Chemical Oxygen Demand

Thirty specimens from two colonies of Azteca trigona collected in Ponte Nova (20°25'S, 42°54'W) and Viçosa (20°45'S, 42°52'W), MG, Brazil were analyzed. The colonies were collected in the field and transferred to a plastic container and maintained in a BOD (Biochemical Oxygen Demand) incubator at 25°C following the protocol described by Cardoso et al. (2011) and fed with honey in order to obtain larvae in the pre-pupa stage (post-defecating larvae). The specimens were identified by specialists and Vouchers of the samples collected in this work were deposited in CEPLAC and MZUSP.
Cytogenetic analysis was performed using cerebral ganglia of the larvae selected. Metaphase chromosomes were obtained according to the methodology proposed by Imai et al. (1988) (link). Preparations obtained from fifteen individuals per colony were analyzed. The preparations were stained with Giemsa diluted in Sörensen buffer at (4%) for 20 minutes. On average, ten metaphases were analyzed per slide and ten slides were submitted to banding techniques. C-banding was performed by BSG method (Barium hydroxide/Saline/Giemsa) according to Sumner (1972) (link). The protocol of Schweizer (1980) (link) was used for preparation of sequential fluorochrome staining (CMA₃/DA/DAPI). Identification of nucleolus organizer regions (NOR) was performed according to Howell and Black (1980) (link). The best metaphases were photographed using an Olympus BX60 microscope equipped with a camera Q color 3 Olympus®. Brightness and contrast of the karyotypes were optimized using Photoshop CS4. The karyotypes were mounted in Corel Draw® 13 image editing software. Karyotype structure was described according to the nomenclature proposed by Imai (1991) (link) and Levan et al. (1964) . For mounting of the karyotypes, the chromosomes were sorted into three groups: metacentric chromosomes (M), acrocentric chromosomes (A) with heterochromatin located across the length of the short arm of the chromosomes, and pseudo-acrocentric chromosomes (A^M) which possess a long heterochromatic arm (Imai 1991 (link)).

A customized flat sheet type membrane module with an effective membrane area of 0.0045 m² (0.09 m × 0.05 m) was submerged in a column type (polymethyl methacrylate) (PMMA) reactor (Fig. S2). The membrane module was manufactured by MemSis Turkey and employed a polysulfone (PS) ultrafiltration (UF) membrane (PHILOS, Korea) with 20 KDa of molecular weight cut-off (MWCO).
The system was operated in a gravity-driven filtration mode where the effluent (or permeate) was collected from the bottom of the tank, and more details can be found elsewhere^{24 (link), 25 (link)}. A synthetic secondary wastewater effluent (SSWE) was pumped continuously into a level regulator to keep the level of the feed water in the tank constant, resulting in a constant pressure head of 45 cm above the membrane (corresponding to a TMP of 4.5 kPa). Only a small quantity of activated sludge (4 mg of MLSS corrected from wastewater treatment plant at KAUST in Saudi Arabia) was initially added to the filtration tank to enhance the formation of a biofilm on the membrane surface. The whole reactor was covered with aluminum foil to elude the growth of algae by light exposure. The experiment was divided into two sets. The first set was conducted for 7 d with the aim to observe the initial stages of biofouling formation, and the second one was continued for 42 d to observe more long-term biofilm developments.
A SSWE was used as feed solution to grow the biofilm on the membrane. The detailed characteristics of feed water can be found elsewhere^{26 (link)}. The feed solution was refreshed every 7 d. Chemical oxygen demand (COD) of the SSWE was 7.5 mg/L for the first set of experiments. In order to enhance the biofilm formation, for the second experiment the COD concentration of the feed water was increased to 15 mg/L.
The permeate flow rate was measured using a flow meter (Sensirion). The permeate flux was calculated by dividing the permeate flow by the membrane area (0.0045 m²). As mentioned above, the GD-SMBR was operated under constant pressure by maintaining a constant water head as shown in Fig. S2. A constant TMP of 4.5 kPa was applied to all experiments.
In this study, the OCT was employed to investigate the biofilm formation (or growth) on a membrane (submerged) in a GD-SMBR. The OCT (Thorlabs GANYMEDE spectral domain OCT system with a central wavelength of 930, Thorlabs, GmbH, Dachau, Germany) equipped with a 5X telecentric scan lens (Thorlabs LSM 03BB) was used.
The time-resolved OCT investigation was performed in three different periods of the filtration runs as given in Table S1. In the first experiment (Experiment 1), a fixed position corresponding to 5.00 mm × 1.35 (width × depth) was monitored. The scan frequency was set to 10 min for the first 42 h (from 12 h to 42 h), and then 5 min from 84 h to 96 h. The second experiment (Experiment 2) was conducted for a period of 42 d, where OCT scans were acquired daily at a fixed position corresponding to 4.00 mm × 1.35 mm.
Serial static images (i.e. video) are commonly used to depict the dynamic process of fouling formation. In this study, three videos (Supplementary Videos 1–3) of the periods monitored (Table S1) are shown in supporting information. The preprocessed OCT scans were assembled into AVI digital movie format using Fiji software.

