Extrapolating toxic effects on individuals to the population level: the role of dynamic energy budgets

The interest of environmental management is in the long-term health of populations and ecosystems. However, toxicity is usually assessed in short-term experiments with individuals. Modelling based on dynamic energy budget (DEB) theory aids the extraction of mechanistic information from the data, which in turn supports educated extrapolation to the population level. To illustrate the use of DEB models in this extrapolation, we analyse a dataset for life cycle toxicity of copper in the earthworm Dendrobaena octaedra. We compare four approaches for the analysis of the toxicity data: no model, a simple DEB model without reserves and maturation (the Kooijman-Metz formulation), a more complex one with static reserves and simplified maturation (as used in the DEBtox software) and a full-scale DEB model (DEB3) with explicit calculation of reserves and maturation. For the population prediction, we compare two simple demographic approaches (discrete time matrix model and continuous time Euler-Lotka equation). In our case, the difference between DEB approaches and population models turned out to be small. However, differences between DEB models increased when extrapolating to more field-relevant conditions. The DEB3 model allows for a completely consistent assessment of toxic effects and therefore greater confidence in extrapolating, but poses greater demands on the available data.     

Plantago lanceolata and Plantago rugelii Extracts are Toxic to Meloidogyne incognita but not to Certain Microbes

Extracts from the plants Plantago lanceolata and P. rugelii were evaluated for toxicity to the root-knot nematode Meloidogyne incognita, the beneficial microbes Enterobacter cloacae, Pseudomonas fluorescens and Trichoderma virens, and the plant-pathogenic fungi Fusarium oxysporum f. sp. gladioli, Phytophthora capsici, Pythium ultimum, and Rhizoctonia solani. Wild plants were collected, roots were excised from shoots, and the plant parts were dried and ground to a powder. One set of extracts (10% w/v) was prepared in water and another in methanol. Treatments included extract concentrations of 25%, 50%, 75% and 100%, and water controls. Meloidogyne incognita egg hatch was recorded after 7-day exposure to the treatments, and second-stage juvenile (J2) activity after 48 hours. All extracts were toxic to eggs and J2, with P. lanceolata shoot extract tending to have the most activity against M. incognita. Numbers of active J2 remained the same or decreased in a 24-hour water rinse following the 48-hour extract treatment, indicating that the extracts were lethal. When data from water- and methanol-extracted roots and shoots of both plant species were combined for analysis, J2 tended to be more sensitive than eggs to the toxic compounds at lower concentrations, while the higher concentrations (75% and 100%) were equally toxic to both life stages. The effective concentrations causing 50% reduction (EC₅₀) in egg hatch and in J2 viability were 44.4% and 43.7%, respectively. No extract was toxic to any of the bacteria or fungi in our assays.     

Statistical cautions when estimating DEBtox parameters

DEBtox (Dynamic Energy Budget in toxicology) models have been designed to analyse various results from classic tests in ecotoxicology. They consist of a set of mechanistic models describing how organisms manage their energy, when they are exposed to a contaminant. Until now, such a biology-based modeling approach has not been used within the regulatory context. However, these methods have been promoted and discussed in recent guidance documents on the statistical analysis of ecotoxicity data. Indeed, they help us to understand the underlying mechanisms. In this paper, we focused on the 21 day Daphnia magna reproduction test. We first aimed to clarify and detail the model building process leading to DEBtox models. Equations were rederived step by step, and for some of them we obtained results different from the published ones. Then, we statistically evaluated the estimation process quality when using a least squares approach. Using both experimental and simulated data, our analyses highlighted several statistical issues related to the fitting of DEBtox models on OECD-type reproduction data. In this case, particular attention had to be paid to parameter estimates and the interpretation of their confidence intervals.     

