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    Root exudate chemical cues of an invasive plant modulate oviposition behavior and survivorship of a malaria mosquito vector

    Experiments using P. hysterophorus root exudate water samplesInsects for oviposition bioassayAnopheles gambiae mosquitoes (Mbita strain) used in this study were from a colony established in 2018 and maintained at the International Centre of Insect Physiology and Ecology (icipe), Duduville Campus, Nairobi, Kenya. The adults were reared using standard insectary conditions at 28 ± 2 °C, and 72% relative humidity (RH) under a photoperiod of 12 h: 12 h (L:D). For egg development, the mosquitoes were offered human blood through arm feeding, one to two times weekly and had ad libitum access to a 6% glucose solution (wt:vol). The blood fed females were kept together with males for 2 days and used on the third day for the oviposition experiment. Eggs were laid in oviposition cups (7 cm top diameter, 4 cm base diameter and 7 cm depth) lined with filter paper (Whatman 90 mm, GE Healthcare UK Ltd, Buckinghamshire, London, UK) and transferred to larval rearing trays (39 × 28 × 4 cm depth). Upon hatching, the larvae were separated into groups of ~ 500 larvae per rearing tray and fed on commercial Tetramin® fish food (Tetra, Germany). The rearing room was maintained at 32 ± 2 °C, and 52% RH during the day, and 24 ± 2 °C and 72% RH at night with a photoperiod of L:D 12:12 h15. The rearing water was changed after every 2 days. Pupae were transferred daily to emergence cups containing 15 mL water and placed in a new cage (15 × 15 × 15 cm). Emerged adults (1 day old) were maintained on 6% glucose solution as described above.Collection of root exudate water samples from wild growing P. hysterophorus
    Root exudate water samples were collected from wild growing P. hysterophorus (~ 30, 60 and 90 cm tall) on the icipe campus and used in various biological assays (i.e., oviposition response, mosquito growth and development assays). Briefly, wild growing P. hysterophorus were transplanted into a plastic pot (23 cm top diameter, 12 cm base diameter and 22 cm depth; batches of ten plants per pot in garden soil from the icipe campus) and watered with rainwater to obtain root exudate water. The root exudate water was collected from the plants after 1 week: to allow the plants to stabilize and to eliminate possible contamination of it with defense compounds released in response to uprooting. Water obtained from potted soil (same volume as used for the potted P. hysterophorus) without P. hysterophorus plants but from the same site, served as control water.Oviposition response assay with root exudate waterRoot exudate water was evaluated for its ability to influence oviposition response of gravid females in a dual-choice assay (Fig. 1A). The gravid females (n = 12 as described in Dieter et al.18; electronic supplementary material, methods) were presented a choice between the treatment and distilled water contained in similar polypropylene cups and the number of eggs laid counted using a microscope (Leica M127, Switzerland) after every 24 h for four consecutive days7. The cups were placed in opposite corners of the experimental cages and their positions interchanged every 24 h to avoid positional bias. Each cup was lined with a filter paper (Whatman 90 mm, GE Healthcare UK Ltd, Buckinghamshire, London, UK) and filled with 30 mL of the test solution (to keep the filter paper moist; as previously described in19) or an equivalent volume of distilled water as the control. A similar experiment was performed using water obtained from soil without P. hysterophorus plants (Fig. 1A). The bioassays were performed in triplicate as previously described in Ilahi et al.20 and repeated once.Figure 1Effect of root exudate water on oviposition response and aquatic stage development of An. gambiae. (A) A schematic representation showing the setup of the oviposition experiment using root exudate water and soil water. (B) Oviposition activity of root exudate water and soil water. (C) Table summarizing median number of eggs laid and the range. (D) Egg hatch rates was low in root exudate water compared to soil water. (E) Time to pupation (days) for larvae exposed to root exudate water relative to soil water.Full size imageHeadspace collection and analysis of root exudate volatilesTo collect headspace volatiles, 500 mL of the root exudate water were placed in air-tight glass jars and activated charcoal-filtered and humidified air passed over it. The volatiles were collected for 24 h on three pre-cleaned (dichloromethane (DCM) and nitrogen-dried) Super Q adsorbent filters (30 mg each, Analytical Research System, Gainesville, Florida, USA) at a flow rate of 170 mL/min. The three Super-Q filters (each treatment), were each eluted with 200 µL GC-grade DCM (Sigma Aldrich, St. Louis, Missouri, USA) into 2 mL clear glass vials, each containing 250 µL conical point glass inserts (Supelco, Bellefonte, PA, USA) and immediately analyzed by coupled gas chromatography/mass spectrometry (GC/MS). Also, natural water from depressions harboring growing P. hysterophorus in the field was sampled during the rainy season (described under parthenin detection) to allow comparison of its volatiles with that of the root exudate water. Volatiles collected from distilled water served as negative controls. Compounds were identified by comparison of their mass spectral data with library data: Adams2, Chemeco and NIST11 and confirmed with those of authentic samples. The relative peak area (%) of each constituent as generated by the NIST11 software, following GC/MS analysis was used to determine the natural ratio of the volatiles in the component blend. The volatiles were stored in glass vials at − 80 °C until used for oviposition assays.Oviposition bioassay with identified volatilesDual choice bioassay was performed to determine the role of root exudate volatiles in egg laying behavior of An. gambiae. Of the volatiles identified, seven compounds: α-pinene, β-pinene, 3-carene, (E)-caryophyllene, camphor, α-phellandrene, β-phellandrene were selected and tested based on results obtained from Random Forest Analysis (RFA) (see electronic supplementary material, methods) and their commercial availability21. In dose–response assays, several tests were carried out: (i) a blend of the seven components (7-component blend) mimicking their naturally-occurring ratio in the root exudate volatile extract based on their GC/MS peak areas, (ii) single compounds, and (iii) a blend of the attractive components (5-component blend) guided by the positive attractive responses obtained from (ii). The compounds were dissolved in dimethyl sulfoxide (DMSO) and serially diluted from a stock solution to generate a concentration