Chelex 100

A set of eight DNA samples of P. falciparum from the Democratic Republic of Sao Tomé & Principe (DRSTP) were used in this study. Blood samples had been collected previously by Passive Case Detection (PCD) as part of an on-going collaboration between Portugal and the DRSTP during the month of February 2004, from suspected malaria carriers attending the Centro Policlínico de Saúde de Água Grande, in the city of São Tomé.
No age restrictions were applied. After confirmation of P. falciparum infection by microscopical observation of thin and thick Giemsa-stained blood films, 1 ml of venous blood was collected into Monovettes containing EDTA, after individual informed consent and local ethical approval. A sub-sample of this was spotted onto Whatman n°4 filter paper and then parasite genomic DNA was obtained from all samples by boiling in Chelex-100 [12 (link)] followed by ethanol precipitation. A similar protocol was used to extract genomic DNA from the references strains P. falciparum 3D7 and Dd2, which were kept in deep frozen stabilates and cultured in vitro at the time of these experiments.

Chloroplast transformation was based on a previously described method (Kindle et al. 1991 (link)) and involved the agitation of an algal/DNA suspension with glass beads of 400–625 μm diameter. A 400-ml culture grown to early log phase (approx. 2 × 10⁶ cells/ml) was concentrated by centrifugation and resuspended in TAP medium to 4 ml. Three hundred microliters of cells were added to a sterilized 5-ml test tube containing 300 mg sterile glass beads, followed by 5–10 μg circular plasmid DNA. The mixture was agitated vigorously at the maximum speed of a Vortex Genie II (Fisher Scientific, Loughborough, UK) for 15 s. The cells were spread on selective agar plates (TAP + spectinomycin at 100 μg/ml for aadA selection; HSM for psbH selection) after mixing with 0.5 % molten (42 °C) agar of the same selective medium. The plates were incubated at 25 °C in dim light (~2 μE/m²/s) overnight then transferred to a moderate light (~50 μE/m²/s) the next day. Transformant colonies were picked after 2–3 weeks and restreaked to single colonies several times on selective media to ensure homoplasmicity, although this was often achieved after the first streaking. Homoplasmy was determined by PCR using a combination of three primers (Table S1). Total genomic DNA was extracted from a loopful of cells using the Chelex 100 method as described by Werner and Mergenhagen (1998 (link)), and PCR amplification was carried out with Phusion DNA polymerase (Thermo Scientific) according to the manufacturer’s instructions. The photosystem II-deficient phenotype of TN72 was confirmed by measuring the photosynthetic capacity through chlorophyll fluorescence (Maxwell and Johnson 2000 (link)). Putative transformant lines were spotted onto TAP agar plates, incubated for 5 days and scanned using a pulse-modulated imaging fluorometer (FluorCam 700MF, Photon Systems Instruments, Czech Republic) as described by Wingler et al. (2004 (link)).

Cross-sectional community surveys were carried out between May to August 2021 when malaria transmission was at its peak in Western Kenya [4 (link), 7 (link), 18 (link)]. The CHVs were trained on recording febrile cases in each household, taking blood samples for RDT, and preparing dry blood spots for real-time-PCR (RT-PCR) analysis. A febrile malaria case was defined as an individual with fever (axillary temperature ≥ 37.5 °C) at the time of examination or complaints of fever and other nonspecific symptoms 1–2 days prior to examination [19 ]. The CHVs used an active case detection (ACD) questionnaire to interview residents about their fever status. Febrile residents’ age, sex, and active fever, fever days, treatment-seeking behaviours, primary occupation, travel history, and bed net usage, health insurance coverage, transportation method to the health facility, and reasons for delaying in treatment were collected in the questionnaire. The questionnaire results were reviewed daily by team supervisors for quality assurance.
Finger-prick blood samples were taken from febrile cases for parasite examination with ultra-sensitive Alere^® malaria RDT (Reference number: 05FK140, Republic of Korea) and RT-PCR on dry blood spots. The samples were then transported to the International Centre of Excellence for Malaria Research (ICEMR) at the University of California Irvine-Tom Mboya University Joint Laboratory in Homa Bay, Kenya [4 (link), 5 (link)], for further analysis. The Chelex resin (Chelex-100) saponin method was used with minor modifications [20 (link)]. Primers and probes specific to Plasmodium species were used to target 18S ribosomal RNA [21 (link)] to confirm the presence of parasite DNA on QuantStudio™ 3 Real-Time PCR.