Compatibility of Soil Amendments with Entomopathogenic Nematodes

The impact of inorganic and organic fertilizers on the infectivity, reproduction, and population dynamics of entomopathogenic nematodes was investigated. Prolonged (10- to 20-day) laboratory exposure to high inorganic fertilizer concentrations inhibited nematode infectivity and reproduction, whereas short (1-day) exposures increased infectivity. Heterorhabditis bacteriophora was more sensitive to adverse effects than were two species of Steinernema. In field studies, organic manure resulted in increased densities of a native population of Steinernema feltiae, whereas NPK fertilizer suppressed nematode densities regardless of manure applications. Inorganic fertilizers are likely to be compatible with nematodes in tank mixes and should not reduce the effectiveness of nematodes used for short-term control as biological insecticides, but may interfere with attempts to use nematodes as inoculative agents for long-term control. Organic manure used as fertilizer may encourage nematode establishment and recycling.     

Use of the Cultex® Radial Flow System as an in vitro exposure method to assess acute pulmonary toxicity of fine dusts and nanoparticles with special focus on the intra- and inter-laboratory reproducibility

Exposure of the respiratory tract to airborne particles (including metal-dusts and nano-particles) is considered as a serious health hazard. For a wide range of substances basic knowledge about the toxic properties and the underlying pathomechanisms is lacking or even completely missing. Legislation demands the toxicological characterization of all chemicals placed on the market until 2018 (REACH). As toxicological in vivo data are rare with regard to acute lung toxicity or exhibit distinct limitations (e.g. inter-species differences) and legislation claims the reduction of animal experiments in general (“3R” principle), profound in vitro models have to be established and characterized to meet these requirements. In this paper we characterize a recently introduced advanced in vitro exposure system (Cultex® RFS) showing a great similarity to the physiological in vivo exposure situation for the assessment of acute pulmonary toxicity of airborne materials.     

Reconsidering the importance of the past in predator–prey models: both numerical and functional responses depend on delayed prey densities

We propose that delayed predator–prey models may provide superficially acceptable predictions for spurious reasons. Through experimentation and modelling, we offer a new approach: using a model experimental predator–prey system (the ciliates Didinium and Paramecium), we determine the influence of past-prey abundance at a fixed delay (approx. one generation) on both functional and numerical responses (i.e. the influence of present : past-prey abundance on ingestion and growth, respectively). We reveal a nonlinear influence of past-prey abundance on both responses, with the two responding differently. Including these responses in a model indicated that delay in the numerical response drives population oscillations, supporting the accepted (but untested) notion that reproduction, not feeding, is highly dependent on the past. We next indicate how delays impact short- and long-term population dynamics. Critically, we show that although superficially the standard (parsimonious) approach to modelling can reasonably fit independently obtained time-series data, it does so by relying on biologically unrealistic parameter values. By contrast, including our fully parametrized delayed density dependence provides a better fit, offering insights into underlying mechanisms. We therefore present a new approach to explore time-series data and a revised framework for further theoretical studies.     

Insect-toxic secreted proteins and virulence of the entomopathogenic fungus Beauveria bassiana

Fungal virulence has been mostly associated with cuticle-degrading enzymes that can be regulated depending on nutrient conditions. However, few studies have related fungal virulence to insect-toxic secreted proteins. Here, we describe how the presence of secreted toxic proteins may be linked to conidial virulence, which can be affected by nutrient factors. In this study we evaluated: (1) the virulence of the conidia of four Beauveria bassiana strains (EABb 01/103-Su, EABb 01/12-Su, EABb 01/88-Su and EABb 01/110-Su) grown on three different media (malt extract agar (MA), Rice (Rice), Sabouraud dextrose agar (SDA) and harvested from the cadavers of fungal-infected Galleria mellonella larvae (CAD) and (2) the toxicity of the crude soluble protein extracts (CSPEs) obtained from Adamek’s liquid medium inoculated with these conidia. Conidial suspensions were obtained from the four media, assessed on G. mellonella larvae and used to produce CSPEs that were injected into healthy G. mellonella larvae. The larvae were also injected with conidia obtained from MA and CAD cultures to expose them to in vivo-secreted proteins. For all isolates, the CAD conidia were by far the most virulent, followed by conidia grown on SDA, Rice and MA. The injected CSPEs showed the same toxicity trends as the conidial suspensions. In addition, the outcomes of injection of the in vivo-secreted proteins showed that the toxic proteins secreted in vitro by the EABb 01/110-Su strain are not produced in vivo. However, the other strains produced toxic proteins both in vivo and in vitro, suggesting that these toxic proteins may be virulence factors involved in invertebrate pathogenesis.     

