Water deficit and potassium affect carbon isotope composition in cassava bulk leaf material and extracted carbohydrates.

Cassava (Manihot esculenta Crantz) is an important root crop, which despite its drought tolerance suffers considerable yield losses under water deficit. One strategy to increase crop yields under water deficit is improving the crop's transpiration efficiency, which could be achieved by variety selection and potassium application. We assessed carbon isotope composition in bulk leaf material and extracted carbohydrates (soluble sugar, starch, and cellulose) of selected leaves one month after inducing water deficit to estimate transpiration efficiency and storage root biomass under varying conditions in a greenhouse experiment. A local and improved variety were grown in sand, supplied with nutrient solution with two potassium levels (1.44 vs. 0.04 mM K+) and were subjected to water deficit five months after planting. Potassium application and selection of the improved variety both increased transpiration efficiency of the roots with 58% and 85% respectively. Only in the improved variety were 13C ratios affected by potassium application (up to - 1.8‰ in δ13C of soluble sugar) and water deficit (up to + 0.6‰ in δ13C of starch and soluble sugar). These data revealed a shift in substrate away from transitory starch for cellulose synthesis in young leaves of the improved variety under potassium deficit. Bulk δ13C of leaves that had fully developed prior to water deficit were the best proxies for storage root biomass (r = - 0.62, r = - 0.70) and transpiration efficiency (r = - 0.68, r = - 0.58) for the local and improved variety respectively, making laborious extractions redundant. Results obtained from the youngest fully developed leaf, commonly used as a diagnostic leaf, were complicated by remobilized assimilates in the improved variety, making them less suitable for carbon isotope analysis. This study highlights the potential of carbon isotope composition to assess transpiration efficiency and yield, depending on the chosen sampling strategy as well as to unravel carbon allocation processes.

Plant invasion causes alterations in Darwin's finch feeding patterns in Galápagos cloud forests.

Invasive species pose a major threat to forest biodiversity, particularly on islands such as the Galapágos. Here, invasive plants are threatening the remnants of the unique cloud forest and its iconic Darwin's finches. We posit that food web disturbances caused by invasive Rubus niveus (blackberry), have contributed to the rapid decline of the insectivourous green warbler finch (Certhidae olivacea). We compared the birds' dietary changes in long-term management, short-term management and unmanaged areas. We measured C:N ratios, and δ15N­nitrogen and δ13C­carbon values in both consumer tissues (bird-blood) and food sources (arthropods), as indicators of resource use change, and collected mass abundance, and arthropod diversity data. We characterised the birds' diets using isotope mixing models. The results revealed that finches in (blackberry-invaded) unmanaged areas foraged more on abundant, yet lower quality, arthropods present in the invaded understory. This suggests that blackberry encroachment leads to a decrease in food source quality with physiological consequences for green warbler finch chicks. Results also implied that blackberry control has a short-term impact on food source quantity, which led to a decrease in chick recruitment that we observed in our previous studies; despite this, in the long-term, these managed systems show signs of recovery within three years of restoration.

13C labeling unravels carbon dynamics in banana between mother plant, sucker and corm under drought stress.

Banana is a perennial crop and typically consists of a mother plant and one or more suckers that will serve as the next generation. Suckers are photosynthetically active, but also receive photo-assimilates from the mother plant. While drought stress is the most important abiotic constraint to banana cultivation, its effect on suckers or banana mats as a whole remains unknown. To investigate whether parental support to suckers is altered under drought stress and to determine the photosynthetic cost to the parental plant, we conducted a 13C labeling experiment. We labeled banana mother plants with 13CO2 and traced the label up to two weeks after labeling. This was done under optimal and drought-stressed conditions in plants with and without suckers. We retrieved label in the phloem sap of the corm and sucker as soon as 24 hours after labeling. Overall, 3.1 ± 0.7% of label assimilated by the mother plant ended up in the sucker. Allocation to the sucker seemed to be reduced under drought stress. The absence of a sucker did not enhance the growth of the mother plant; instead, plants without suckers had higher respiratory losses. Furthermore, 5.8 ± 0.4% of the label was allocated to the corm. Sucker presence and drought stress each led to an increase in starch accumulation in the corm, but when both stress and a sucker were present, the amount was severely reduced. Furthermore, the second to fifth fully open leaves were the most important source of photo-assimilates in the plant, but the two younger developing leaves assimilated the same amount of carbon as the four active leaves combined. They exported and imported photo-assimilates simultaneously, hence acting as both source and sink. 13C labeling has allowed us to quantify source and sink strengths of different plant parts, as well as the carbon fluxes between them. We conclude that drought stress and sucker presence, respectively causing a reduction in supply and an increase in carbon demand, both increased the relative amount of carbon allocated to storage tissues. Their combination, however, led to insufficient availability of assimilates and hence a reduced investment in long-term storage and sucker growth.