This study was conducted with free-living Adélie penguins during the guard stage of chick rearing when adults alternate between taking foraging trips or guarding the young (21st December 2018-14th January 2019) at Dumont d'Urville station, Terre Adélie, East Antarctica (66°40′S; 140°01′E). We captured 58 adults (24 females and 34 males) on the nest, when both adults were attending the nest prior to a changeover. Only one member of a pair was ever sampled, to reduce disturbance time to the nest.
This study took advantage of a blood sample protocol required for a doubly labelled water (DLW) experiment that necessitated two initial blood samples, one taken after capture and a second one taken after a calibration period in which the animals must be held in captivity (Hicks et al., 2020 (link)). Specifically, upon capture, individuals were blood sampled within 3 min of first handling from the tarsus vein. This sample served as a background sample providing the unstressed situation. After blood sampling, birds were weighed to the nearest gram, flipper measured to the nearest millimetre, and injected with 0.3 ml of DLW per kg of body weight into the pectoral muscle [see Hicks et al. (2020) (link) for details]. For future identification, birds were marked with a unique identifying code printed on a piece of marine tape rolled around their back feathers. This entire handling period took about 15 min (study mean) and several previous studies have shown that 15 min of handling evokes a stress response as measured by increases in corticosterone in this species (Cockrem, 2013 (link); Cockrem et al., 2006 (link), 2008 (link)) and also in this population (Marciau et al. under review ). After this handling period, birds were placed in a contained area outside the lab for the DLW to equilibrate (for between 1.6 and 2.7 h). The contained area measured 2×2.80×5.20 m, birds were always placed in the contained area with another penguin to calm them and the space was filled with a layer of snow for comfort and cooling. A second blood sample was taken after the equilibrium period. This sample served as our handling time sample. Hold duration was calculated as the time between the first capture and the second blood sample. Whole blood was kept on ice in Eppendorf tubes for up to 10 min before being centrifuged (10,000 rpm, 10 min) and plasma stored at −80°C until analysed. The molecular sexing of all individuals was carried out at the service Analyses Biologiques of the Centre d'Etudes Biologiques de Chizé (CEBC). DNA extraction was conducted with 2 µl of pellet (red blood cells) and using a chelex resin (Chelex 100 Molecular Biology Resin, Bio-Rad; 10%) associated with Proteinase K (PK) as written in the manufacturer's instructions. We then performed a polymerase chain reaction (PCR) with amplification of the CHD gene following a standard procedure validated on penguins (Lee et al., 2010 (link)).

According to the results of the high-throughput sequencing, we performed regression analysis between the plant biomass and fungal or bacterial OTU abundances. The OTUs, whose abundances ranked in the top 10 and showed a positive relationship with the plant biomass, were selected as the target. To identify the closest taxonomy, the representative sequence of each targeted OTU was identified by sequence alignment at the National Center for Biotechnology Information (NCBI). Finally, one AM fungal OTU (OTU4, the second highest abundance and the maximum regression coefficient) and one bacterial OTU (OTU3, the first highest abundance and the maximum regression coefficient) were picked out, taxonomically belonging to Gipaspora and Sphingomonas, respectively.
To isolate the target AM fungus, trap culturing was first conducted to enrich spores for 5 months (Liu and Wang, 2003 (link)), and AM fungal spores were then collected with the wet-sieving and decanting method (Gerdemann and Nicolson, 1963 (link)). AM fungi were identified according to the spore morphology² in combination with a molecular technique. Briefly, a single spore was rinsed in sterile distilled water three times in a PCR tube and crushed with a pipette tip under sterile conditions. DNA was extracted with 30 μL of TE buffer solution containing 20% Chelex 100 (Sigma) at 100°C for 5 min. After centrifugation, the supernatant was stored at −20°C. Nest PCR was then performed with the two primer pairs AML1/AML2 and AMV4.5NF/AMDGR. These sequences were aligned with that of Gigaspora OTU4, and spores corresponding to the matching sequence were considered as the target AM fungal taxa for later culture.
Sphingomonas was isolated on R2A solid medium with the dilution plating method according to Vanbroekhoven et al. (2004) (link). Yellow-pigmented colonies were picked out and cultured in R2A liquid medium. Bacterial cells were then collected, and DNA was extracted with 100 μL of TE buffer solution containing 20% Chelex 100 (Sigma) at 100°C for 5 min. RCR reaction was performed with the universal 16S primer pair 27f/1492r (Luo et al., 2019 (link)), which covers the full length of products amplified with the primer pair 338F and 806R employed in high-throughput sequencing. The PCR products were sequenced, and the resultant sequences were aligned with that of Sphingomonas OTU3. The isolate corresponding to the matching sequence was considered to be the target bacterial taxa for later proliferation.