The genetics and evolution of obligate reproductive parasitism in Trichogramma pretiosum infected with parthenogenesis-inducing Wolbachia

Parthenogenesis-inducing (PI) Wolbachia belong to a class of intracellular symbionts that distort the offspring sex ratio of their hosts toward a female bias. In many PI Wolbachia-infected species sex ratio distortion has reached its ultimate expression-fixation of infection and all-female populations. This is only possible with thelytokous PI symbionts as they provide an alternative form of reproduction and remove the requirement for males and sexual reproduction. Many populations fixed for PI Wolbachia infection have lost the ability to reproduce sexually, even when cured of the infection. We examine one such population in the species Trichogramma pretiosum. Through a series of backcrossing experiments with an uninfected Trichogramma pretiosum population we were able to show that the genetic basis for the loss of female sexual function could be explained by a dominant nuclear effect. Male sexual function had not been completely lost, though some deterioration of male sexual function was also evident when males from the infected population (created through antibiotic curing of infected females) were mated to uninfected females. We discuss the dynamics of sex ratio selection in PI Wolbachia-infected populations and the evolution of non-fertilizing mutations.     

Evaluation of Microcystis as food for zooplankton in a eutrophic lake

Four experiments were conducted to evaluate Microcystis as food for zooplankton in Lake Kasumigaura, and the following results were obtained. (1) Moina micrura (Cladocera) showed little growth and no reproduction when the animal was reared with Microcystis cultured in the laboratory. The animal did not grow nor reproduce well when Chlorella was mixed with Microcystis as food. (2) Moina micrura assimilated Microcystis much less than Chlorella when the animal fed on single species of Microcystis or a mixture with Chlorella. (3) Microcystis collected from Lake Kasumigaura could not be utilized by Moina micrura even though the colonies were broken up into edible sizes. However, the alga turned into utilizable food when it was decomposed. (4) No inhibitors of Moina micrura population growth could be found in the non-filtered water of Lake Kasumigaura where Microcystis was blooming heavily. Decomposed Microcystis seemed to be utilized by zooplankton as an important food source in Lake Kasumigaura.     

The Potential of Thiarubrine C as a Nematicidal Agent against Plant- parasitic Nematodes

Thiarubrine C, a polyacetylenic 1,2-dithiin isolated from the roots of Rudbeckia hirta (Asteraceae), exhibited strong nematicidal activity in in vitro and growth chamber assays. Thiarubrine C was toxic, in the absence of light, to the plant-parasitic nematodes Meloidogyne incognita and Pratylenchus penetrans at LC₅₀s of 12.4 ppm and 23.5 ppm, respectively. A minimum exposure time between 12 and 24 hours was the critical period for nematode mortality due to thiarubrine C. Although thiarubrine C was not totally dependent on light for toxicity, activity was enhanced in the presence of light, especially with the microbivorous nematode, Teratorhabditis dentifera. Upon exposure of M. incognita juveniles to 20 ppm thiarubrine C for 1 hour, infection of tomato plants was greatly reduced compared to untreated checks. Thiarubrine C was also effective in reducing plant infection when mixed with soil 24 hours prior to or at planting, unlike other related compounds such as δ-terthienyl.     

Effects of saxitoxin- and non-saxitoxin-producing strains of the cyanobacterium Cylindrospermopsis raciborskii on the fitness of temperate and tropical cladocerans

Life-table experiments were performed with two strains of Cylindrospermopsis raciborskii, a saxitoxin-producing strain (T3) and a non-saxitoxin-producing strain (NPLP-1) combined with the green algae Ankistrodesmus falcatus, aiming to detect effects on the reproduction of three cladocerans species: Daphnia pulex, Daphnia gessneri and Moina micrura. Survivorship, age at first reproduction, clutch size and population growth rate during 12–15 days were recorded. Cladocerans showed different susceptibility to saxitoxin-producing strain (T3), with D. pulex and M. micrura being negatively affected, followed by and D. gessneri, which did not show any decrease in fitness and performed even better than the controls with this strain. A reverse response was found in experiments with the non-saxitoxin-producing strain (NPLP-1): while D. pulex and M. micrura had an increase, D. gessneri showed a significant decrease in fitness, suggesting that this strain may produce some bioactive compound. The contrasting responses of the cladoceran species to saxitoxins and non-saxitoxin-producing strains of C. raciborskii suggest that the presence of this cyanobacterium may be a selective factor in determining the composition of zooplankton communities.     