Carbon allocation in cassava is affected by water deficit and potassium application - A 13 C-CO2 pulse labelling assessment.

RATIONALE: Cassava production faces challenges in a changing climate. Pulse labelling cassava with 13 C-CO2 has the potential to elucidate carbon allocation mechanisms of cassava under drought stress and with potassium application. Understanding these mechanisms could guide efforts to mitigate effects of drought in cassava cropping systems. METHODS: Forty-eight cassava plants received a nutrient solution high or low in potassium. Water deficit was imposed on half of the plants at bulk root initiation stage, after which they were labelled for 8 h with 13 C-CO2 in a 15 m3 growth chamber. Plants were harvested 8 h, 9 days and 24 days after labelling, and separated into leaves, stems and roots. δ13 C values of the different parts were measured using an isotope ratio mass spectrometer, from which 13 C excess was calculated. RESULTS: Water deficit decreased transpiration (P < 0.001) and increased carbon respiration (P < 0.05). Potassium application increased assimilate distribution to the roots (P < 0.05) at 9 days after labelling, more strongly for plants under water deficit. The opposite was found at 24 days (P < 0.05) with the legacy of water deficit additionally increasing assimilate distribution to roots (P < 0.05). Youngest, fully expanded leaves contained up to 47% of initial 13 C excess at 24 days after labelling. CONCLUSIONS: Pulse labelling proved to be successful in shedding light on carbon allocation in relation to water and potassium availability. This technique, once adapted to field conditions, could further be used to improve fertilizer recommendations or change agronomic practices to cope with plant stress.

Remediating Garden Soils: EDTA-Soil Washing and Safe Vegetable Production in Raised Bed Gardens.

Soil remediation is an important practice in the restoration of heavy metal-contaminated soils and reduce the heavy metal exposure of the local population. Here, we investigated the effect of an ex-situ soil washing technique, based on ethylenediaminetetraacetic acid (EDTA) as a chelating agent, on a contaminated Cambisol. Lead, Cd and Zn were investigated in different soil fractions, drainage water and four vegetables from August 2019 to March 2021. Three treatments consisting of (C) contaminated soil, (W) washed soil and (WA) washed soil amended with vermicompost and biochar were investigated in an outdoor raised bed set up. Our results showed that the total and bioavailable metal fractions were significantly reduced but failed to meet Austrian national guideline values. Initial concentrations in the soil leachate increased significantly, especially for Cd. Vegetables grown on the remediated soil took up significantly lower amounts of all heavy metals and were further reduced by the organic amendment, attaining acceptable values within EU guideline values for food safety. Only spinach exceeded the thresholds in all soil treatments. The increase in soil pH and nutrient availability led to significantly higher vegetable yields.

Tracking global invasion pathways of the spongy moth (Lepidoptera: Erebidae) to the United States using stable isotopes as endogenous biomarkers.

The spread of invasive insect species causes enormous ecological damage and economic losses worldwide. A reliable method that tracks back an invaded insect's origin would be of great use to entomologists, phytopathologists, and pest managers. The spongy moth (Lymantria dispar, Linnaeus 1758) is a persistent invasive pest in the Northeastern United States and periodically causes major defoliations in temperate forests. We analyzed field-captured (Europe, Asia, United States) and laboratory-reared L. dispar specimens for their natal isotopic hydrogen and nitrogen signatures imprinted in their biological tissues (δ2H and δ15N) and compared these values to the long-term mean δ2H of regional precipitation (Global Network of Isotopes in Precipitation) and δ15N of regional plants at the capture site. We established the percentage of hydrogen-deuterium exchange for L. dispar tissue (Pex = 8.2%) using the comparative equilibration method and two-source mixing models, which allowed the extraction of the moth's natal δ2H value. We confirmed that the natal δ2H and δ15N values of our specimens are related to the environmental signatures at their geographic origins. With our regression models, we were able to isolate potentially invasive individuals and give estimations of their geographic origin. To enable the application of these methods on eggs, we established an egg-to-adult fraction factor for L. dispar (Δegg-adult = 16.3 ± 4.3‰). Our models suggested that around 25% of the field-captured spongy moths worldwide were not native in the investigated capture sites. East Asia was the most frequently identified location of probable origin. Furthermore, our data suggested that eggs found on cargo ships in the United States harbors in Alaska, California, and Louisiana most probably originated from Asian L. dispar in East Russia. These findings show that stable isotope biomarkers give a unique insight into invasive insect species pathways, and thus, can be an effective tool to monitor the spread of insect pest epidemics.

