples (4 cases), resulting in a final analytical sample of 632 participants.A cross-sectional survey was conducted in three county-level cities of Shandong Province: Chengyang District in Qingdao city, Zhoucun District in Zibo city, and Qingzhou in Weifang city. Participants were recruited continuously from the Detention Center of Qingzhou Public Security Bureau, Shandong Women’s Detoxification Centre in Zhoucun, and Qingdao First Detention Center, from April, 11 to December, 5, 2017. A detention center (detention house) refers to a public security agency used for temporarily detaining a criminal suspect before the court makes a judicial decision for the suspected criminal suspect. According to the relevant laws and regulations in China, criminal suspects detained for drug offences and suspects arrested for other criminal offences are detained in detention centers. Inclusion criteria were as follows: (1) individuals ≥ 18 years of age; (2) using MA at least once a month in the past 6 months before being arrested, and having a positive result on the urine test; (3) being compulsorily detoxified by a public security agency owing to drug use; (4) voluntarily give informed consent to participate in the study, provide blood and urine specimens, and complete the questionnaire; (5) free of any psychiatric illness and learning disability during the survey. First, we calculated the sample size according to the following formula: a two-side inspection level α = 0.05 was required, and the expected prevalence of STIs among MA users was estimated to be 14% . Within Before this survey, fieldwork staff was trained by epidemiology and clinical laboratory professionals. The investigation team included two laboratory technicians for urine and blood collection, one male and one female questionnaire interviewers, and one financial staff for distribution of the interview allowance. In addition, one trained supervisor was designated for informed consent and quality control of the investigation process. The study was approved by the Ethics Committee of Shandong Provincial Institute of Dermatology and Venereology. Participants sat separately in a quiet room to ensure accuracy, privacy and anonymity of the information collected. Data collected from questionnaires that we designed included: demographic characteristics , behaviors of MA use  and related sexual behaviors  during MA use? condom use in sex with commercial sexual partners during MA use, whether had sex with MSM during MA use? condom use in sex with MSM during MA use), and perception of sexual health knowledge . Demographic information was collected by face-to-face interviews, and the other information in the questionnaire was completed independently by the participants themselves to ensure confidentiality. “Have been acquainted with” was defined as they have met each other before and have contacts sometimes, but do not regularly meet. “During MA use” in “primary sexual partner during MA use” was defined as sexual encounters during a period of time when the participant was using MA.First pass urine specimens were collected from all participants with disposable urine preservation tube and detected for deoxyribonucleic acid (DNA) of NG and CT using Nucleic Acid Amplification Technology (NAAT) on cobas4800 platforms . Venous whole blood specimens were collected with 2 mL of vacuum EDTA anticoagulant blood tubes, and serums were recovered after centrifugation for detecting for HIV antibodies, HSV-2 human IgG class antibodies, and syphilis-specific antibodies with enzyme-linked immune sorbent assay (ELISA) procedure. To ensure the accuracy of the test result, ELISA-positive syphilis specimens underwent confirmatory testing with treponema pallidum particle assay (TPPA) and toluidine red unheated serum test (TRUST). Active syphilis was defined as both TPPA and TRUST positive, and treated syphilis as TPPA positive but TRUST negative. ELISA-positive HIV and HSV-2 specimens were repeat tested using the ELISA procedure for confirmation. According to the manufacture instructions, the sensitivity and specificity of the syphilis ELISA test were 96.70% and 100.00% respectively, whereas for the NAAT of CT and NG they were 96.6% and 100% respectively. The participants with positive results were notified in time by the local skin disease prevention and treatment health station or the centers for disease control and prevention (CDC). Patients were arranged to be treated by the prison doctor or recommended to the local hospital or CDC to receive standardized treatment under the guard of prison guards.p-value < 0.1 in univariate analysis that were biologically plausible and without missing values were included in the multivariate regression model. Multivariate analysis was performed by forward stepwise multiple logistic regression to control for confounding factors such as gender and age. In addition, multicollinearity among associated factors was examined using variance inflation factors, condition index, and variance proportions. Both adjusted odds ratio (AOR) and 95% CI were calculated for each explanatory variable in the final models. All statistical tests were two-sided, and variables with p-value < 0.05 were considered statistical significance.Epidata 3.1  was used for data entry and validation and SAS 9.4  for data management and analyses. Prevalence and 95% confidence interval (CI) of STIs were estimated based on the binomial distribution. Chi-square test was used to compare categorical variables between STIs positive and negative participants. Univariate analysis was performed to explore the association between STIs infection and related variables with logistic analysis and crude odds ratio (COR). All the variables with a We enrolled 632 MA users made up of 464 (73.42%) males, and 168 (26.58%) females, with a median age of 33.70 ± 8.62 years (range 18–69). 374 (59.18%) participants were single; 400 (63.29%) had only received middle school or a lower level of education; 93.67% were of Han descent; 232 (36.71%) were self-employed; 546 (86.39%) were Shandong residents; and nearly half of them (45.57%) have been acquainted with at least 10 MA users  reportedly used MA more than once per week. Majority (72.15%) of participants chose their own or friends’ home as their regular MA use site. In addition, 8 (1.27%) participants admitted being infected with HIV.Of the 632 participants, nearly one-third (32.91%) of participants reported that their primary sexual partners during MA use were casual partners. About 33.86% (n = 214) of participants reported they have had sex with multiple sexual partners during MA use, but only 20.09% (n = 43) reported consistent condoms use. More than half (53.45%) of male participants have had sex with FSWs during MA use, whereas about 38.69% (n = 65) of the females reported they had exchanged sex for drugs or money. However, among the 307 participants who had engaged in commercial sex, only 51.47% (n = 158) reported consistent condom use. 7.97% (n = 37) the male participants admitted that they have had sex with MSM during MA use, but only 21.62% (n = 8) of them were reported consistent condom use. Majority (71.52%) of the participants thought that the duration of sexual episodes during MA use was increased. Nearly half (49.37%) of the participants reported that their regular partners did not know of their behavior of MA use, and regular partners of majority (71.99%) of the participants did not use drugs.Several questions examined perception of sexual health knowledge among participants. Less than half (42.72%) of participants had a clear understanding of the routes of transmission of STIs, nearly one-third (32.91%) of participants reported that it is possible for babies born to women with STIs to have STIs, and 231 (36.55%) participants believed that STIs would not increase the risk of HIV transmission. Furthermore, among all the participants, 338 (53.48%) acquired sexual health knowledge primarily from television or broadcasts, 337 (53.32%) primarily from newspapers or books, 193 (30.54%) primarily from medical personnel, 257 (40.66%) primarily from health publicity materials, 54 (8.54%) from family, 168 (26.58%) primarily from friends, 117 (18.51%) primarily from school education, and 263 (41.61%) primarily from the Internet.Among the 632 participants, 317  were positive for at least one kind of STIs, including 242  for HSV-2, 107  for active syphilis, 46  for treated syphilis, 40  for CT, 6  for HIV, and 3  for NG infection. 99  participants were co-infected with two kinds of STIs, including 91  participants were co-infected with HSV-2 and syphilis. 14  participants were co-infected with three kinds of STIs, and 4 HIV positive participants were co-infected with both syphilis and HSV-2 . Significant differences in prevalence of STIs were observed in terms of both age  and educational level . Positive for STIs was less among MA users < 35 years of age (44.42%) than ≥ 35 years of age (59.11%), and highest among participants with below primary school education (70.37%). In addition, prevalence of STIs also significantly differed by household registration , and was less among Shandong residents (47.80%) than non-Shandong (65.12%).Results of the univariate analysis are shown in Table 2 = 7.92, p = 0.02) and those who thought that the duration of sexual episodes during MA use has no change (64.84%) . Significant differences in STIs prevalence were observed between whether their regular partners knew of their behavior of MA use , and whether their regular partners used drugs . The prevalence among participants who chose their own or friends’ home (53.07%) as their regular MA use site was higher than those who chose hotels, guesthouse, dance halls, nightclubs, etc. (42.61%) , who admitted they had been infected with HIV was higher than those denied , who had a clear understanding of the routes of transmission of STIs (55.90%) was higher than those did not know (38.46%) , and higher among those thought that it is possible for babies born to women with STIs to have STIs (55.90%) than those had an opposite view (38.46%) . Moreover, STIs prevalence also differed statistically by primary ways of acquiring sexual health knowledge. Participants that acquired sexual health knowledge primarily from the Internet had the lowest prevalence (41.44%) , while primarily from health propaganda (45.53%)  and family (66.67%)  had higher prevalence.The prevalence of STIs was highest among those who have had sex primarily with their regular partner during MA use (54.52%)  than in males. This may be due to a variety of factors. First, in our sample population, when compared with male MA users, female MA users were more likely to report that their primary sexual partner during MA use was their regular partner (45.47% vs. 91.67%). Several studies reported that use of condoms with a regular partner was less frequent than with a casual partner, because condom use with regular partners was viewed more often as a sign of disrespect and a barrier to intimacy and affection, rather than as a positive means of showing respect –44. SecoOur findings also found that MA users ≥ 35 years of age may be related to the risk of STIs. Increased odds of seropositivity of HSV-2 and syphilis with age would be expected, given that the risk of ever being infected by STIs would accumulate over time, especially for those with more sexual partners and having unprotected encounters. In addition, previous research found that older participants had an increased risk of HIV infection in Cambodia . A possiThe current study also found that the ways to acquire sexual health knowledge have an important impact on SITs among MA users. Logistic regression model showed that the acquisition of sexual health knowledge primarily from the Internet may be a protective factor. These suggested that compared with traditional media such as TV and broadcasts, the Internet may better cater for MA users . The effThere were several limitations to this study. First, this study was conducted in Shandong, a province of Eastern Coast of China. Therefore, our findings should be interpreted with caution as they may not be applicable to MA users in other areas of China. Second, prevalence of STIs among individuals less than 18 years old and older than 69 years old was not estimated in our study. Hence, further study to investigate the prevalence of STIs among these population is needed. Third, the data collection of this study relied on self-reporting and involved some sensitive questions which may cause recall bias and information bias. In spite of these limitations, our data still present some basis for the development of targeted interventions.This study found that the prevalence of HSV-2 and syphilis are alarming high among non-injecting MA users in Shandong Province in Eastern China. The prevention and control intervention of STIs among MA users in Shandong were needed, especially for females and MA users ≥ 35 years of age.