A conserved class of queen pheromones? Re-evaluating the evidence in bumblebees (Bombus impatiens)

The regulation of reproductive division of labour is a key component in the evolution of social insects. Chemical signals are important mechanisms to regulate worker reproduction, either as queen-produced pheromones that coercively inhibit worker reproduction or as queen signals that honestly advertise her fecundity. A recent study suggested that a conserved class of hydrocarbons serve as queen pheromones across three independent origins of eusociality. In bumblebees (Bombus terrestris), pentacosane (_C25) was suggested to serve as a queen pheromone. Here, we repeat these studies using a different species of bumblebee (Bombus impatiens) with a more controlled experimental design. Instead of dequeened colonies, we used same-aged, three-worker queenless groups comprising either experienced or naive workers (with/without adult exposure to queen pheromone). We quantified three hydrocarbons (_C23, _C25 and _C27) on the cuticular surfaces of females and tested their effects on the two worker types. Our results indicate differences in responses of naive and experienced workers, genetic effects on worker reproduction, and general effects of hydrocarbons and duration of egg laying on ovary resorption rates. However, we found no evidence to support the theory that a conserved class of hydrocarbons serve as queen pheromones or queen signals in Bombus impatiens.     

Effects of Meloidogyne spp. and Rhizoctonia solani on the Growth of Grapevine Rootings

A disease complex involving Meloidogyne incognita and Rhizoctonia solani was associated with stunting of grapevines in a field nursery. Nematode reproduction was occurring on both susceptible and resistant cultivars, and pot experiments were conducted to determine the virulence of this M. incognita population, and of M. javanica and M. hapla populations, to V. vinifera cv. Colombard (susceptible) and to V. champinii cv. Ramsey (regarded locally as highly resistant). The virulence of R. solani isolates obtained from roots of diseased grapevines also was determined both alone and in combination with M. incognita. Ramsey was susceptible to M. incognita (reproduction ratio 9.8 to 18.4 in a shadehouse and heated glasshouse, respectively) but was resistant to M. javanica and M. hapla. Colombard was susceptible to M. incognita (reproduction ratio 24.3 and 41.3, respectively) and M. javanica. Shoot growth was suppressed (by 35%) by M. incognita and, to a lesser extent, by M. hapla. Colombard roots were more severely galled than Ramsey roots by all three species, and nematode reproduction was higher on Colombard. Isolates of R. solani assigned to putative anastomosis groups 2-1 and 4, and an unidentified isolate, colonized and induced rotting of grapevine roots. Ramsey was more susceptible to root rotting than Colombard. Shoot growth was inhibited by up to 15% by several AG 4 isolates and by 20% by the AG 2-1 isolate. AG 4 isolates varied in their virulence. Root rotting was higher when grapevines were inoculated with both M. incognita and R. solani and was highest when nematode inoculation preceded the fungus. Shoot weights were lower when vines were inoculated with the nematode 13 days before the fungus compared with inoculation with both the nematode and the fungus on the same day. It was concluded that both the M. incognita population and some R. solani isolates were virulent against both Colombard and Ramsey, and that measures to prevent spread in nursery stock were therefore important.     