All relevant clinical and laboratory data were collected from electronic medical records. Data pertaining to the ventilator settings of the patients on IMV support and of the laboratory tests of all the patients were collected according to the index date of each patient. In the case group, for patients who did not receive IMV support, the index date was defined as the day with the highest recorded oxygen demand before the development of PNX/PNM. For those who received IMV support, the index date was set as the day with the highest recorded peak pressure before the development of PNX/PNM. In the matched-control group, for the patients who did not receive IMV support during admission, the index date was defined as the day with the highest recorded oxygen demand, and for those who received IMV support, the index date was set as the day with the highest recorded peak pressure during admission. Disease severity was classified according to the worst National Institute of Allergy and Infectious Disease Ordinal Scale during admission.
The Charlson Comorbidity Index was calculated at admission to classify patients according to overall comorbidity. The sequential organ failure assessment (SOFA) score was used to measure the severity of organ dysfunction. Superimposed pneumonia was defined as follows: (1) new or worsening infiltrates on chest radiography, (2) positive bacterial culture from the respiratory specimen or positive PCR results for other respiratory pathogens, and (3) administration of antimicrobial agents against newly identified pathogens. COVID-19-associated pulmonary aspergillosis was defined as proven or probable invasive aspergillosis based on the definition proposed by the EORTC/MSGERC ICU Working Group [17 (link)].
The objective of this study was identifying risk factors associated with PNX/PNM for patients with COVID-19.

Linear mixed effects models were employed to assess the role of environmental factors as a driver of carbon and nitrogen stable isotopes values fluctuation over the years. For each species or functional group, a separate analysis was performed. Given that the values of stable isotopes in the animal body are gathered over time, we used environmental data from the end of April to the end of July of each respective year. To omit the effects of monthly variability on stable isotopes value, we used month as a random effect. The final model was determined by sequential deletion of the least significant explanatory variables from the full model. Parameter significance was evaluated using F-tests from analysis of deviance. The final model included only parameters with significant p-values. Temperature, transparency, flooded area, oxygen, pH, NH₄⁺, chemical oxygen demand by manganese (COD_Mn), and Chlorophyl a were used as explanatory variables of stable isotopes values changes (Table 1, Fig. S1). These parameters were chosen using correlation matrix where parameters with high correlation with used parameters were omitted (value 0.6 consider as threshold among parameters used in analyses). The significant parameters of the final model, a simple linear regression was applied to test the biological effect of given environmental variables. We compared the slopes of given variables among consumers using the lstrends function of the ‘lsmean’ package and ran generalized linear models to reveal differences in each consumer carbon and nitrogen stable isotopes over the years. For all statistical tests, p-values < 0.05 were considered significant. Analyses were performed in R-software²⁶ (4.05).