Here we show that patients have increased cortical activity related to arousal and lower activity related to sleep homeostasis compared to controls. These results give additional insights in sleep pathophysiology of BC survivors and suggest sleep homeostasis disruption in non-advanced stages of BC.Complaints of sleep disturbance are prevalent among breast cancer (BC) patients and are predictors of quality of life. Still, electrophysiological measures of sleep are missing in patients, which prevents from understanding the pathophysiological consequences of cancer and its past treatments. Using polysomnography, sleep can be investigated in terms of macro-  and micro-  structure. We aimed to characterize sleep complaints, and macro- and microstructure in 33 BC survivors untreated by chemotherapy and that had finished radiotherapy since at least 6 months (i.e. out of the acute effects of radiotherapy) compared to 21 healthy controls (HC). Compared to HC, BC patients had a larger number of awakenings ( Insomnia is defined as a dissatisfaction with sleep quality caused by difficulties to falling asleep, maintaining sleep, early morning awakening, or non-restorative sleep leading to daytime impairments, at least three times a week and for at least 3 months4. Sleep complaints are now well recognized in non-central nervous system (CNS) cancer patients, not only following chemotherapy6 but also before the occurrence of treatments11. Despite significant sleep complaint from patients13, it has not been fully attested by objective evaluations such as polysomnography (PSG) that is considered as the gold standard of sleep evaluation15. Previous experimental studies dedicated to evaluate sleep using PSG in BC patients are scarce and have shown either a lack of sleep alterations following chemotherapy18 or deleterious effects of chemotherapy and/or radiotherapy20. Regarding radiotherapy, one study reported larger duration of NREM Stage 2 following radiotherapy in breast cancer patients compared to healthy controls19. However, in this study, the delay between the PSG recording and the end of radiotherapy was unclear.Breast cancer (BC) is the most frequently diagnosed in women in the majority of countries worldwide21, to sleep fragility and environmental awareness (i.e. Alpha band)23, or to arousal particularly during Non-Rapid Eye Movement—NREM sleep (i.e. Beta band)24. With healthy ageing, previous reports have shown that Delta power decreases and Alpha and Sigma power increase, indicating that sleep becomes lighter with age26. In insomnia disorder, a recent meta-analysis reported increased Alpha, Sigma and Beta power and lower Delta power compare to healthy adults, notably during NREM sleep27.PSG allows to go further in the understanding of sleep modifications, not only in its macrostructure , but also in its microstructure as reflected by spectral analyses of the electroencephalography (EEG). Especially, power spectrum analyses consist of computing Fast Fourier Transforms on EEG signal in order to reveal the frequency and the amplitude of its waves. Results are combined by averaging the data in different frequency bands that have been previously related to sleep electrophysiological modifications. For examples, sleep spectral analyses provide additional information related to deepening of sleep (i.e. Delta band), to emotion and dreaming (i.e. Theta band)28. In contrast, greater cortical arousal (i.e. increased Beta power spectrum) has been successfully revealed during sleep in insomnia using spectral analyses24. Indeed, as in aging, sleep spectral analysis can thus be a more sensitive measure of sleep disruption than sleep architecture29, making such analysis potentially useful in highlighting slight sleep disruptions and allowing deepen understanding of electrophysiological modifications associated with sleep in BC patients. To our knowledge, no study until now has investigated sleep spectral analysis in non-CNS cancers patients. Still, previous reports suggested that tumors disrupt the homeostatic processes of sleep30, a process that is related to the Delta power spectrum during sleep. Sleep is well-known to be under the regulation of two processes (i.e. S and C), that interact continuously31. Process S refers to sleep pressure accumulated during the day, while process C is related to circadian rhythms over 24 h.In the context of cancer, it may be that, despite subtle modification in sleep architecture, alterations in the sleep electroencephalogram can be more visible using spectral analysis of sleep as previously shown in patients suffering from insomnia disorder. Indeed, in these patients suffering from insomnia disorder, without other comorbidities such as sleep-apnea or cancer, subjective complaints of sleep disturbance assessed by self-rating questionnaires are often discordant with sleep measured objectively using PSGThe current cross-sectional study aimed at quantifying sleep macro- and microstructure modifications with PSG in BC survivors untreated by adjuvant chemotherapy and evaluated at least 6 months after radiotherapy (i.e. out of the acute effects of radiotherapy) compared to healthy controls. Overall, we expected greater sleep complaints and sleep structure modifications in BC patients compared to healthy controls.Demographics and psychological characteristics of HC and patients and clinical characteristics of patients are presented in Table M = 8.1, SD = 4.5) and insomnia symptoms  than HC . According to the ISI, 63% of BC patients and 48% of HC had complaints of sleep disturbance related to insomnia (i.e. ISI score ≥ 8). This rate of participants having sleep disturbance did not differ significantly between the two groups (p=0.43).Results of subjective sleep disturbance are reported Table M = 9.6, SD = 4.3) than HC .Sleep architecture measures are reported in Table No significant difference between BC patients and HC was observed neither on other sleep parameters , nor on the percentage of sleep stages.Results are described in Table ps < 0.01) and Alpha (ps < 0.01) frequency bands differed between groups. BC patients had significantly lower Delta  than HC . BC patients also had higher Alpha power spectrum  than HC . In addition, during Stage 2, Beta power differed between groups with BC patients having higher Beta power  than HC .During Stages 1 and 2, power spectra for the Delta (ps < 0.01). BC patients had lower Delta  and higher Beta  and Alpha  power spectrum than HC .During Stage 3, a significant effect of group was observed for Delta, Alpha, and Beta frequency bands  than HC .During REM sleep, Theta power spectrum differed between groups, with BC patients having lower Theta power  based on EEG signal and thus reflecting cortical effects. These results are close to those reported in healthy ageing and insomnia disorder, which questioned about the contribution of insomnia symptoms and age to these modifications. Still, because our groups did not differ in terms of insomnia symptoms rate and in terms of age, we can suppose that it reflects an effect of cancer and its past treatments. To our knowledge, this is the first study to report spectral analyses results during sleep in BC patients, allowing a deepen and finer understanding of sleep pathophysiology in BC patients.Regarding sleep architecture, results revealed greater number of awakenings in BC patients than in HC. Regarding spectral analyses of cortical EEG during sleep, results revealed modifications of power spectrum in Delta, Alpha and Beta bands during NREM sleep  in BC patients compared to HC. During REM sleep, BC patients differed from HC for the Theta power spectrum. Of note, no significant topographical difference was highlighted between patients and controls suggesting that the main sources of sleep related EEG activity did not differ between groups but was rather a more widespread effect over the scalp.33. One may argue that the greater number of awakenings in patients could be driven by the effects of radiotherapy as most of our sample of patients was treated with radiotherapy following surgery. However, we ensure a period of at least 6 months between the end of radiotherapy and the PSG recordings in order to avoid acute effects of radiotherapy on sleep. In addition, previous studies that have measured sleep, either with PSG or actigraphy, following radiotherapy did not report significantly larger number of awakenings related to radiotherapy either compared to before radiotherapy or compared to HC35. Although further studies are needed to confirm this result, we may assume that sleep modifications reported in the current study are not specifically related to the long-term effects of radiotherapy.Despite a lack of significant difference in sleep efficiency , BC patients had larger number of awakenings (longer than one minute) than HC. These results suggest a more fragmented sleep with short arousals in BC patients that did not affect sleep efficiency. Overall, BC patients seem to have subtle but still present modifications of sleep architecture compared to HC. These results are in accordance with those of previous studies suggesting effects of cancer and/or diagnostic on sleep36 that was accompanied by aberrant activity of hypocretin/orexin neurons that are involved in the sleep/wake cycle regulation. Therefore, our results confirm previous findings and help to understand pathophysiology of BC during sleep. The Delta band during NREM sleep depends on the precise timing of thalamo-cortical activity37 and is known to reflect the homeostatic process of sleep 38. These results give rationale for altered activity in brain circuits regulating sleep homeostasis (i.e. hypocretin/orexin neurons) in BC patients. These results thus support a previous hypothesis about tumor disrupting sleep homeostasis in rodents’ model of non-metastatic breast cancer30. This result is also in accordance with a previous report suggesting sleep homeostasis disruption in advanced stages BC patients (stages III and IV)20. It may thus be possible that sleep homeostasis is disrupted in BC, even in non-advanced stages as represent by the current sample (i.e. mostly stages 0 and I).Regarding sleep microstructure, BC patients had lower Delta power spectrum than HC during NREM sleep. Previous reports in rats model of non-metastatic BC have shown Delta power reduction39. Alpha during NREM sleep has also been associated with cortical hyperarousal in insomnia patients40. Seemly, the greater Beta power in BC patients compared to HC during NREM sleep suggests greater cortical arousal in BC patients. This greater cortical arousal could be related to non-restorative sleep as the Beta EEG band has been previously linked to cortical arousal during sleep in insomnia patients (e.g.43). Greater cortical hyperarousal in insomnia has been previously linked to greater risk for developing depressive symptoms44, highlighting the need for further light over sleep disturbance in BC patients in order to prevent depressive symptoms. During REM sleep, BC patients had reduction