Resistance of Interspecific Arachis Breeding Lines to Meloidogyne javanica and an Undescribed Meloidogyne Species

Resistance to a peanut-parasitic population of Meloidogyne javanica and an undescribed Meloidogyne sp. in peanut breeding lines selected for resistance to Meloidogyne javanica was examined in greenhouse tests. The interspecific hybrid TxAG-7 was resistant to reproduction of Meloidogyne javanica, M. javanica, and Meloidogyne sp. An Meloidogyne javanica-resistant selection from the second backcross (BC) of TxAG-7 to the susceptible cultivar Florunner also was resistant to M. javanica but appeared to be segregating for resistance to the Meloidogyne sp. When reproduction of M. javanica and Meloidogyne javanica were compared on five BC4F3 peanut breeding lines, each derived from Meloidogyne javanica-susceptible BC4F2 individuals, all five lines segregated for resistance to M. javanica, whereas four of the lines appeared to be susceptible to Meloidogyne javanica. These data indicate that several peanut lines selected for resistance to Meloidogyne javanica also contain genes for resistance to populations of M. javanica and the undescribed Meloidogyne sp. that are parasitic on peanut. Further, differences in segregation patterns suggest that resistance to each Meloidogyne sp. is conditioned by different genes.     

Ecological Trade-offs between Migration and Reproduction Are Mediated by the Nutrition-Sensitive Insulin-Signaling Pathway

Crowding and changes in food availability are two critical environmental conditions that impact an animal''s trajectory toward either migration or reproduction. Many insects facing this challenge have evolved wing polyphenisms. When conditions favor reproduction, wing polyphenic species produce adults that either have no wings or short, non-functional wings. Facultative wing growth reflects a physiological and evolutionary trade-off between migration and reproduction, triggered by environmental conditions. How environmental cues are transduced to produce these alternative forms, and their associated ecological shift from migration to reproduction, remains an important unsolved problem in evolutionary ecology. The brown planthopper, a wing polymorphic insect exhibiting strong trade-offs in investment between migration and reproduction, is one of the most serious rice pests in Asia. In this study, we investigated the function of four genes in the insulin-signaling pathway known to couple nutrition with growth, PI3 Kinase (PI3K), PDK1, Akt (Protein Kinase B), and the forkhead gene FOXO. Using a combination of RNA interference and pharmacological inhibitor treatment, we show that all four genes contribute to tissue level regulation of wing polymorphic development in this insect. As predicted, silencing of the NlPI3K, NlAkt and NlPDK1 through dsRNA and with the pharmacological inhibitor Perifosine resulted in short-winged brown planthoppers, whereas knockdown of NlFOXO resulted in long-winged planthoppers. Morphometric analyses confirm that phenotypes from our manipulations mimic what would be found in nature, i.e., major parameters such as bristle number, wing area and body weight are not significantly different from non-experimental animals. Taken together, these data implicate the insulin-signaling pathway in the transduction of environmental factors into condition-dependent patterns of wing growth in insects.     

The alignment between phenotypic plasticity,the major axis of genetic variation and the response to selection

Phenotypic plasticity is the ability of a genotype to produce more than one phenotype in order to match the environment. Recent theory proposes that the major axis of genetic variation in a phenotypically plastic population can align with the direction of selection. Therefore, theory predicts that plasticity directly aids adaptation by increasing genetic variation in the direction favoured by selection and reflected in plasticity. We evaluated this theory in the freshwater crustacean Daphnia pulex, facing predation risk from two contrasting size-selective predators. We estimated plasticity in several life-history traits, the G matrix of these traits, the selection gradients on reproduction and survival, and the predicted responses to selection. Using these data, we tested whether the genetic lines of least resistance and the predicted response to selection aligned with plasticity. We found predator environment-specific G matrices, but shared genetic architecture across environments resulted in more constraint in the G matrix than in the plasticity of the traits, sometimes preventing alignment of the two. However, as the importance of survival selection increased, the difference between environments in their predicted response to selection increased and resulted in closer alignment between the plasticity and the predicted selection response. Therefore, plasticity may indeed aid adaptation to new environments.     