ranging from 0.25 to 4 µg/µL. Thereafter, for each concentration tested, 50 µL of each compound or blend solution were dispensed into the oviposition cup containing distilled (50 mL) water and monitored for egg laying. Each concentration was tested against a control (distilled water (50 mL) containing 50 µL of DMSO). The bioassays were performed in triplicate.Mosquito growth and development assaysThe root exudate water and the control water (water from soil) were used separately for mosquito rearing. First, mosquitoes were provided with root exudate water to lay eggs in a no-choice oviposition bioassay. Thereafter, a total of 200 eggs were counted and placed into each rearing tray (24 × 34 × 4 cm) and the number of hatched eggs was determined by counting the first instars larvae that emerged in each tray. The larvae were fed daily on a standardized regimen of ground fish food (Tetramin, Tetra, Germany) and water was changed every other day. Daily survival of larvae (from first instar (L1) to pupation) was recorded, and pupae transferred into cups in experimental cages (30 × 30 × 30 cm) until adult emergence. The conditions of the bioassay room were the same as that of the rearing room described above. All experiments were performed in four replicates.Experiments using partheninDetection of parthenin in root exudate waterOne liter (1 L) of water was collected from flooded depressions/open puddles in which wild P. hysterophorus was growing on the icipe campus and pooled (the field collection was repeated twice, 1 week apart). The habitat had other non P. hysterophorus plants such as grasses. The water was filtered using a muslin cloth and stored at − 80 °C overnight and then freeze-dried (VirTis SP scientific, Model Advantage EL-85) for 72 h to obtain 38 mg of root exudate. This was extracted with dichloromethane (DCM) and analyzed by GC/MS as described under chemical analysis. A similar analysis was carried out on the root exudate water (four replicates) obtained from potted plants described above.Preparation of parthenin stock solutionA sample of parthenin obtained from a methanolic extract of P. hysterophorus from a previous study conducted in our laboratory22, was used as a standard. A sample of this isolate (300 mg) was dissolved in 0.3 mL DMSO and then diluted to 300 mL with distilled water to obtain a stock solution of 1000 ppm.Oviposition response assay with partheninParthenin was evaluated at a concentration of 0.13 µg/µL corresponding to the estimated amount of the root exudate tested (described above). The amount of parthenin in the root exudate was estimated by comparing the relative peak area (%) of parthenin in the root exudate to that recorded for the standard parthenin of known concentration. The oviposition assays were performed as described for root exudate. However, the distilled water which served as a negative control was prepared in 0.1% DMSO. A similar experiment was performed using parthenin water spiked with 50 µL of headspace volatiles collected for 24 h from the plant root exudate. All experiments were performed in triplicates.Mosquito growth and development assays using partheninParthenin prepared as described for the oviposition bioassay was used for mosquito rearing. Mosquitoes reared on distilled water prepared in 0.1% DMSO served as the control group. The experiment was performed as described for root exudate water.To determine the effect of exposure to parthenin on mosquito adult survival, the female mosquitoes that emerged from parthenin treated water and controls were monitored separately in different cages. The mosquitoes were maintained on P. hysterophorus potted plant until their natural death (survival). The conditions in the bioassay rooms were the same as those of the rearing room described above.Chemical analysisGas chromatography/mass spectrometry (GC/MS)To detect parthenin in the root exudate, the sample was freeze-dried and prepared at a concentration of 600 ng/µL in dichloromethane (DCM) and dried over anhydrous Na2SO4 (Sigma Aldrich, St Louis, MO USA). The standard parthenin sample was prepared at a concentration of 300 ng/µL in DCM. For GC/MS analysis, 1 µL of each sample (parthenin and root exudate) was analyzed on a 7890B gas chromatograph (Agilent Technologies, Inc., Santa Clara, CA, USA) linked to a 5977A mass selective detector under the following conditions: Inlet temperature 270 °C, transfer line temperature 280 °C, and column oven temperature programmed from 35 to 285 °C, with the initial temperature maintained for 5 min then 10 °C/min to 280 °C for 5.5 min and finally 5 °C/min to 285 °C for 34.9 min. The GC was fitted with a HP-5 MS low bleed capillary column (30 m × 0.25 mm i.d., 0.25 µm) (J&W, Folsom, CA USA). Helium at a flow rate of 1.2 mL/min served as the carrier gas. The mass selective detector was maintained at ion source temperature of 230 °C and a quadrupole temperature of 180 °C. Electron impact (EI) mass spectra were obtained at the acceleration energy of 70 eV. Compounds were injected in the splitless mode using an auto-sampler 7693 (Agilent Technologies, Inc., Beijing, China). Fragment ions were analyzed over 38–550 m/z mass range in the full scan mode. The filament delay time was set at 3.0 min. Parthenin was identified based on its general fragmentation pattern and compared also with previously published results23. The root exudate samples were analyzed in triplicate, with each replicate collected from a different batch of plants.Statistical analysisThe oviposition activity index (OAI) for dual choice oviposition assay data was calculated according to the formula described in19;$$OAI=frac{mathrm{Nt}-mathrm{Nc}}{mathrm{Nt}+mathrm{Nc}}$$
    where Nt is number of eggs laid in the treatment and Nc the number of eggs laid in the control. The OAI ranges from − 1 to + 1, with 0 indicating neutral response, positive value indicating an attraction towards the treatment and a negative value indicating the converse. The oviposition data was analyzed by generalized linear model using negative binomial. The model validity was assessed by inspection of residuals24: Number of eggs deposited served as the response variable while the treatments were used as the predictor variable.Hatch rate of mosquito eggs exposed to different treatments was calculated as follows:$$% hatchability=left(frac{number ; of ; hatched ; eggs}{total ;number ; of ; eggs}right) times 100$$The % hatchability data was also analyzed using generalized linear model with negative binomial error structure. The variation in survival was analyzed by the Kaplan-Meir method and statistical significance comparisons made using log-ranks test. All statistical analyses were performed using SPSS 23.0 software (IBM SPSS Statistics) and results considered significance at p  More