of the Theta power spectrum compared to HC. The Theta band is the central activity of REM sleep and has been associated with dreaming and emotion45, suggesting alteration in emotion regulation related to sleep in BC patients. Still, this hypothesis remains to be tested.This result is in line with recent hypotheses proposing that tumors disrupt homeostatic processes. In addition, the current results suggest less deepen sleep in BC patients with potential deleterious effects on daytime functioning. BC patients had increase in the Alpha power spectrum during NREM sleep, referring to the Alpha-Delta sleep. As NREM sleep deepened, Alpha disappears and the re-appearance of Alpha during slow-wave sleep (Stage 3) has been associated with non-restorative sleepThis study is an exploratory one with a small number of participants, further studies using larger sample sizes are warranted. Nevertheless, it paves the road for further investigation in order to better understand the pathophysiological modifications associated with BC and its treatments.Altogether, our results highlighted modifications of sleep structure in BC patients compared to HC. BC patients had a greater number of awakenings during sleep as well as greater arousal and a less deepen sleep than HC during NREM sleep as reflected by cortical activity. During REM sleep, BC patients seem to have deregulation of EEG bands related to emotional regulation and dreaming. Although we had a small sample size, our results give additional insights in sleep pathophysiology of BC survivors and suggest sleep homeostasis disruption in non-advanced stages of BC.All participants provided written informed consent for the study. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the ethics committee (CPP Ile de France III), n°ID-RCB: 2017-A02778-45.Participants were recruited between January 2018 and March 2020 at the François Baclesse regional cancer center . Among the patients approached, 50 agreed to participate. Eleven patients finally declined to participate. An additional six patients were removed from the analyses due to PSG recording issues (i.e. holter shut down prematurely) during the ambulatory home-based PSG. Among the 25 healthy controls (HC) included, four finally refused to participate. Thus, groups consisted of 33 patients and 21 HC. Informed consent was obtained from all subjects involved in the study.Patient inclusion criteria were : (i) less than 70 years old, (ii) no metastatic BC, (iii) already undergone surgical or radiotherapy treatment, (iv) radiotherapy finished since at least six months ago and no chemotherapy treatment, (v) menopausal status since at least one year ago at the time of inclusion, (vi) no personality disorder nor progressive psychiatric disorder (vii) no neurological sequelae, (viii) no drug use or alcohol abuse, (ix) be a native French speaker, (x) have at least a primary school level of education.Inclusion criteria for HC were the same as for patients, including no history of cancer and no global cognitive impairment according to the Montreal Cognitive Assessment. HC were matched in age and education with BC patients.46 and anxiety was measured with the State Trait Anxiety Inventory (STAI)47. For both questionnaires higher values indicate higher depression and trait-anxiety symptoms. The STAI questionnaire was not fulfilled at the same time that sleep measurement, thus the State score was not representative of their anxiety state at the time of sleep and only Trait anxiety score was reported here.Depression was assessed with the Beck Depression Inventory (BDI)48. The cut-off of 8 has been validated in cancer patients50, representing the best specificity and sensitivity ratio in this population.Perceived sleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI), a 19-item questionnaire measuring sleep habits  over the previous month. Scores range from 0 to 21, with higher scores reflecting poorer sleep quality. Insomnia’s symptoms were measured with the Insomnia Severity Index (ISI) composed of seven items evaluating difficulty falling asleep, maintaining sleep and the frequency of early morning awakenings, as well as the degree of dissatisfaction with current sleep and its impact on daily activities. Scores range from 0 to 28, with higher scores indicating higher insomnia related symptoms. The interpretation of the total score is as follows: lack of insomnia (0–7); sub-threshold insomnia (8–14); moderate insomnia (15–21); and severe insomnia (22–28)51 using Compumedics software. Artifacts were detected visually by JP, and epochs with artifacts  were rejected from analyses. Sleep onset latency  was determined as the time from lights out to the first epoch (i.e. 30 s) of Stage 2 sleep, according to AASM Scoring Manual51. Then, sleep architecture parameters included: total time in bed , total sleep time , sleep efficiency , latency to Stage 3 (min), number of awakenings, number of stage transition, time (min) and percentage  of Stages 1, 2, 3 and rapid eye movement (REM). AHI (Apnea–Hypopnea Index) is defined as the sum of apneas and hypopneas  divided by the total sleep time in hours. This index has been visually scored by a sleep technician (SR) according to AASM criteria51.A full night of sleep was recorded using ambulatory PSG at home. Twenty electroencephalography (EEG) electrodes were placed over prefrontal (FP1/FP2), frontal (F3/F4/F7/F8/Fz), central (C3/C4/Cz), temporal (T3/T4), parietal (P3/P4/Pz), and occipital (O1/O2) sites, according to the international 10–20 system. Ag/Au electrodes with a vertex ground and a bi-mastoids reference were used. Two electrodes were placed at each place: above and under the eyes, on the chin, and under each clavicle. This montage included thoracic and abdominal belts, nasal and oral thermistors, nasal airflow and a finger pulse oximeter. All sensors were connected to the Compumedics Siesta sleep system and placed by an EEG technician (SP). The hardware EEG filter band pass was 0.15–121 Hz and the sample rate was set to 256 Hz. Sleep stages  were visually scored by an electrophysiology technician (SR) in 30 s epochs according to the AASM Scoring Manual proceduresComputerized spectral analysis was performed with fast Fourier transformation (FFT) on the all-night filtered EEG. Spectral analysis was performed on 4-s epochs and on the 17 channels recorded to investigate the difference in areas EEG power spectra. Before computing the FFT, the data were tapered with the Hamming window. The FFT was computed on artifact-free epochs. The FFT was realized for each sleep stage on the total number of epochs corresponding to the maximal number of artifact-free epochs observed in all subjects. The relative power spectrum was obtained in the following frequency bands: Delta (1.5–4 Hz); Theta (4–7.5 Hz); Alpha (7.5–12.5 Hz); Sigma (12.5–14 Hz); Beta (14–30 Hz). All night power averages were obtained for Stages 1, 2, 3 and REM sleep separately. For spectral analyses, groups of electrodes were averaged together to increased statistical power into prefrontal (FP1/FP2), frontal (F3/F4/F7/F8/Fz), central (C3/C4/Cz), temporal (T3/T4), parietal (P3/P4/Pz), and occipital (O1/O2) areas.p < 0.05. The nonparametric Wilcoxon-Mann–Whitney test was used instead of Student’s t test, if the normality hypothesis was rejected according to Shapiro–Wilk test, to evaluate whether demographic, psychological and subjective sleep differed between groups. Pearson chi-squared test was used to determine whether sleep disturbance rate differed between BC patients and HC.Analyses were performed with R software, with statistical significance set at 53. Effects sizes were considered as follows: η2, small effect: η2 ≥ 0.01, medium effect: η2 ≥ 0.06, and large effect: η2 ≥ 0.1454.We performed ANOVA to evaluate whether architecture parameters differed between groups, with sleep architecture parameters as dependent variable, group as the independent variable and AHI as co-variable. To evaluate whether the sleep microstructure differed between groups and across cortical areas, we realized an ANOVA for each frequency band  in each sleep stage  with EEG power as dependent variable, group and cortical areas  as independent variables and AHI as co-variable. Effect size were calculated using eta squared values


This review provides a summary of the preclinical evidence supporting the use of carbapenem/BLI combinations and presents the clinical evidence across a range of MDR Gram-negative infections, with a focus on the use of meropenem/vaborbactam. All three BLIs have shown in vivo activity against Klebsiella pneumoniae carbapenemase and other class A carbapenemases. In 2019, meropenem/vaborbactam was listed in the WHO’s list of essential medicines, because of its activity against priority 1 antibiotic-resistant pathogens. Meropenem/vaborbactam has considerable in vitro and in vivo activity against CRE, and in vitro evidence showing a low potential for resistance at clinically relevant doses. In randomized trials, meropenem/vaborbactam was non-inferior to piperacillin/tazobactam in patients with complicated urinary tract infection and more effective than the best-available treatment in patients with serious CRE infections. Meropenem/vaborbactam is well tolerated and, based on clinical experience, demonstrated lower toxicity compared with the combination regimens that have previously been the standard of care. In conclusion, carbapenem/BLI combinations represent an important therapeutic strategy in patients with MDR Gram-negative infections.Multi-drug resistant (MDR) Gram-negative bacteria represent a growing threat, with an increasing prevalence of carbapenem-resistant Enterobacterales (CRE) infections, for which treatment options are limited. New treatment combinations composed of a β-lactam antibiotic plus a potent β-lactamase inhibitor (BLI) with anti-carbapenemase activity have been developed, including two carbapenem/BLI combinations that are commercially available—meropenem/vaborbactam (Vabomere Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacterales), and all but two of the nine priority 2 and 3 pathogens are Gram-negative MDR.Major public health organizations around the world, including the WHO, US CDC, and the ECDC, recognize the growing threat of MDR Gram-negative bacteria.,® in the US (Melinta); Vaborem® in Europe (Menarini)], imipenem/cilastatin/relebactam  and meropenem/nacubactam .Carbapenem antibiotics are the mainstay of treatment of MDR Gram-negative infections; they are recommended for serious infections caused by extended-spectrum β-lactamase (ESBL)-producing Enterobacterales, and are the drugs of choice for the empirical treatment of sepsis caused by MDR Gram-negative organisms.This article reviews the rationale for this type of combination in the treatment of patients with MDR Gram-negative infections, and describes the combinations currently available or in late-stage development, with a focus on the preclinical and clinical data for meropenem/vaborbactam.Carbapenems act by inducing the lysis of