Application of leaf analysis in addition to growth assessment to evaluate the suitability of greywater for irrigation of Tilia cordata and Acer pseudoplatanus.

Water is the key resource in fulfilling the cooling function of plants in urban environments and needs to be supplied reliably and adequately, especially during dry periods. To avoid an unsustainable use of high-quality drinking water for irrigation, the reuse of greywater should be implemented for Green Infrastructure irrigation in the sense of the circular economy. In this study, the influence of greywater irrigation on vitality of two trees species, Tilia cordata and Acer pseudoplatanus, was determined by investigating the effect of irrigation with raw or treated greywater in comparison to municipal tap water. Plant growth parameters were measured, including leaf area, number of leaves, average leaf area and annual growth. In addition, the relative chlorophyll content was determined and image analysis was used to identify vital and necrotic leaf parts. While treatment did not affect growth after one growing season A. pseudoplatanus had significantly higher leaf necrosis (34.8%) when irrigated with raw greywater compared to treated greywater (15.5%) and tap water (5.8%). Relative chlorophyll content of T. cordata irrigated with tap water decreased over time until it was significantly lower (28.5) then the greywater treatments (34.5 and 35). Image analysis of leaves to quantify necrosis proved to be a sensitive method to quantify plant health and showed negative effects earlier than an analysis of growth. Anionic surfactants and electrical conductivity had a significant influence on plant vitality. Therefore, plant selection should take these parameters into account, when planning green infrastructure irrigated with greywater.

Effect of Chelant-Based Soil Washing and Post-Treatment on Pb, Cd, and Zn Bioavailability and Plant Uptake.

The remediation of Pb, Cd, and Zn contaminated soil by ex situ EDTA washing was investigated in two pot experiments. We tested the influence of (i) 0, 0.5, 1.0, and 1.5%wt zero-valent iron (ZVI) and (ii) a combination of 5%wt vermicompost, 2%wt biochar, and 1%wt ZVI on the metal availability in EDTA-washed soil using different soil extracts (Aqua regia, NH4NO3) and plant concentrations. We found that EDTA soil washing significantly reduced the total concentration of Pb, Cd, and Zn and significantly reduced the Cd and Zn plant uptake. Residual EDTA was detected in water extracts causing the formation of highly available Pb-EDTA complexes. While organic amendments had no significant effect on Pb behavior in washed soils, an amendment of ≥ 1%wt ZVI successfully reduced EDTA concentrations, Pb bioavailability, and plant uptake. Our results suggest that Pb-EDTA complexes adsorb to a Fe oxyhydroxide layer, quickly developing on the ZVI surface. The increase in ZVI application strongly decreases Zn concentrations in plant tissue, whereas the uptake of Cd was not reduced, but even slightly increased. Soil washing did not affect plant productivity and organic amendments improved biomass production. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11270-021-05356-0.

New Ag3 PO4 comparison material for stable oxygen isotope analysis.

RATIONALE: A silver phosphate reference material (Ag3 PO4 ) for the measurement of stable oxygen isotope compositions is much needed; however, it is not available from the authorities distributing reference materials. This study aims to fill this gap by calibrating a new Ag3 PO4 stable isotope comparison material produced by the University of Natural Resources and Life Sciences (BOKU). METHODS: Aliquots of Ag3 PO4 were distributed to four laboratories who frequently measure the δ18 O value in Ag3 PO4 ; the University of Natural Resources and Life Sciences (BOKU), the University of Western Australia (UWA), the University of Helsinki (UH), and the Helmholtz Centre for Environmental Research (UFZ). The instruments used to perform the measurements were high-temperature conversion elemental analysers coupled with continuous flow isotope ratio mass spectrometers. The working gas δ18 O value was set to 0‰ and the normalization was done by a three-point linear regression using the reference materials IAEA-601, IAEA-602, and NBS127. RESULTS: The mean δ18 O value of the new BOKU Ag3 PO4 comparison material on the VSMOW-SLAP scale is 13.71‰ and the combined uncertainty is estimated as ±0.34‰. This estimated uncertainty is within the range typical for comparison materials of phosphates and sulphates. Consistent results from the different laboratories probably derived from similar instrumentation, and use of the same reference materials and normalization procedure. The matrix effect of the different reference materials used in this study was deemed negligible. CONCLUSIONS: The BOKU Ag3 PO4 can be used as an alternative comparison material for stable oxygen isotope analysis and is available for stable isotope research laboratories to facilitate calibration.