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    Temperature heterogeneity correlates with intraspecific variation in physiological flexibility in a small endotherm

    Field data and analysisField samplingWe captured adult juncos by mist net or potter trap at sites in Arizona (breeding season), Colorado (breeding), Illinois (non-breeding), Montana (breeding), New Mexico (non-breeding), New York (breeding), South Dakota (non-breeding), and Wyoming (breeding), spanning 16° in latitude and 37° in longitude (Fig. 1). This work was completed with approval from the U.S. Fish and Wildlife Service (MB84376B-1 to M.S.; MB01543B-0 to Z.A.C.; MB758442 to D.L.S.; MB06336A-4 to M.D.C.; MB757670-1 to David Winkler; and MB094297-0 to Christopher Witt,), the State of Arizona Game and Fish Department (SP590760 and SP707897 to D.L.S.), Colorado Parks and Wildlife (10TRb2030A15 to M.D.C.), the Illinois Department of Natural Resources (NH13.5667 to Z.A.C.), the Montana Department of Fish Wildlife and Parks (2016-013 and 2017-067-W to M.S.), the New Mexico Department of Game & Fish (3217 to Christopher Witt), the New York State Division of Fish, Wildlife, & Marine Resources (LCP 1477 to David Winkler), the State of South Dakota Department of Game, Fish, and Parks (06-03, 07-02, 08-03 to D.L.S.), Wyoming Game and Fish (754 to M.D.C.), and the Institutional Animal Care and Use Committees at Cornell University (2001-0051 to David Winkler), the University of Illinois (13385 to Z.A.C.), the University of Montana (010-16ZCDBS-020916 to Z.A.C.), the University of South Dakota (03-08-06-08B to D.L.S), and the University of Wyoming (A-3216-01 to M.D.C.). We classified individuals to taxonomic unit based on plumage (J. h. caniceps, J. h. hyemalis, J. h. mearnsi, J. h. oreganus group, and J. p. palliatus). The J. h. oreganus group encompasses seven subspecies (J. h. montanus, J. h. oreganus, J. h. pinosus, J. h. pontilis, J. h. shufeldti, J. h. thurberi, and J. h. townsendii) with similar plumages and overlapping nonbreeding ranges26,27. For this reason, we were unable to distinguish subspecies of nonbreeding individuals within this group, though all breeding individuals were collected from the J. h. montanus range.Field metabolic assaysBirds were transported from the site of capture to a nearby laboratory ( More