bacterial cells. These agents do not easily cross the outer membrane of Gram-negative organisms, but rely on transport by porins.Klebsiella pneumoniae carbapenemase (KPC)].Many antibiotics have the same mechanism of action. but are broken down by β-lactamase enzymes in the periplasmic space before they can cause appreciable cell lysis. β-Lactamases fall into several categories based on their structure and hydrolytic activity Table .8 Class The recently developed combination antibiotics include both a carbapenem and a BLI with inhibitory activity against a range of β-lactamase enzymes, including several carbapenemases.Currently, there are two carbapenem/BLI combinations commercially available: meropenem/vaborbactam and imipenem/cilastatin/relebactam. Meropenem/vaborbactam was approved by the US FDA in 2017 for the treatment of complicated urinary tract infection (cUTI) in adults,,,,in vivo activity against KPC and other class A carbapenemases.The BLIs in these three combination agents do not have a β-lactam structure. Relebactam and nacubactam are diazabicyclooctane molecules, both structurally related to avibactam (approved for use in combination with ceftazidime),The carbapenems in the combinations are similarly effective in the management of serious infections, but meropenem shows greater activity than imipenem against Gram-negative bacilli, whereas imipenem is generally more active than meropenem against Gram-positive cocci.In 2019, meropenem/vaborbactam was added to WHO’s list of essential medicinesIn vitro data show that meropenem/vaborbactam is highly active against a range of KPC-positive CRE at concentrations of ≤4 mg/L (the susceptibility breakpoint defined by the US FDA) or ≤8 mg/L (the breakpoint defined by EUCAST).K. pneumoniae, Escherichia coli, Enterobacter spp. (including Enterobacter cloacae), Klebsiella oxytoca, Serratia marcescens and Citrobacter spp.90) for meropenem/vaborbactam was ≤1 mg/L for all isolates,,,in vitro antimicrobial activity than meropenem alone, ceftazidime/avibactam, tigecycline, ceftazidime alone, minocycline, gentamicin or polymyxin B against clinical isolates of KPC-positive Enterobacterales from a global collection.90 of meropenem/vaborbactam was 1 mg/L, which was four times more potent than ceftazidime/avibactam and >64 times more potent than meropenem alone.in vitro hollow-fibre model, meropenem/vaborbactam at concentrations equivalent to those achieved by administration of the approved human dosage showed significant activity against a range of KPC-producing CRE strains, including K. pneumoniae, E. cloacae and E. coli isolates  ≤39 mL/min.,CR of 20–39 mL/min should receive 1 g/1 g every 8 h, those with a CLCR of 10–19 mL/min should receive 1 g/1 g every 12 h, and those with a CLCR of <10 mL/min should receive 0.5 g/0.5 g every 12 h.The closely matched pharmacokinetic profiles of meropenem and vaborbactam in humans (including the shared route of excretion) result in comparable changes in pharmacokinetics in patients with renal impairment and similar recommended changes in dose in these patients.The combination shows good penetration of lower respiratory tract tissues after intravenous (IV) administration, with epithelial lining fluid concentrations of 65% for meropenem and 79% for vaborbactam (based on free drug).Based on the well-established pharmacokinetic profile of meropenem and the pharmacokinetic profile of vaborbactam defined during Phase I development, the clinical development of meropenem/vaborbactam was able to proceed without the need for Phase II (dose-finding) studies.Because Phase II studies were not required for meropenem/vaborbactam, the efficacy and safety of the combination was able to be investigated in two sequential Phase III studies: TANGO I in patients with cUTI and acute pyelonephritis (AP)TANGO I was the first study to investigate the efficacy of the proposed meropenem/vaborbactam dosage in a population of patients with complicated infections. This multicentre, randomized, double-blind, non-inferiority study compared meropenem/vaborbactam with piperacillin/tazobactam in 550 patients with cUTI or AP, with Enterobacterales as the most common causative pathogens.4 cfu in urine). For the EMA, the primary endpoint was microbial eradication (<103 cfu in urine) at the test-of-cure visit (7 days after the end of treatment).Patients in both groups were treated with IV agents for 1–15 days (mean 8 days), but the overall mean antibiotic treatment duration was 10 days when oral step-down therapy was included. Two different primary endpoints were defined for the two key regulatory bodies. For the US FDA, the primary endpoint was overall success at the end of IV treatment, defined as a composite of clinical cure (complete resolution or significant improvement in symptoms) and microbial eradication (<10P < 0.001). Additionally, because the lower limit of the 95% CI was also greater than 0%, meropenem/vaborbactam was also shown to be superior to piperacillin/tazobactam (P = 0.01).The non-inferiority of meropenem/vaborbactam was demonstrated for both primary endpoints. The rate of overall success (US FDA criterion) was 98.4% in the group receiving meropenem/vaborbactam and 94.0% in the group receiving piperacillin/tazobactam (difference 4.5%). Because the lower limit of the 95% CI was greater than the prespecified non-inferiority margin of −15%, meropenem/vaborbactam was shown to be significantly non-inferior to piperacillin/tazobactam (P < 0.001 for non-inferiority). Similar rates of overall success were seen in subgroups of patients with AP and cUTI with or without a non-removable source of infection. Patients with bacteraemia all showed negative cultures after treatment.Microbial eradication (EMA criterion) was seen in 66.7% of patients treated with meropenem/vaborbactam and 57.7% of patients treated with piperacillin/tazobactam .TANGO II was the first randomized and controlled clinical trial that tested the efficacy and safety of an antibiotic in a population with infections caused by CRE and CRE-KPC pathogens, including immunocompromised patients with several comorbidities, who are usually excluded from clinical trials.Patients were randomized 2:1 to open-label treatment with meropenem/vaborbactam or BAT selected by the investigator; this could include any monotherapy or combination of polymyxins, carbapenems, aminoglycosides or tigecycline, or monotherapy with ceftazidime/avibactam.P = 0.03) and 59.4% versus 26.7% at test of cure (P = 0.02)]. The 28 day mortality rate was numerically lower with meropenem/vaborbactam (15.6%) than with BAT  .Although TANGO II was a randomized study, patients with prior antibiotic failure, who are expected to have a lower response to salvage therapy, were all enrolled in the meropenem/vaborbactam group. Therefore, a n = 26) or ceftazidime/avibactam (n = 105) between February 2015 and October 2018.Importantly, three real-world observational studies conducted in the USA have confirmed the effectiveness and safety of meropenem/vaborbactam during real-world clinical use.n = 8), pneumonia , tracheobronchitis , skin and soft tissue infections (n = 2), pyelonephritis (n = 1) and peritonitis with intra-abdominal abscess (n = 1). This was a vulnerable group of patients: 14 (70%) were in the ICU at the onset of the infection, and seven (35%) required renal replacement therapy with intermittent or continuous haemodialysis. In addition, the median Acute Physiology and Chronic Health Evaluation (APACHE II) score was 20 and median Charlson comorbidity index (CCI) was 4. The most common pathogen was K. pneumoniae, and 19 of the 20 isolates (95%) were resistant to ertapenem.In the single-centre study, patients had bacteraemia (n = 13), followed by UTI (n = 8), intra-abdominal infections (n = 5) and skin and soft tissue infections (n = 5); 11 patients had bacteraemia—primary in 2 and secondary in 9. Overall, 45 pathogens were isolated, most commonly K. pneumoniae (47%), E. cloacae (20%) and E. coli (13%).Klebsiella spp. (69%) and other Enterobacterales (21%).Patients in the multicentre studies were similarly vulnerable. In the study of patients with Gram-negative infections, median APACHE II score was 17, median CCI was 6, 70% were in the ICU, and 90% had at least one risk factor for developing an MDR infection.All three studies defined clinical success as the composite of 30 day survival, absence of recurrence at 30 days after starting treatment, and resolution of signs and symptoms of infection while receiving treatment.,et al.S. marcescens and carbapenem-resistant Enterobacter aerogenes in an asplenic patient with HIV infection and renal failure requiring dialysis. In another case, Athans and colleaguesK. pneumoniae in a liver transplant recipient who developed hepatic artery thrombosis and graft failure. The infection had not responded to courses of ceftazidime/avibactam or polymyxin B, initially administered as monotherapy, then with added gentamicin and tigecycline. This combination was discontinued because of renal toxicity. Once meropenem/vaborbactam was started, the patient’s renal function improved and their infection cleared, allowing the patient to undergo a successful second transplantation.,The management of Gram-negative infections is often complicated by the presence of multiple morbidities and need of venous access devices, catheters or ventilators in fragile subjects. Real-world case reports indicate that meropenem/vaborbactam can be effective even in these vulnerable individuals.in vitro and in vivo activity against these pathogens, with a low potential for resistance at clinically relevant doses. The available clinical data show its efficacy and tolerability in patients with complicated CRE infections, with a low potential for toxicity compared with the combination regimens that have been the standard of care until now. In addition, observational studies and case reports have confirmed the efficacy of meropenem/vaborbactam in a real-world setting, including in very clinically complex patients with immunosuppression, renal dysfunction, or extensively drug-resistant organisms. Therefore, it is not surprising that this agent is now listed by the WHO as an essential medicine. With time, further data will emerge about how to optimally use meropenem/vaborbactam and other carbapenem-BLI combinations in the treatment of CRE.Given the increasing threat posed by carbapenem-resistant pathogens and the limited treatment options for patients with CRE infections, the development of these new combinations of carbapenems and BLIs represents an important therapeutic advance. Meropenem/vaborbactam demonstrates considerable E.B. has participated in advisory boards and received payment for conferences from Menarini.Catherine Rees of Springer Healthcare Communications wrote the first draft of this manuscript. This medical writing assistance was funded by A. Menarini Farmaceutica Internazionale. This paper was published as part of a Supplement sponsored and financially supported by A. Menarini Farmaceutica Internazionale.