Successive and automated stable isotope analysis of CO2 , CH4 and N2 O paving the way for unmanned aerial vehicle-based sampling.

RATIONALE: Measurement of greenhouse gas (GHG) concentrations and isotopic compositions in the atmosphere is a valuable tool for predicting their sources and sinks, and ultimately how they affect Earth's climate. Easy access to unmanned aerial vehicles (UAVs) has opened up new opportunities for remote gas sampling and provides logistical and economic opportunities to improve GHG measurements. METHODS: This study presents synchronized gas chromatography/isotope ratio mass spectrometry (GC/IRMS) methods for the analysis of atmospheric gas samples (20-mL glass vessels) to determine the stable isotope ratios and concentrations of CO2 , CH4 and N2 O. To our knowledge there is no comprehensive GC/IRMS setup for successive measurement of CO2 , CH4 and N2 O analysis meshed with a UAV-based sampling system. The systems were built using off-the-shelf instruments augmented with minor modifications. RESULTS: The precision of working gas standards achieved for δ13 C and δ18 O values of CO2 was 0.2‰ and 0.3‰, respectively. The mid-term precision for δ13 C and δ15 N values of CH4 and N2 O working gas standards was 0.4‰ and 0.3‰, respectively. Injection quantities of working gas standards indicated a relative standard deviation of 1%, 5% and 5% for CO2 , CH4 and N2 O, respectively. Measurements of atmospheric air samples demonstrated a standard deviation of 0.3‰ and 0.4‰ for the δ13 C and δ18 O values, respectively, of CO2 , 0.5‰ for the δ13 C value of CH4 and 0.3‰ for the δ15 N value of N2 O. CONCLUSIONS: Results from internal calibration and field sample analysis, as well as comparisons with similar measurement techniques, suggest that the method is applicable for the stable isotope analysis of these three important GHGs. In contrast to previously reported findings, the presented method enables successive analysis of all three GHGs from a single ambient atmospheric gas sample.

Stable Isotope Probing of Microbial Phospholipid Fatty Acids in Environmental Samples.

Phospholipid fatty acid (PLFA) extracted from environmental samples describe the microbial community pattern and are sensitive to monitor and quantify shifts in the microbial community. Linkage with the stable isotope technique adds a functional perspective and is frequently used to quantify carbon turnover in microbial communities and detect physiological changes. Here we present a PLFA extraction method by using an organic solvent water mixture, followed by lipid separation based on solid-phase extraction and an alkaline methylation. Finally, we provide a protocol for the carbon stable isotope measurements of the extracted fatty acid methyl esters (FAMEs) by gas chromatograph-isotope ratio mass spectrometer (GC-IRMS) and calculation of concentration and δ13CVPDB values.

Biophysical potential of crop residues for biochar carbon sequestration, and co-benefits, in Uganda.

Increasing organic matter/carbon contents of soils is one option proposed to offset climate change inducing greenhouse gas (GHG) emissions, under the auspices of the UNFCC Paris Agreement. One of the complementary practices to sequester carbon in soils on decadal time scales is amending it with biochar, a carbon rich byproduct of biomass gasification. In sub-Saharan Africa (SSA), there is a widespread and close interplay of agrarian-based economies and the use of biomass for fuel, which makes the co-benefits of biochar production for agriculture and energy supply explicitly different from the rest of the world. To date, the quantities of residues available from staple crops for biochar production, and their potential for carbon sequestration in farming systems of SSA have not been comprehensively investigated. We assessed the productivity and usage of biomass waste from maize, sorghum, rice, millet, and groundnut crops; specifically quantifying straw, shanks, chaff, and shells, based on measurements from multiple farmer fields and household surveys in eastern Uganda. Moreover, allometric models were tested, using grain productivity, plant height, and density as predictors. These models enable rapid and low-cost assessment of the potential availability of feedstocks at various spatial scales: individual cropland, farm enterprise, region, and country. Ultimately, we modeled the carbon balance in soils of major cropping systems when amended with biochar from biomass residues, and up-scaled this for basic scenario analysis. This interdisciplinary approach showcases that there is significant biophysical potential for soil carbon sequestration in farming systems of Uganda through amendment of biochar derived from unused residues of cereals and legume crops. Furthermore, information about these biomass waste flows is used for estimating the rates of biochar input that could be made to farmlands, as well as the amounts of energy that could be produced with gasifier appliances.