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    Andes foothills protected by carbon-offset fund

    CORRESPONDENCE
    20 July 2021

    Andes foothills protected by carbon-offset fund

    Evert Thomas

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    Marleni Ramirez

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    Lily Rodriguez

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    Manuel Glave

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    Evert Thomas

    CIMA Cordillera Azul, Lima, Peru

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    Marleni Ramirez

    CIMA Cordillera Azul, Lima, Peru

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    Lily Rodriguez

    CIMA Cordillera Azul, Lima, Peru

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    Manuel Glave

    CIMA Cordillera Azul, Lima, Peru

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    At its creation in 2008, the United Nations REDD (‘reducing emissions from degradation and deforestation’) programme was hailed as a way to finance conservation with tools such as carbon offsets. Thirteen years on, little of that promise has been realized. A REDD transaction signed in March for the Cordillera Azul National Park, in the foothills of the Andes in Peru, offers hope.The deal will support the conservation in perpetuity of the 13,500-square-kilometre park and its rich and pristine biodiversity. A trust fund to cover all expenses is being set up by the non-governmental organization CIMA Cordillera Azul, which manages the park, and the Peruvian Service for Natural Protected Areas.Investments in sustainable livelihoods will strengthen efforts to curb and reverse deforestation in the 23,000‑km2 buffer zone around the park — home to more than 300,000 people. Notably, they will boost development of sustainable products from forest restoration and agroforestry. For example, CIMA has built a cacao-processing plant to promote cacao agroforestry as an alternative to land use that relies on deforestation.To our knowledge, this is the first REDD transaction to ensure that all conservation costs for a national park of this size are financed by private-sector carbon-credit sales, with minimal transaction costs. Similar deals around the globe could help to catalyse the carbon market.

    Nature 595, 494 (2021)
    doi: https://doi.org/10.1038/d41586-021-01958-0

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    The authors declare no competing interests.

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    Seasonal niche differentiation among closely related marine bacteria

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    Effects of nitrogen fertilization on protein and carbohydrate fractions of Marandu palisadegrass

    In our studies with Marandu palisadegrass, a grazing management strategy with continuous stocking where 95% of the light is intercepted by the canopy resulted in forage at a height of 25 cm, a high green leaf proportion, and low amounts of dead material during the growing season23,24. The use of N fertilization4, different stocking rates, and supplementation25,26,27 are crucial for obtaining forage with a high nutritional value, resulting in a high weight gain per animal and area, and a reduction in slaughter age and greenhouse gas emissions. In the present study we did not find any interection N doses with years. Therefore, only the significative effects of N fertilization or variation within year are presented and discussed.Total and non-fibrous carbohydrates and total digestible nutrientsTotal carbohydrate concentrations decreased linearly with increasing N levels (P  More

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    A monkey researcher fights to protect threatened and endangered primates

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    I was a ten-year-old in Singapore when I received a vervet monkey (Chlorocebus pygerythrus) as a pet. By the time I was 15, I knew that my family did not have the necessary international permit to own it legally. With help from local agencies, we sent the monkey to a rehabilitation sanctuary in Zambia, which ultimately released it into the wild. That experience got me interested in learning more about wild monkeys and how to help them.I research threatened and endangered leaf-eating primates known as Asian colobinae. They have specialized, multi-chambered stomachs, as do cows, and need a long rest after meals. They are shy and hard to find, so there has been less research on them than on orangutans or the great apes.One species I study is the critically endangered Raffles’ banded langur (Presbytis femoralis). Globally, there are just 320 individuals: 70 in Singapore and 250 in Malaysia. I work with national agencies, educational and non-governmental organizations and local communities to help protect these and other monkeys — especially those living between forest and urban areas. For example, in an area prone to roadkill, we installed a rope bridge to let langurs and other animals cross the road safely.One of the biggest threats to these and other monkeys is inbreeding as their numbers shrink. We hope to exchange animals between Singapore and Malaysia to boost their population’s genetic health.In this picture from 2017, I was monitoring primate populations in a reserve in central Singapore when I saw these long-tailed macaques (Macaca fascicularis). Here, we are observing one another — and respecting each other’s space.I’ve started a website called Primate Watching to help observers learn about these primates and where to see them. People think monkeys are aggressive, but really they are just naturally curious. Still, the public should always keep a safe distance, not put a camera in their faces.

    Nature 595, 618 (2021)
    doi: https://doi.org/10.1038/d41586-021-01995-9

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