The prevalence of obesity is considered to be increased worldwide. Lack of mineral elements is one of the essential side effects of bariatric surgery as a trending treatment for obesity. We aimed to assess zinc deficiency among morbidly obese patients before and following different types of bariatric surgical procedures.In the present retrospective cohort study, 413 morbidly obese patients (body mass index (BMI) ≥ 40 kg/m2 or BMI ≥ 35 kg/m2 with a complication or risk factor, e.g., diabetes mellitus) were enrolled who received bariatric surgery, aged between 18 and 65 years old, and had a negative history of active consumption of alcohol and illicit drugs. Patients were assigned into three groups of bariatric surgeries: mini-gastric bypass, Roux-en-Y gastric bypass (RYGB), and sleeve gastrectomy (SG). We recorded baseline clinical and demographic characteristics and zinc serum levels during the preoperative and postoperative follow-up periods at three, six, and 12 months after the operation.All patients with a mean age of 40.57 ± 10.63 years and a mean preoperative BMI of 45.78 ± 6.02 kg/m2 underwent bariatric surgery. 10.2% of the bariatric patients experienced zinc deficiency before the surgery, and 27.1% at 1 year after the surgery. The results showed that 27.7% of mini-gastric bypass patients, 29.8% of RYGB, and 13.3% of SG experienced zinc deficiency 12 months following surgery. We observed no statistical differences in the preoperative and postoperative zinc deficiency between different types of surgeries.A high prevalence of preoperative zinc deficiency among morbidly obese patients who underwent bariatric surgery was observed, which increased during the postoperative periods. We recommend assessing zinc serum levels and prescribing zinc supplements before the bariatric operation to alleviate the prevalence of zinc deficiency after the operation.The online version contains supplementary material available at 10.1186/s12902-021-00763-0. Obesity, as one of the most crucial health issues worldwide, is considered a modifiable and preventable risk factor for many diseases. The increased rate of obesity raises the mortality and morbidity rates in populations. The main consequences are cardiovascular diseases, cerebrovascular accidents and strokes,, and cancer types . ApproxiThe trace elements, including zinc, copper, selenium, and iron, playan essential role in biomedical pathways for cell function and metabolism in cells . Zinc isThis study aimed to assess the zinc deficiency in morbidly obese patients after bariatric surgeries and its changes according to the types of surgical procedures among the Iranian population.2 or BMI ≥ 35 kg/m2 with a complication or risk factor, e.g., DM) who underwent bariatric surgery, aged between 18 and 65 years old, and did not actively consume alcohol and illicit drugs. Patients who had a history of bariatric surgery were excluded. The Ethics Committee of Iran University of Medical Sciences approved the present study (Ethics code: IR.IUMS.REC 1396.31866).We conducted a retrospective cohort study at the obesity center of RasoulAkramHospital, Tehran, Iran, fromDecember2018 to December2019. We recruited 413 morbidly obese patients (body mass index (BMI) ≥ 40 kg/mWe assigned the patients into three groups according to the type of bariatric surgery . We recorded zinc serum levels during the preoperative period and follow-up measurements at three, six, and 12 months after the surgery. The reference value for zinc serum levels is 70-120 μg/dl . BaselinAt the first postoperative visit (2–3 weeks after the operation), we encouraged the patients to take multivitamin and mineral supplementations postoperatively , which contained30 mg zinc as zinc oxide per tablet alongside other vitamins and micronutrients, presented in Table p values of < 0.05 statistically significant.We performed statistical analyses using IBM SPSS statistics version 19. Continuous variables were demonstrated as mean ± standard deviation (SD) or median and categorical values as percentages or absolute values. To compare continuous variables, we used ANOVA or Kruskal-Wallis test based on the assumptions of the former. In case the ANOVA or Kruskal-Wallis test met the significant levels, we performed Tukey post hoc test. We made a comparison of categorical values by Chi-square or Fisher’s exact tests. We considered 2-tailed 2underwent bariatric surgery. Significantly more patients were female , and more underwent mini-gastric bypass than RYGB or SG . The ANOVA test for age between surgical groups was not significant . The frequency of comorbidities, including hypertension, DM type II, hyperlipidemia, and hypothyroidism, were summarized in Table Four hundred thirteen 413) patients met the inclusion criteria, and their demographic information is shown in Table 3 patientP-value< 0.001), with statistically significant differences between the preoperative period and the six and 12 months after the operation, as well as between the third postoperative month and the six and 12 months after the operation. There was an association between BMI groups and zinc serum levels , with a statistical difference between 40 ≤ BMI ≤ 50 and BMI > 50 groups. Furthermore, the association between sex and zinc serum levels was significant . The data weresummarized in Table The mean preoperativezinc plasma concentration was 89.96 ± 21.4 μg/dl. Thezincserum level after surgery was significantly decreased in patients  [Comparing zinc deficiency among different surgical techniques, we found that 27.7% of the patients who received mini-gastric bypass, 29.8% of RYGB, and 13.3% of SG experienced zinc deficiency 12 months after surgery. It can be also interpreted that zinc deficiency increased 18% among mini-gastric bypass and 19.2% among RYGB patients from the preoperative period to 12 months after the surgery. However, no significant difference was observed in zinc deficiency between groups of bariatric surgery. Similar to our findings, Ferraz et al. observed no significant difference in zinc deficiency between RYGB and SG after 12 months of surgery . Nevertheless, they presented a substantial difference between the surgeries at 24 months of follow-up  . Consiste< 0.05) .It has been reported that zinc deficiency could cause different disorders, including skin disorders , and be fatal in severe cases , 26, 27.Collectively, in the present cohort study, we assessed the zinc status in a population of morbidly obese patients during the pre- and post-bariatric periods and compared zinc deficiency between different groups of bariatric surgeries within the first year of operation. We observed a high prevalence of preoperative zinc deficiency among morbidly obese patients who underwent bariatric surgery, which increased during the postoperative period. Besides, we found that mini-gastric bypass and RYGB, compared to SG, showed higher zinc deficiency rates at the end of the 12-month follow-up. However, we recommend an assessment of zinc serum levels and prescribing zinc supplementation before bariatric surgery to alleviate the prevalence of zinc deficiency after the operation.Our study’s strengths included: 1. cohort study design and the large study population, 2. we compared different bariatric operations regarding zinc deficiency during the 12 months of follow-up that helped us extend the currently available data concerning zinc status in different bariatric surgical procedures. However, we encountered some limitations. First, we were unsure of whether patients take their multivitamin and mineral supplementations on a daily and regular basis after the surgery. Second, we did not evaluate confounding variables, including albumin and C-reactive protein (CRP) serum levels, which might affect the status of trace elements. Third, different distribution of patients among bariatric surgery categories, as well as the small number of patients in SG group, which could explain no significant difference in zinc deficiency between bariatric surgery groups. Forth, some modifiable variables might change the zinc serum levels, including dietary intakes and food habits. Fifth, patients’ compliance with the multivitamin and mineral supplementations following the operation was not recorded, which could possibly have an impact on zinc serum levels.To track the exact changes in the prevalence of zinc deficiency among different types of bariatric surgeries during the pre- and postoperative period, we suggest further prospective studies with long-term follow-ups, assigning a similar number of patients into each group of surgery and matching study participants regarding nutritional habits, monitoring the patients to receive their multivitamin and micronutrient supplementations regularly after the operation, and evaluating the albumin and CRP serum levels as potential confounders.Additional file 1: Table S1. Zinc serum levels among bariatric surgery groups during the pre- and postoperative periods. Table S2. Zinc deficiency among bariatric surgery groups during the pre- and postoperative periods.


Artemia have an unequalled ability to endure extreme salinity and complete anoxia. This study aims to elucidate its strategies to cope with these stressors.Brine shrimp A. franciscana Kellogg, 1906. We identified 21,828 genes of which, under high salinity, 674 genes and under anoxia, 900 genes were differentially expressed . Under high salinity, relevant stress genes and pathways included several Heat Shock Protein and Leaf Embryogenesis Abundant genes, as well as the trehalose metabolism. In addition, based on differential gene expression analysis, it can be hypothesized that a high oxidative stress response and endocytosis/exocytosis are potential salt management strategies, in addition to the expression of major facilitator superfamily genes responsible for transmembrane ion transport. Under anoxia, genes involved in mitochondrial function, mTOR signalling and autophagy were differentially expressed. Both high salt and anoxia enhanced degradation of erroneous proteins and protein chaperoning. Compared with other branchiopod genomes, Artemia had 0.03% contracted and 6% expanded orthogroups, in which 14% of the genes were differentially expressed under high salinity or anoxia. One phospholipase D gene family, shown to be important in plant stress response, was uniquely present in both extremophiles Artemia and the tardigrade Hypsibius dujardini, yet not differentially expressed under the described experimental conditions.Here, we present the genome of an inbred Artemia was assembled, annotated and analysed, facilitating research on its extremophile features, and providing a reference sequence for crustacean research.A relatively complete genome of The online version contains supplementary material available at 10.1186/s12864-021-07937-z. Artemia are small planktonic crustaceans found in hypersaline environments worldwide vacuolar H + -ATPase); 3) six different mitochondrially coded genes .Under anoxia, many genes involving mitochondrial function were significantly upregulated, indicating that enhancement of the mitochondrial metabolism may be involved in coping with anoxic circumstances: 1) genes responsible for mitochondrial protein translation (leucine--tRNA ligases), also part of the significantly enriched GO “leucine-tRNA ligase activity” Fig. ; 2) eighmTOR gene, which in its turn further activates autophagy [Artemia under anoxia with 10 DEG  of all DEG were differentially expressed under both high salinity and anoxia, with 11 having no functional annotation. There were no common DEG among the top 20 DEG under high salinity and anoxia  compared to other arthropods. This was overcome by using tailor-made gene modelling for Artemia. As a result, and in spite of its unprecedented repeat content, unusual gene structure and limited budget, the Artemia genome assembly had a GC content of 34% in correspondence with