Production, characterization and adsorption studies of bamboo-based biochar/montmorillonite composite for nitrate removal.

Biochar is a promising immobilization tool for various contaminants in liquid wastes, aqueous solutions and soils. To further improve the sorption characteristics, a biochar/montmorillonite composite was produced and synthesized in an experimental pyrolysis reactor, using bamboo as biomass feedstock. The composite was characterized by physico-chemical and structural methods (FTIR, SEM, SEM/EDX, SSA, Low temperature nitrogen adsorption method). Based on these methods, the successful preparation of a bamboo based biochar/montmorillonite composite preparation has been demonstrated. The particles of montmorillonite were distributed across the biochar surface. The adsorption studies for removal nitrates from aqueous solutions were investigated by a batch method at laboratory temperatures. The experimental data were fitted by three adsorption models (Langmuir, Freundlich and DR; R2 > 0.93). The maximum adsorption capacity achieved by biochar at pH 4, was about 5 mg g-1 and by biochar/montmorillonite composite 9 mg g-1. The results suggest that the bamboo-based biochar/montmorillonite composite can be used effectively in the treatment of industrial effluents or waste water containing anionic pollutants such as nitrates.

Reverse osmosis and ultrafiltration for recovery and reuse of larval rearing water in Anopheles arabiensis mass production: Effect of water quality on larval development and fitness of emerging adults.

BACKGROUND: Countries around the world are showing increased interest in applying the sterile insect technique against mosquito disease vectors. Many countries in which mosquitoes are endemic, and so where vector control using the sterile insect technique may be considered, are located in arid zones where water provision can be costly or unreliable. Water reuse provides an alternate form of water supply. In order to reduce the cost of mass rearing of Anopheles arabiensis mosquitoes, the possibility of recycling and reusing larval rearing water was explored. METHODS: The used rearing water ('dirty water') was collected after the tilting of rearing trays for collection of larvae/pupae, and larvae/pupae separation events and underwent treatment processes consisting of ultrafiltration and reverse osmosis. First-instar An. arabiensis larvae were randomly assigned to different water-type treatments, 500 larvae per laboratory rearing tray: 'clean' dechlorinated water, routinely used in rearing; dirty water; and 'recycled' dirty water treated using reverse osmosis and ultrafiltration. Several parameters of insect quality were then compared: larval development, pupation rate, adult emergence, body size and longevity. Water quality of the samples was analyzed in terms of ammonia, nitrite, nitrate, sulphate, dissolved oxygen, chloride, and phosphate concentrations after the larvae had all pupated or died. Surface water temperatures were also recorded continuously during larval development. RESULTS: Pupation rates and adult emergence were similar in all water treatments. Adult body sizes of larvae reared in recycled water were similar to those reared in clean water, but larger than those reared in the dirty larval water treatment, whereas the adult longevity of larvae reared in recycled water was significantly increased relative to both 'clean' and 'dirty' water. Dirty larval water contained significantly higher concentrations of ammonium, sulfate, phosphate and chloride and lower levels of dissolved oxygen than clean water. These parameters significantly varied during the period of larval development. After dirty water was recycled by ultrafiltration and reverse osmosis, all the parameters measured were the same as those in clean water. CONCLUSION: This study demonstrated the potential for using recycled larval rearing water to supplement clean dechlorinated water supplies. Recycling used water improved its quality and of the reared mosquitoes. As water demands and environmental pressures grow, recycling of larval rearing water will improve the sustainability and affordability of mosquito mass-rearing.

Darwin's finches treat their feathers with a natural repellent.

