Rebecca Hood-Nowotny

Stable isotope methods in biological and ecological studies of arthropods

This is an eclectic review and analysis of contemporary and promising stable isotope methodologies to study the biology and ecology of arthropods. It is augmented with literature from other disciplines, indicative of the potential for knowledge transfer. It is demonstrated that stable isotopes can be used to understand fundamental processes in the biology and ecology of arthropods, which range from nutrition and resource allocation to dispersal, food‐web structure, predation, etc. It is concluded that falling costs and reduced complexity of isotope analysis, besides the emergence of new analytical methods, are likely to improve access to isotope technology for arthropod studies still further. Stable isotopes pose no environmental threat and do not change the chemistry or biology of the target organism or system. These therefore represent ideal tracers for field and ecophysiological studies, thereby avoiding reductionist experimentation and encouraging more holistic approaches. Considering (i) the ease with which insects and other arthropods can be marked, (ii) minimal impact of the label on their behaviour, physiology, and ecology, and (iii) environmental safety, we advocate more widespread application of stable isotope technology in arthropod studies and present a variety of potential uses.

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.

Methylparaben in Anopheles gambiae s.l. sugar meals increases longevity and malaria oocyst abundance but is not a preferred diet

The antimicrobial and antifungal chemical methylparaben (methyl-4-hydroxybenzoate) was added to the adult sucrose diet of Anopheles gambiae and Anopheles arabiensis, and its effect on longevity was determined. In all cases, significant increases in longevity were observed when 0.2% (w/v) methylparaben was added to meals that were refreshed weekly. When fresh sugar diet was refreshed daily, no increase in longevity was observed due to methylparaben suggesting that the effect of methylparaben is to preserve the quality of the sugar diet. No longevity effect of providing pure water in addition to sugar- or methylparaben-supplemented meals was observed. Feeding preference tests were performed to determine whether meals containing methylparaben were preferred, and whether, when given no choice but the less-preferred diet, mosquitoes would consume less sugar. Using the stable carbon isotope (13)C in paired tests, we show that the sugar diet containing methylparaben was clearly avoided by A. gambiae but not A. arabiensis. Little effect of methylparaben on the total amount of sugar consumed was observed when mosquitoes were given no diet choice. Methylparaben effects on Plasmodium cynomolgi B oocyst formation and encapsulation were observed in a normal A. gambiae stock and one which encapsulates at a high frequency. Nearly two-fold increases in the number of both normal and encapsulated oocysts were observed as a result of methylparaben in the diet. Because of its longevity effects, we have implemented methylparaben use for all mosquitoes in our holdings and recommend it as a routine sugar meal supplement.

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

ABSTRACT 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 &dgr;15N or &dgr;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.

Routine Isotope Marking for the Mediterranean Fruit Fly (Diptera: Tephritidae)

ABSTRACT A simple method of marking Mediterranean fruit fly Ceratitis capitata (Wiedemann) using stable isotopes is described. This species is economically important and is a target species of many successful area-wide integrated pest management (AW-IPM) programs using the sterile insect technique (SIT). Program monitoring in the field relies on being able 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 fluorescent dust, 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 caused by discrimination against the heavier isotopes during some biological processes. 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. The majority of fruit fly species feed on C3 plants in the wild, which have a carbon isotope signature of around -28‰. However, almost all mass-rearing facilities use cane sugar in the larval and adult diet, which is a C4 sugar source (with a signal of around -11‰), and this could provide an easy signature to differentiate released flies from wild flies. To test this approach, samples of flies from several mass-rearing facilities and wild populations were analyzed. It was clearly shown that using C4 sugar in the larval-rearing diet was an effective and economic way of intrinsically labeling Mediterranean fruit flies, and it was possible to distinguish mass-reared from wild populations with >95% confidence. The C4 marker was detectable and distinguishable from wild populations up to 12 d after “release.” This technique could be adopted for use in any other SIT program with similar rearing protocols to Mediterranean fruit fly.

Nutritional status and the foraging behaviour of Bactrocera tryoni with particular reference to protein bait spray

Poisoned protein baits comprise a recognized method for controlling tephritid fruit flies in the form of a ‘lure‐and‐kill’ technique. However, little is known about how a flys internal protein and carbohydrate levels (i.e. nutritional status) might influence the efficacy of this control. In the present study, the relationships between the internal levels of protein (as measured by total body nitrogen) and carbohydrate (as measured by total body carbon) of the fruit fly Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) are investigated, as well as its foraging behaviours in response to protein, fruit and cue‐lure (a male‐specific attractant) baits. Small cage behavioural experiments are conducted using flies from cultures of different nutritional status and wild flies sampled from the field during the fruiting cycle of a guava crop. For female flies, increasing total body nitrogen is correlated with decreased protein foraging and increased oviposition activity; increasing total body carbon levels generate the same behavioural changes except that the oviposition response is not significant. For males, there are no significant correlations between changes in total body nitrogen and total body carbon and protein or cue‐lure foraging. For wild flies from the guava orchard, almost all of them are sexually mature when entering the crop and, over the entire season, total body nitrogen and total body carbon levels are such that protein hunger is unlikely for most flies. The results infer strongly that the requirements of wild, sexually mature flies for protein are minimal and that flies can readily gain sufficient nutrients from wild sources for their physiological needs. The results offer a mechanistic explanation for the poor response of male and mature female fruit flies to protein bait spray.

Attractiveness and competitiveness of irradiated light brown apple moths

The sterile insect technique (SIT) potentially provides a socially acceptable approach for insect eradication of new pest incursions. The light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), was discovered in Berkeley (CA, USA) in 2006, leading to an incursion response that included this technology. In this study, we assessed factors affecting mating success from a bisex release of irradiated moths: effects of radiation dose on male multiple mating, male flight competition, female sex pheromone titre and attractiveness of irradiated females to males, and identification of successful mating in vineyards of either irradiated or wild males (identified by isotope analysis of spermatophores from sentinel females). There was a significant negative relationship between male radiation dose and mating frequency. In head‐to‐head flights of irradiated males against non‐irradiated males to a pheromone lure in a wind tunnel, irradiated males reached the lure first only 31% of the time. With increasing radiation dose, the production of the major sex pheromone component in females, (E)‐11‐tetradecenyl acetate, dropped, from 0.7 ± 0.1 ng per female in non‐irradiated females to 0.2 ± 0.07 ng per female when irradiated at 300 Gy. Male catch was reduced to 11% of control females in traps containing females irradiated at 300 Gy. Isotope analysis of spermatophores found in the bursa copulatrix of females indicated that mating success of irradiated males inside the live (entry‐only) traps containing virgin females was lower (13.1 ± 3.3%) than suggested by male catch (21.2 ± 3.8%) in pheromone traps, the current standard for assessing field competitiveness. Impacts of irradiation on male and female moth fitness should be taken into account to improve estimates of irradiated to wild male E. postvittana overflooding ratios needed for population suppression.

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.

Intrinsic and Synthetic Stable Isotope Marking of Tsetse Flies

Abstract 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.