literature, a genome assembly contiguity of the same order of magnitude as other similar-sized crustacean genomes (scaffold N50 of 112 Kb), and a 21,828 gene genome annotation, containing 75.5% of the BUSCO reference genes found in other arthropods. While BUSCO analysis results are often not included in crustacean genome publications, this is in a similar range as the 88% BUSCO reference genes found in the E. texana genome, a highly contiguous crustacean assembly considered as a new standard for crustacean genomes [This is the first time that an Anostracan genome is assembled. Unique challenges were overcome for assembly and annotation of the  genomes .Artemia has been described as an extremophile, with nauplii and adults able to survive and thrive at extreme salinities in lakes and saltworks and producing cysts able to survive anoxic conditions. Artemia involving EIF (eukaryotic initiation factor), Heat shock 70 kDa protein cognate, the chitin metabolic pathway, DNA repair, ubiquitination, cell cycle arrest (La-related protein), lipid metabolism and  ion transport [EIF was involved (significant upregulation of genes NAT1A and EIF4AIII), 2) a HSC70-like gene was significantly upregulated, 3) DNA repair-related genes were significantly downregulated, 4) the gene La-related protein 6A was significantly upregulated, and 5) the GO terms “cellular response to lipid”, “lipid transporter activity” and “heme binding” as well as the cellular component GO “structural constituent of cuticle” were significantly enriched.Genome-wide research on halophiles has so far largely been limited to microbial organisms and a few eukaryotes, such as fungi, algae and land plants –11. As fransport . The curArtemia under high salinity. Under salinities as high as 200 g/L, growth in Artemia is substantially slowed down due to its increased energy needs for osmoregulation, whereas at lower salinities (e.g. the 30 g/L control in our experiment), the brine shrimp larva moults every few hours into a subsequent developmental stage until maturity [Portunus trituberculatus [Euphausia superba [L. vannamei [Artemia nauplii to high salinity thus corresponds with a prolonged state of inter-moult.All genes related to “structural constituents of the cuticle”, including several cuticle proteins were downregulated in maturity . Moultinmaturity . Transcrrculatus ; Euphaus superba  and L. vvannamei  at diffevannamei . ExposurArtemia and these were significantly enriched in the DEGs under the Artemia salt stress response in this study. In plants, salt stress causes genotoxicity in which DNA alteration/damage can arise as a consequence of errors in DNA replication and DNA repair [Artemia. In plants as in Artemia, MCM family genes show salt stress-responsive behaviour [Suaeda salsa under high (30 g/L) salinity [For several biological processes previously reported in plant salt stress, similar processes were found in A repair , which aehaviour  and the salinity .+ and Cl− ions as a feature of salt tolerance. During endocytosis, parts of the plasma membrane enclose extracellular substances in vesicles eventually fusing with lysosomes and vacuoles to isolate the plant from the excess salt. During exocytosis, these vesicles export excess ions via plant salt glands [Artemia showed significant GO enrichment of processes in the clathrin-coated vesicles, including clathrin upregulation. Clathrin was also part of an expanded gene family in Artemia compared to other non-extremophile branchiopods. Hence, it appears possible that Artemia would transport salt via clathrin-dependent vesicular transport (endocytosis) for final salt excretion (exocytosis). It remains to be established if exocytosis is ongoing via the Artemia salt gland [In halophytic plant species, there is strong evidence in favour of clathrin-dependent vesicular transport mechanisms of Nat glands . Intereslt gland , which iArtemia salt stress responses in this study, play a key role in targeting and degrading misfolded proteins by ATP-dependent heat shock proteins. Both the use of ATP-dependent HSP and, potentially, endo-and exocytosis of salt, are energy-intensive processes, which may explain the enhanced carbon metabolism in Artemia under high salinity.Protein processing in the endoplasmic reticulum and chaperones, both found to be important Artemia also showed a redox response similar to that observed in plants [Macrostomum lignano [Artemia under salt stress, suggesting that they were directly responsible for ROS control. Transition metals are known to function as balancing factors and signalling for ROS in plants [Artemia. Salt stress most likely induced a strong oxidative stress in Artemia, which was then balanced with transition metal binding proteins.Salt-stressed n plants  and in t lignano , all per lignano . Catalasn plants . TransitArtemia compared to other branchiopods , where they contribute to desiccation tolerance [Artemia genome (results not shown). Tardigrades and Artemia thus share some common strategies relative to osmotic stress and/or desiccation , but they probably also harbour species-specific genes.LEA proteins were part of an expanded gene family in olerance , 83. On olerance , were noHIF) pathway are known master regulators of oxygen sensitivity and distribution inside cells, and are therefore considered highly conserved in most animal groups. Homologs of the Marsupenaeus japonicus hypoxia-inducible factor (HIF-1α [Artemia genome (results not shown) but were not differentially expressed under anoxia in this study. Homologs of VHL (Von Hippel-Lindau tumor suppressor), regulating HIF-α degradation and FIH-1, hydroxylating a specific residue of HIF-α under normoxic conditions, were both absent in the Artemia genome. Within the Crustacea, even if the HIF pathway can be found in several species, it has been lost in other crustaceans, such as the intertidal Tigriopus californicus, where alternative hypoxic stress response mechanisms are believed to play a major role, such as genes involved in cuticle reorganization and ion transport [Artemia has partially lost its HIF pathway and uses an alternative hypoxia response through cuticle reorganization and ion transport. This seems plausible, as Artemia is a halophile and the gene cuticle protein was one of the top 20 differentially expressed genes in Artemia under anoxia. Moreover, it could be that the HIF pathway is involved in regulating low-oxygen (hypoxic) conditions, but not full anoxia such as Artemia was exposed to in this study.The genes in the hypoxia-inducible factor ( (HIF-1α ) were idransport . It is pOrconectes virilis under severe and prolonged anoxia (5–20 h), cAMP-dependent protein kinases strongly decrease and phosphatase activity is strongly increased [mTOR, activating autophagy [Artemia, many protein kinases were up- and downregulated, while most (protein) phosphatase genes were upregulated  cytosolic organelles for nutrients.In the freshwater crayfish ncreased , while hutophagy . In ArteArtemia, mitochondrial function was clearly enhanced with significant upregulation of genes responsible for ATP production in the mitochondrial membrane, cristae morphogenesis, mitochondrial transport and intermembrane chaperoning.Mitochondria are major oxygen consumers and a potential source of ROS. In response to hypoxia, the mitochondrial metabolism often becomes more efficient . In ArteArtemia had the highest number of unique gene families compared to other Branchiopods, containing gene expansions in domains important for salt stress tolerance, such as redox response and endocytosis-related mechanisms, as well as for anoxia tolerance, such as protein phosphorylation. The PLD gene family, while its genes were not differentially expressed under high salinity or anoxia in Artemia, was expanded compared to other, non-extremophilic branchiopods. The PLD gene family consists of lipid metabolic genes that degrade membranes, thus triggering a signalling cascade molecule influencing the mTOR pathway towards survival or inflammation responses, thus initiating proliferation, transcription, cytoskeletal organization and NADPH oxidase and vesicle formation for exo- or endocytosis [In this study, comparative genomics showed that ocytosis . PLD genocytosis . Artemia were identified, still 32% of DEG under salt stress and 21% under anoxia had no known function. Since functional gene annotation is based on homology with other organisms, it is possible that mechanisms unique to Artemia are still hidden within these unknown genes.While many mechanisms underlying the salt stress and the anoxia responses of Artemia has been used in the study of host-microbial interactions (e.g. Vibrio infection), uniquely allowing Vibrio challenge tests under gnotobiotic conditions [Artemia is also, through its short generation time and large population size, susceptible to truncation-based genetic selection. Cysts generated in such experiments can be stored and hatched simultaneously, allowing for common garden experiments [Artemia are instrumental in the study of transgenerational phenotype inheritance and hence in the research of epigenetic processes [Artemia shows a relatively large similarity with the recently published L. vannamei genome . It is anticipated that with both annotated genomes currently now available, it will be possible to fully explore the potential of Artemia as a model species for commercial aquatic crustaceans in the fields of host-microbial interaction, genetics and epigenetics.nditions . Artemiaeriments . Moreoverocesses . Finally, contaA. franciscana, as well as comparative genomics with other animals and functional genomics under high salinity and anoxia. Compared with other branchiopods, the Artemia genome has many contracted and expanded gene families involved in different strategies for coping with stressors including high salt and anoxia. This means that the differences between Artemia and other branchiopods can probably be largely attributed to the stress coping abilities of Artemia. Under high salinity, Artemia potentially uses plant-like, energy-intensive strategies to enclose excess salt in the cell, as well as DNA repair, extensive molecular chaperoning, redox and osmotic balancing, and LEA proteins and trehalose against desiccation. Under anoxia, mitochondrial function was enhanced, while the mTOR pathway activated the autophagy pathway, potentially for recycling of damaged cell components as nutrients.Here, we present the genome assembly and annotation for an inbred All bioinformatics up until genome characterization with DBG2OLC were performed on the VIB-PSB Biocomputing Linux cluster, all subsequent steps were performed on the HPC-UGent, supported by the Flemish Supercomputer Center.A. franciscana Kellogg, 1906 , Utah, USA) were reared at the Department of Medical Technology, University of Occupational and Environmental Health, Kitakyushu, Japan: a 48 generation inbred female parent , the nauplii were harvested. Considering the hatching time frame, these nauplii should be instar I or II, which are difficult to separate during the hatching process. Nauplii were reared until sexual maturity in aerated seawater with added sea salt  and fed with microalgae Tetraselmis suecica. Mature animals were rinsed with sterile distilled water, the phenotypic sex of each individual offspring was determined visually and females were selected for further processing. Females were favoured since they are the heterogametic sex, thus ensuring sequencing of both the Z and the W chromosomes. For gut evacuation before DNA extraction, the females were kept overnight in a cellulose solution  [Inbred r “GSL2” ) and a 6r “GSL2” ), were cich USA) , followeDNA was individually extracted from seven female individuals grown from Specimen 9 cysts according to a modified CTAB-method for shrimp tissue . BrieflyDNA from inbred females served as the template for PacBio (Pacific Biosciences USA) and