Darwin's finches are highly innovative. Recently we recorded for the first time a behavioural innovation in Darwin's finches outside the foraging context: individuals of four species rubbed leaves of the endemic tree Psidium galapageium on their feathers. We hypothesised that this behaviour serves to repel ectoparasites and tested the repellency of P. galapageium leaf extracts against parasites that negatively affect the fitness of Darwin's finches, namely mosquitoes and the invasive hematophagous fly Philornis downsi. Mosquitoes transmit pathogens which have recently been introduced by humans and the larvae of the fly suck blood from nestlings and incubating females. Our experimental evidence demonstrates that P. galapageium leaf extracts repel both mosquitoes and adult P. downsi and also inhibit the growth of P. downsi larvae. It is therefore possible that finches use this plant to repel ectopoarasites.

Biochar decelerates soil organic nitrogen cycling but stimulates soil nitrification in a temperate arable field trial.

Biochar production and subsequent soil incorporation could provide carbon farming solutions to global climate change and escalating food demand. There is evidence that biochar amendment causes fundamental changes in soil nutrient cycles, often resulting in marked increases in crop production, particularly in acidic and in infertile soils with low soil organic matter contents, although comparable outcomes in temperate soils are variable. We offer insight into the mechanisms underlying these findings by focusing attention on the soil nitrogen (N) cycle, specifically on hitherto unmeasured processes of organic N cycling in arable soils. We here investigated the impacts of biochar addition on soil organic and inorganic N pools and on gross transformation rates of both pools in a biochar field trial on arable land (Chernozem) in Traismauer, Lower Austria. We found that biochar increased total soil organic carbon but decreased the extractable organic C pool and soil nitrate. While gross rates of organic N transformation processes were reduced by 50-80%, gross N mineralization of organic N was not affected. In contrast, biochar promoted soil ammonia-oxidizer populations (bacterial and archaeal nitrifiers) and accelerated gross nitrification rates more than two-fold. Our findings indicate a de-coupling of the soil organic and inorganic N cycles, with a build-up of organic N, and deceleration of inorganic N release from this pool. The results therefore suggest that addition of inorganic fertilizer-N in combination with biochar could compensate for the reduction in organic N mineralization, with plants and microbes drawing on fertilizer-N for growth, in turn fuelling the belowground build-up of organic N. We conclude that combined addition of biochar with fertilizer-N may increase soil organic N in turn enhancing soil carbon sequestration and thereby could play a fundamental role in future soil management strategies.

An analysis of diet quality, how it controls fatty acid profiles, isotope signatures and stoichiometry in the malaria mosquito Anopheles arabiensis.

BACKGROUND: Knowing the underlying mechanisms of mosquito ecology will ensure effective vector management and contribute to the overall goal of malaria control. Mosquito populations show a high degree of population plasticity in response to environmental variability. However, the principle factors controlling population size and fecundity are for the most part unknown. Larval habitat and diet play a crucial role in subsequent mosquito fitness. Developing the most competitive insects for sterile insect technique programmes requires a "production" orientated perspective, to deduce the most effective larval diet formulation; the information gained from this process offers us some insight into the mechanisms and processes taking place in natural native mosquito habitats. METHODOLOGY/PRINCIPAL FINDINGS: Fatty acid profiles and de-novo or direct assimilation pathways, of whole-individual mosquitoes reared on a range of larval diets were determined using pyrolysis gas chromatograph/mass spectrometry. We used elemental analysis and isotope ratio mass spectrometry to measure individual-whole-body carbon, nitrogen and phosphorous values and to assess the impact of dietary quality on subsequent population stoichiometry, size, quality and isotopic signature. Diet had the greatest impact on fatty acid (FA) profiles of the mosquitoes, which exhibited a high degree of dietary routing, characteristic of generalist feeders. De-novo synthesis of a number of important FAs was observed. Mosquito C:N stoichiometry was fixed in the teneral stage. Dietary N content had significant influence on mosquito size, and P was shown to be a flexible pool which limited overall population size. CONCLUSIONS/SIGNIFICANCE: Direct routing of FAs was evident but there was ubiquitous de-novo synthesis suggesting mosquito larvae are competent generalist feeders capable of survival on diet with varying characteristics. It was concluded that nitrogen availability in the larval diet controlled teneral mosquito size and that teneral CN ratio is a sex- and species-specific fixed parameter. This finding has significant implications for overall mosquito competitiveness and environmental management.

Evaluation of a stable isotope method to mark naturally-breeding larval mosquitoes for adult dispersal studies.