Interactions of Concomitant Species of Nematodes and Fusarium oxysporum f. sp. vasinfectum on Cotton

Meloidogyne incognita, Hoplolaintus galeatus, and North Carolina and Georgia populations of Belonolaimus longicaudatus were introduced singly and in various combinations with Fusarium oxysporum f. sp. vasinfectum on wilt-susceptible ''Rowden'' cotton. Of all the nematodes, the combination of the N. C. population of B. longicaudatus with Fusarium promoted greatest wilt development. H. galeatus had no effect on wilt. With Fusarium plus M. incognito or B. longicaudatus, high nematode levels promoted greater wilt than low levels. The combination of either population of B. longicaudatus with M. incognita and Fusarium induced greater wilt development than comparable inoculum densities of either nematode alone or where H. galeatus was substituted for either of these nematodes. Nematode reproduction was inversely related to wilt development. Without Fusarium, however, the high inoculum level resulted in greater reproduction of all nematode species on cotton. Combining M. incognita with B. longicaudatus or H. galeatus gave mutually depressive effects on final nematode populations. The interactions of H. gateatus with B. longicaudatus varied with two populations of the latter.     

Cloning, Expression, Purification and Toxicity Evaluation of Helicobacter pylori Outer Inflammatory Protein A

The Helicobacter pylori outer membrane proteins play an important role in pathogenesis; the outer inflammatory protein A (OipA) is one of these proteins which play the main role in the development of inflammation. In this study, purification of recombinant H. pylori OipA was performed by Ni–NTA affinity chromatography. Gastric carcinoma epithelial cells (AGS cell) were treated by different concentrations of recombinant OipA for various lengths of time and cell viability was evaluated by the viability assay. Statistical analysis showed that OipA had toxic effects on AGS cells in a concentration of 500 ng/ml after 24 and 48 h, and this toxic dose was 256 ng/ml after 72 h. OipA had direct toxic effects on gastric epithelial cells and the toxicity was observed to depend on time and dose of H. pylori exposure. Attachment of H. pylori to gastric epithelial cells is a key part in the pathogenesis and enables H. pylori to damage the epithelial cells with OipA.     

Nest fidelity is driven by multi-scale information in a long-lived seabird

Although the reproductive success of most organisms depends on factors acting at several spatial scales, little is known about how organisms are able to synthesize multi-scale information to optimize reproduction. Using longitudinal data from a long-lived seabird, Monteiro''s storm-petrel, we show that average breeding success is strongly related to oceanic conditions at the population level, and we postulate that (i) individuals use proximal information (their own reproduction outcome in year t) to assess the qualities of their mate and nest and to decide to retain them or not in year t + 1; (ii) the intensity of these responses depends on the quality of the oceanic environment in year t, which affects the predictability of reproduction outcome in year t + 1. Our results confirm that mate and nest fidelities are higher following successful reproduction and that the relationship between the success of a given pair and subsequent nest fidelity is stronger in years with unfavourable oceanic conditions, suggesting that individuals rely on distant information to modulate their use of proximal information and adjust their breeding strategy.     

Recycling Potential and Fitness of Steinernematid Nematodes Cultured in Curculio caryae and Galleria mellonella

The recycling potential of entomopathogenic nematodes in the pecan weevil, Curculio caryae, following inundative applications is an important factor in considering whether nematodes could be incorporated into a C. caryae management strategy. Our objective was to determine the recycling potential and fitness of Steinernema carpocapsae and S. riobrave cultured in C. caryae. To estimate fitness and quality, we reared nematodes in larvae of C. caryae and in the commonly used standard host, the greater wax moth, Galleria mellonella. Nematode lipid content, infectivity (power to invade), virulence (power to kill), and reproductive capacity (yield per insect) in C. caryae larvae were compared with G. mellonella data. Lipid content was higher in S. carpocapsae cultured in C. caryae than in G. mellonella, but S. riobrave lipid content was not affected by host source. Host source did not affect subsequent infectivity or virulence to C. caryae (P > 0.05) but did affect reproductive capacity (P < 0.0001). Both nematode species produced more progeny in C. caryae when they were first cultured in G. mellonella than when they were first passed through C. caryae. In terms of potential to recycle under field conditions, we predict that nematodes resulting from one round of recycling in C. caryae larvae would be equally capable of infecting and killing more weevils, but the potential to continue recycling in C. caryae would diminish over time due to reduced reproduction in that host.