Illumina (USA) sequencing technologies.DNA from one female individual (sample name F2) from Specimen 9 was sequenced with several technologies, as described below.One library (500–800 bp) was prepared for the Illumina sequencing: Illumina NextSeq library prep was done with the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® . The library was paired-end sequenced with Illumina NextSeq 500 High 300 Medium technology  and with Illumina MiSeq 600 v3 technology . Read quality was tested using the software FastQC v0.11.2 . IlluminA total of 10X intended sequencing output by PacBio technology was generated: 3–20 kb DNA from four female individuals from Specimen 9  was sequenced on 8 SMRT cells using PacBio RS technology with 2X intended coverage , and 10 kb DNA from two other Specimen 9 females  was sequenced on 12 SMRT cells using PacBio RSII technology with 8X intended coverage . The data collected from the PacBio RS instrument were processed and filtered using the SMRT Analysis software suite . The Continuous Long Read (CLR) data were filtered by Read-length (> 35), Subread-length (> 35) and Read quality (> 0.75).Read quality was tested using the software FastQC v0.11.2 . PacBio A. franciscana (SFB x VC) genome (SUB5823124) was assembled at the ARC as a side product of a bulked segregant analysis [Artemia. Since it was constructed from Illumina sequences of a pooled sample of non-inbred diploid animals, and the genome contained many repeats (44%), heterozygosity was high at almost 4 M SNPs in the 1310 Mbp genome assembly, redundancy was 41% compared to the published genome size of 0.93 Gb [Previously to this publication, a non-inbred analysis , out of analysis  Illumina 0.93 Gb , and the 0.93 Gb .A. franciscana (GSL) genome described in this publication was subsequently assembled, based on Next-Generation Sequencing (NGS) data from one female and scaffolded with PacBio data from six additional females. Various assemblies were generated using different combinations of software and subsets of data. The best assembly was selected according to scaffold N50 and assembly size. The foundation of the chosen assembly was assembled using the SPAdes genome assembler v3.10.0  [Therefore, the new inbred careful)  with 3 iArtemia DNA sequencing reads: a first scaffolding round with the inbred Illumina PE and SE data, a second scaffolding round with the inbred PacBio data, and a third scaffolding round with the inbred Illumina PE and SE data, according to the following command lines :DBG2OLC v20150519  was useddbg2olc k 50 KmerCovTh 0 MinOverlap 70 LD1 0 PathCovTh 3 Contigs contigs.fasta f illumina_PE.fastq f illumina_SE.fastq.dbg2olc MinLen 100 k 17 KmerCovTh 2 MinOverlap 10 AdaptiveTh 0.001 LD1 0 RemoveChimera 0 Contigs. /DBG2OLC_Consensus.fasta f pacbio.fastq.dbg2olc k 50 KmerCovTh 0 MinOverlap 70 LD1 0 PathCovTh 3 Contigs. /backbone_raw.fasta f illumina_PE.fastq f illumina_SE.fastq.2)A. franciscana transcriptome [L_RNA_scaffolder v20141124  was usedcriptome  and the blat /dev/null /dev/null -makeOoc = 11.ooc -repMatch = 1024, blat -t = dna -q = rna -ooc = 11.ooc -fine -noHead, sh L_RNA_scaffolder.sh -d. -l 0.9 -p 0.9 -e 100,000 -f 1.3)SSPACE_Basic v2.1.1  was used-× 0 -g 3 -k 3.The SPAdes genome assembly result with K-mer size 127 was scaffolded using three different scaffolding software packages:Gaps in the scaffolded genome were filled with GapFiller v1.10  using thThe gap-filled genome sequence was polished with 10 iterations of BWA v0.7.15 . mappingArtemia transcriptome assembled for that purpose.The genome was structurally annotated using several data sources, including a new inbred 2 progeny from the inbred A. franciscana  was further interbred in one tank for 2 months during which cysts were continuously harvested, after which they were stored in saturated NaCl-brine at 7 °C. These cysts were transferred to a Petri dish  and sampled after 0 h (quiescent), 4 h, 8 h, 12 h, 16 h and 20 h. After 24 h, a sample of nauplii was taken. The remaining animals were further reared  and the RNeasy Mini Kit  as described previously , except All samples were pooled, and one library (500 bp) was prepared for the Illumina sequencing (Illumina Truseq stranded mRNA). The library was paired-end sequenced with Illumina MiSeq v2 500 technology . Read quality was tested using the software FastQC v0.11.2 . IlluminTrimmomaticPE 0.32, ILLUMINACLIP: adaptors.fa:2:30:10 LEADING:3 TRAILING:3 MINLEN:36.bbmerge-auto.sh rem k = 62 extend2 = 50 ecct vstrict.then merged with BBMap v36.62 to produce 250 bp long single reads , commandMerged reads were assembled to a transcriptome with SPAdes v3.10.1  using the protein homology and RNA-seq provided as hints, to obtain a first draft annotation from which gene models were extracted and manually inspected to create a sufficiently large training set for EUGENE v4.2a [Artemia , transc samples , and RNAplatform  with Genplatform . Genome platform .Artemia genome, with RepeatModeler v1.0.8(−engine ncbi) [To explain the difference in genome size between branchiopods, the genomes analysed for comparative genomics were analysed for genome size, repeats, transposable elements, as well as exon and intron length and small RNAs as follows. As only for a subset of the investigated organisms, repeat data sets exist and as we aimed to have homogeneous repeat data to be able to compare repeats between species, we searched and collected repeats “de novo” in each branchiopod species mentioned in Table ne ncbi) . The colne ncbi)  data forA. franciscana mitochondrial genes [Artemia proteome obtained by translation of the nuclear inbred genome assembly annotation. Scaffolds of equal or shorter length than the A. franciscana mitochondrial genome , containing only mitochondrial genes were considered “mitochondrial” to retain only nuclear genome sequences.Scaffolds of mitochondrial origin were identified by BLAST+ v2.9.0  of the al genes  as indical genes  onto theScaffolds originating from alien microbial, fungal or viral organisms were eliminated from the assembly after performing BLAST + v2.9.0 (blastx) on the RefSeq non-redundant protein databases “archaea”, “bacteria”, “protozoa”, “fungi”, “viral” and “invertebrate” , removinFunctional genome content and repeats were determined as described (Methods sections 1.5.2. and 1.5.3.) based on arthropod proteome databases (see Table Tetranychus urticae), two insects with high-quality genome assemblies, one tardigrade as the representative extremophile, and 7 crustaceans including Amphipoda, Decapoda, Copepoda and Branchiopoda, thus including all four branchiopod lineages [Artemia as compared to three other (non-extremophile) branchiopod species. The second aim was to find similarities between extremophilic tardigrade H. dujardini and Artemia on the one hand, and non-extremophile crustaceans on the other hand. The third aim was to find similarities between Artemia and L. vannamei pointing to biological processes in L. vannamei for which Artemia would be a suitable model.Based on genome contig N50 , and taxonomic group, 11 arthropod proteomes were selected  was performed onto expanded, contracted and common gene families to identify which biological processes were involved in 1) gene family expansion/contraction in Artemia compared to other Branchiopoda; 2) unique gene families in extremophiles Artemia and Tardigrada; 3) the non-expanded/contracted gene families in Artemia compared to L. vannamei. Enrichment was done with default settings: remove double ids was unchecked, rendering the Fisher’s Exact Test sensitive in the direction of the test: the sequences that are present in both test-set and reference set will be deleted from the reference, but not from the test set, which can lead to zero values in the reference set. For comparative genomics with Branchiopoda, results were reduced to the most specific GO’s (FDR < 0.05), and a bar chart with GO enrichment of up-and downregulated genes was made. For comparative genomics, with L. vannamei, a combined graph was made to show functional hierarchy of the non-expanded/contracted gene families in Artemia compared to L. vannamei.Large and small gene family expansions were analysed between A. franciscana of the strain San Francisco Bay (SFB), cyst bank ID code ARC1767. SFB was chosen and not GSL, because we were still working on the non-inbred (SFB x VC) genome at the time, and we had not yet sequenced the genome of the inbred Artemia (GSL).All following experiments were subsequently done with 2 to flash-freeze. The remainder of the hydrated cysts was 1) put in filtered, non-autoclaved sea water brought to 30 g/L in a cone with aeration in the dark at 28 °C for 24 h to remove cysts hatching in the dark, the remaining cysts were left to hatch for 24 h under the same conditions, but in light, or 2) left to hydrate longer for a total of 6 h with aeration, and then used for testing under anoxic conditions.In order to synchronize the cyst metabolic stages, the following protocol was followed: cysts were hydrated in water, dehydrated in brine, rinsed and hydrated with tap water for 1 h with aeration, sieved and rinsed with mQ. Samples were taken with a small spatula cleaned with ethanol and put in labelled Eppendorfs, previously put in liquid NNauplii were separated from cysts by removing aeration for a few min, pipetting the nauplii (25 ml pipette) over a sieve and adding them to a new, clean, aerated cone with Instant Ocean® USA 30 g/L.Artemia mix per sample with a 1-ml pipette with cut tips. Samples were flash-frozen with liquid N2. Between sampling times, the sieve was washed with tap water and rinsed with autoclaved mQ.Samples were taken after 15, 30, 45 and 60 min in new 30 g/L Instant Ocean® USA artificial sea water by transferring 50 ml (with 25 ml pipette) from a cone of nauplii over a sieve per sample time, rinsing with mQ over the sieve and taking 100 μl of mQ/To check the cysts/nauplii ratio in our samples  and the number of animals per sample, we observed 1 ml of the 50-ml samples and counted 93 instar I/II nauplii/ml and 9 cysts (full or empty)/ml, corresponding with 1550 nauplii per sample, each sample containing approximately 10% cysts (full or empty).Nauplii were separated from cysts by removing aeration for a few min, pipetting the nauplii (25-ml pipette) over a sieve and adding them to a new, clean, aerated cone with Instant Ocean® artificial brine at 200 g/L salinity.Artemia mix per sample with a 1 ml-pipette with cut tips. Samples were flash-frozen with liquid N2. Between sampling times, the sieve was washed with tap water and rinsed with autoclaved mQ.Samples were taken after 15, 30, 45 and 60 min in new 200 g/L Instant Ocean® USA seawater by putting 50 ml (25 ml-pipette) from a cone of nauplii over a sieve per sample time, rinsing the sieve with mQ, and taking 100 μl of mQ/2 gas was flushed through the vial to remove the oxygen in the vial, then the vial was capped and set aside, allowing the N2 to seep into the porous parts of the outer cyst shell. N2 was bubbled through buffer  during a minimum of 4 h. With the N2 still bubbling, 6 ml of the anoxic solution was transferred into each vial without any air leaking into the vial and Parafilm was wrapped around the tightly shut vial cap. Vials were oscillated on a rotating platform shaker (50 revolutions/min) for the first 24 h to hydrate the cysts completely and to ascertain that even traces of oxygen (if present) were consumed. The vials containing the anoxic, hydrated cysts were stored at room temperature and in ambient light.Two scintillation vials with cysts hydrated for 6 h in 30 g/L sea water at 28 °C were put under anoxic conditions in the light on a rotor. Anoxic conditions were reached according to a modified protocol . In scinArtemia mix was taken with a 1-ml pipette with cut tips. Samples were flash-frozen with liquid N2. Between sampling times, the sieve was washed with tap water and rinsed with autoclaved mQ.Samples were taken after 25 h of anoxia (1 supplementary hour after metabolic activity stops). The content of each vial was sieved and rinsed with mQ. Per sample, 100 μl of mQ/The anoxic state in the scintillation vials was verified indirectly by observing the cysts under a binocular microscope after 3 days of anoxia. In a sample of 500 cysts, there was one clear umbrella and a few , 3 cystsA. franciscana from the San Francisco Bay strain (SFB) were sampled under four different conditions, including different life cycle stages, and analysed (Table http://gc.uzleuven.be) on an Illumina HiSeq 2000 instrument . Reads of each RNAseq library were quality-trimmed with CLC Assembly Cell v4.06 software [ed Table . RNA extFastQC v 0.11.2 and MultiqQC, v1.7  were useThe “high salinity” and “anoxia” conditions Table  were exaSamples were annotated based on life cycle stage, time point at which the sample was taken, salinity level of the treatment, light environment of the treatment and the batch effect, a technical variation source introduced by necessity in high-throughput studies Table . SamplesFor high salinity, after filtering of non-informative genes, i.e., counts per million equivalent < 3 for over 14 out of 16 samples, the following model was fitted to the expression data :B: batch effect of second batch.With βS: high salinity treatment effect.