Understanding mosquito dispersal is critically important for vector-borne disease control and prevention. Mark-release-recapture methods using various marking techniques have made substantial contributions to the study of mosquito biology. However, the ability to mark naturally breeding mosquitoes noninvasively and with life-long retention has remained problematic. Here, we describe a method to mark naturally breeding mosquitoes with stable isotopes. Culex pipiens f. molestus mosquitoes were provisioned as larvae in laboratory experiments with 15N-labeled potassium nitrate and 13C-labeled glucose. Larval enrichment was sufficient to differentiate marked adult mosquitoes from unmarked control mosquitoes and the natural source population from Chicago Illinois, using either delta 15N or delta 13C. Isotopic retention lasted for at least 55 d for adult male and females mosquitoes. There were no consistent effects of isotopic enrichment on immature mosquito survival or adult mosquito body size. We then applied this marking technique to naturally breeding Culex pipiens mosquitoes in suburban Chicago, IL, and for the first time, report successful isotopic enrichment of mosquitoes in the field. This stable isotope marking technique will facilitate studies of mosquito dispersal.

Intrinsic and synthetic stable isotope marking of tsetse flies.

The sterile insect technique has been successfully used to eliminate tsetse populations in a number of programs. Program monitoring in the field relies on the ability to accurately differentiate released sterile insects from wild insects so that estimates can be made of the ratio of sterile males to wild males. Typically, released flies are marked with a dye, which is not always reliable. The difference in isotopic signatures between wild and factory-reared populations could be a reliable and intrinsic secondary marker to complement existing marking methods. Isotopic signatures are natural differences in stable isotope composition of organisms due to discrimination against the heavier isotopes during some biological processes. As the isotopic signature of an organism is mainly dependent on what it eats; by feeding factory-reared flies isotopically different diets to those of the wild population it is possible to intrinsically mark the flies. To test this approach unlabeled samples of Glossina pallidipes (Austen) (Diptera: Glossinidae) from a mass rearing facility and wild populations were analyzed to determine whether there were any natural differences in signatures that could be used as markers. In addition experiments were conducted in which the blood diet was supplemented with isotopically enriched compounds and the persistence of the marker in the offspring determined. There were distinct natural isotopic differences between factory reared and wild tsetse populations that could be reliably used as population markers. It was also possible to rear artificially isotopically labeled flies using simple technology and these flies were clearly distinguishable from wild populations with greater than 95% certainty after 85 days of "release". These techniques could be readily adopted for use in SIT programs as complimentary marking techniques.

Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon fly Bactrocera cucurbitae: effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast.

The application of methoprene, and providing access to diet including hydrolyzed yeast, are treatments known to enhance mating success in the male melon fly Bactrocera cucurbitae Coquillett (Diptera: Tephritidae), supporting their use in mass rearing protocols for sterile males in the context of sterile insect technique (SIT) programmes. The objective of the present laboratory study was to investigate the effect of methoprene application and diet supplementation with hydrolyzed yeast (protein) on the turnover of body lipids and protein to confirm the feasibility of their application in melon fly SIT mass-rearing programmes. While females had access to a diet that included hydrolyzed yeast (protein), males were exposed to one of the following treatments: (1) topical application of methoprene and access to diet including protein (M+P+); (2) only diet including protein (M-P+); (3) only methoprene (M+P-) and (4) untreated, only sugar-fed, control males (M-P-). Total body carbon (TBC) and total body nitrogen (TBN) of flies were measured at regular intervals from emergence to 35 days of age for each of the different treatments. Nitrogen assimilation and turnover in the flies were measured using stable isotope ((15)N) dilution techniques. Hydrolyzed yeast incorporation into the diet significantly increased male body weight, TBC and TBN as compared to sugar-fed males. Females had significantly higher body weight, TBC and TBN as compared to all males. TBC and TBN showed age-dependent changes, increasing until the age of sexual maturity and decreasing afterwards in both sexes. Methoprene treatment did not significantly affect TBC or TBN. The progressive increase with age of TBC suggests that lipogenesis occurs in adult male B. cucurbitae, as is the case in other tephritids. Stable isotope dilution was shown to be an effective method for determining N uptake in B. cucurbitae. This technique was used to show that sugar-fed males rely solely on larval N reserves and that the N uptake rate in males with access to diet including hydrolyzed yeast was higher shortly after emergence and then stabilized. The implications of the results for SIT applications are discussed.