βT30: time effect after 30 min (compared to 15 min).βT45: time effect after 45 min (compared to 15 min).βT60: time effect after 60 min (compared to 15 min).βp-value.This way, the effect of the salinity could be assessed at each time point separately. As the design is rather complex, a stage-wise testing approach as described by Van Den Berghe et al. was performed, thus boosting power for the interaction effects . BrieflyFor anoxia, after filtering of non-informative genes, i.e., counts per million < 3 for over 3 out of 6 samples, a simple model only considering the effect of anoxia compared to hydrated cysts was used. No batch effect was present in samples studied under anoxia.Normalization was performed using trimmed mean of M-values (TMM), and FDR adjustment by Benjamini-Hochberg’s method, as default for edgeR v3.24.3, which was used for both omnibus test and post-hoc testing . Genes wArtemia proteome  as the reference set. Enrichment was done with default settings: remove double ids was unchecked, rendering the Fisher’s Exact Test sensitive in the direction of the test: the sequences that are present in both test-set and reference set will be deleted from the reference, but not from the test set, which can lead to zero values in the reference set. Results were reduced to the most specific GO’s (FDR ≤ 0.05), and a bar chart with GO enrichment of differentially expressed genes was made. Pathway enrichment was performed with the online GUI software STRING v11.0 using default settings and model organism Daphnia pulex [D. pulex proteins in its database, only the best match was kept. Subsequently, STRING performed pathway enrichment with a stringent cut-off (FDR ≤ 0.05) on each D. pulex gene. Interesting enriched pathways were drawn with the online GUI software KEGG Mapper Search Pathway function [For each contrast, GO names , were analysed for enrichment (FDR ≤ 0.05) with Fisher’s exact test in OmicsBox  using thia pulex  and usinia pulex . STRING function  in the oAdditional file 1. Assembly characteristics of all assembled crustacean genomes. Characteristics listed are: species, whether the species genome is annotated yes or no, N50 of the fragments with the highest assembly hierarchy, number of fragments with the highest assembly hierarchy in the assembly, haploid genome size, assembly size, completeness of the assembly (=haploid GS/assembly size), taxonomic lineage (NCBI taxonomy), reference for the genome paper.Additional file 2. Evolution of Artemia assembly quality metrics throughout the assembly steps. Evolution of the scaffold N50, the number of fragments and the genome completeness (assembly size/genome size) in the subsequent Artemia assembly stagesAdditional file 3 BUSCO analysis results for the A. franciscana genome assembly and annotation.Additional file 4. BLAST results for mitochondrial genes in the Artemia genome. Listed: Query accession and gene name, presence of a (significant) BLAST hit in the Artemia proteome with the highest bit score, E-value and bit score of the hit, scaffold length of the scaffold on which the hit lies, percentage of mitochondrial genes on this scaffold.Additional file 5. Taxonomic groups of alien genomes identified in the Artemia genome.Additional file 6 Expanded or contracted Artemia orthogroups compared to other Branchiopoda. Listed: Orthogroup ID, number of genes in this orthogroup in A. franciscana, D. pulex, L. arcticus, and E. texana, expanded or contracted status of the orthogroup in Artemia compared to D. pulex, L. arcticus and E. texana, conservation in Branchiopoda (whether this orthogroup contains genes for each branchiopod), comma-separated IPR description of Artemia genes in this orthogroup, Artemia genes in this orthogroup.Additional file 7 GO enrichment of Artemia compared to other Branchiopoda. Listed: GO ID, name and category, false discovery rate (FDR) and P value of the Fisher’s exact test enrichment analysis in Blast2GO, number of Artemia genes from expanded/contracted orthogroups in this GO ID, number of whole Artemia genome genes in this GO category, number of Artemia genes from expanded/contracted orthogroups in this GO ID without GO annotation. The Fisher’s Exact Test is sensitive in the direction of the test: the genes that are present in the test-set and also in the reference genome set will be deleted from the reference, but not from the test set, resulting in zero sequences in the reference set and values above zero in the test set. Significantly enriched GOs  of Artemia genes in expanded or contracted orthogroups compared to Branchiopoda are given.Additional file 8 Expanded or contracted Artemia and H. dujardini orthogroups compared to other Arthropoda. Listed: Orthogroup ID, number of genes in this orthogroup in A. franciscana and in the other arthropod species, expanded or contracted status of the orthogroup in Artemia compared to the other arthropod species, comma-separated IPR description of Artemia genes in this orthogroup, H. dujardini genes in this orthogroup, Artemia genes in this orthogroup.Additional file 9. STAR mapping statistics for differential expression analysis in Artemia. Listed: sample name, total number of reads for this sample, percentage of uniquely mapped reads, absolute number of uniquely mapped reads, percentage of multi mapped reads, absolute number of multi mapped reads.Additional file 10. Summarization statistics for differential expression analysis in Artemia. Listed: sample name, total counts, percentage of counts assigned to a gene annotation, absolute counts assigned to a gene annotation. * notice that this amount can be more than the sum of uniquely mapped + multi-mapped in the mapping statistics since multi-mapped reads are considered.Additional file 11 Differentially expressed genes under high salinity (p < 0.05). Listed: functional annotation of the differentially expressed gene, gene ID in the genome annotation and on the ORCAE platform, p value, average log fold change of gene expression under high salinity, gene regulation of the differentially expressed gene (up or down), InterPro description of the gene family to which the gene belongs.Additional file 12 Differentially expressed genes under anoxia (p < 0.05). Listed: functional annotation of the differentially expressed gene, gene ID in the genome annotation and on the ORCAE platform, p value, log fold change of gene expression under anoxia, gene regulation of the differentially expressed gene (up or down), InterPro description of the gene family to which the gene belongs.Additional file 13 GO enrichment in Artemia under high salinity. Significantly Enriched GOs (FDR ≤ 0.05) of Artemia genes differentially expressed under high salinity. Listed: GO ID, name and category, false discovery rate (FDR) and P value of the Fisher’s exact test enrichment analysis in Blast2GO, number of DEG under high salinity in this GO category, number of whole Artemia genome genes in this GO category, number of DEG under high salinity without GO annotation. The Fisher’s Exact Test is sensitive in the direction of the test: the genes that are present in the test-set and also in the reference genome set will be deleted from the reference, but not from the test set, resulting in zero sequences in the reference set and values above zero in the test set.Additional file 14 Pathway enrichment in Artemia under high salinity. Significantly enriched  pathways of Artemia genes differentially expressed under high salinity. Listed in first worksheet (STRING annotation): gene number, ORCAE gene ID, STRING Daphnia pulex gene ID, BLAST identity and bit score, gene name and gene annotation. Listed in second worksheet (STRING pathway enrichment): KEGG Daphnia pulex pathway name, pathway description, number of DEG under high salinity in this pathway, number of genes in the D. pulex genome that belong to this pathway, enrichment FDR, matching D. pulex gene IDs, matching gene names in pathways shown in figures and additional files, matching D. pulex gene ID labels.Additional file 15. Consolidation of DEG analysis, GO enrichment and pathway enrichment in Artemia under high salinity.Additional file 16. The enriched Carbon metabolism pathway in Artemia under high salinity. Up- and downregulated genes are indicated on the KEGG map dpx01200.Additional file 17. GO enrichment in Artemia under anoxia. Significantly enriched GOs (FDR ≤ 0.05) of Artemia genes differentially expressed under anoxia. Listed: GO ID, name and category, false discovery rate (FDR) and P value of the Fisher’s exact test enrichment analysis in Blast2GO, number of DEG under anoxia in this GO ID, number of whole Artemia genome genes in this GO ID, number of DEG under anoxia without GO annotation. The Fisher’s Exact Test is sensitive in the direction of the test: the genes that are present in the test set and also in the reference genome set will be deleted from the reference, but not from the test set, resulting in zero sequences in the reference set and values above zero in the test set.Additional file 18 Pathway enrichment in Artemia under anoxia. Significantly enriched  pathways of Artemia genes differentially expressed under anoxia. Listed in first worksheet (STRING annotation): gene number, ORCAE gene ID, STRING Daphnia pulex gene ID, BLAST identity and bit score, gene name and gene annotation. Listed in second worksheet (STRING pathway enrichment): KEGG Daphnia pulex pathway name, pathway description, number of DEG under anoxia in this pathway, number of genes in the D. pulex genome that belong to this pathway, enrichment FDR, matching D. pulex gene IDs, matching gene names in pathways shown in figures and additional files, matching D. pulex gene ID labels.Additional file 19. Consolidation of DEG analysis, GO enrichment and pathway enrichment in Artemia under anoxia.Additional file 20. The enriched N-glycan biosynthesis pathway in Artemia under anoxia. Up- and downregulated genes are indicated on the KEGG map dpx00510.Additional file 21. The enriched Basal transcription factors pathway in Artemia under anoxia. Up- and downregulated genes are indicated on the KEGG map dpx03022.Additional file 22. Augustus custom training files for Artemia. Includes probabilities, parameters and weights used for Augustus training for annotation of the Artemia genome.Additional file 23. EuGene custom parameter file for Artemia. Includes parameters used for EuGene training for annotation of the Artemia genome.Additional file 24. Sequence GC-content profiles for all samples used for differential expression analysis.