Vincenzo Girolami

Translocation of Neonicotinoid Insecticides From Coated Seeds to Seedling Guttation Drops: A Novel Way of Intoxication for Bees

ABSTRACT The death of honey bees, Apis mellifera L., and the consequent colony collapse disorder causes major losses in agriculture and plant pollination worldwide. The phenomenon showed increasing rates in the past years, although its causes are still awaiting a clear answer. Although neonicotinoid systemic insecticides used for seed coating of agricultural crops were suspected as possible reason, studies so far have not shown the existence of unquestionable sources capable of delivering directly intoxicating doses in the fields. Guttation is a natural plant phenomenon causing the excretion of xylem fluid at leaf margins. Here, we show that leaf guttation drops of all the corn plants germinated from neonicotinoid-coated seeds contained amounts of insecticide constantly higher than 10 mg/1, with maxima up to 100 mg/1 for thiamethoxam and clothianidin, and up to 200 mg/1 for imidacloprid. The concentration of neonicotinoids in guttation drops can be near those of active ingredients commonly applied in field sprays for pest control, or even higher. When bees consume guttation drops, collected from plants grown from neonicotinoid-coated seeds, they encounter death within few minutes.

Assessment of the environmental exposure of honeybees to particulate matter containing neonicotinoid insecticides coming from corn coated seeds.

Since seed coating with neonicotinoid insecticides was introduced in the late 1990s, European beekeepers have reported severe colony losses in the period of corn sowing (spring). As a consequence, seed-coating neonicotinoid insecticides that are used worldwide on corn crops have been blamed for honeybee decline. In view of the currently increasing crop production, and also of corn as a renewable energy source, the correct use of these insecticides within sustainable agriculture is a cause of concern. In this paper, a probable--but so far underestimated--route of environmental exposure of honeybees to and intoxication with neonicotinoid insecticides, namely, the atmospheric emission of particulate matter containing the insecticide by drilling machines, has been quantitatively studied. Using optimized analytical procedures, quantitative measurements of both the emitted particulate and the consequent direct contamination of single bees approaching the drilling machine during the foraging activity have been determined. Experimental results show that the environmental release of particles containing neonicotinoids can produce high exposure levels for bees, with lethal effects compatible with colony losses phenomena observed by beekeepers.

Conclusions of the Worldwide Integrated Assessment on the risks of neonicotinoids and fipronil to biodiversity and ecosystem functioning

The side effects of the current global use of pesticides on wildlife, particularly at higher levels of biological organization: populations, communities and ecosystems, are poorly understood (Kohler and Triebskorn 2013). Here, we focus on one of the problematic groups of agrochemicals, the systemic insecticides fipronil and those of the neonicotinoid family. The increasing global reliance on the partly prophylactic use of these persistent and potent neurotoxic systemic insecticides has raised concerns about their impacts on biodiversity, ecosystem functioning and ecosystem services provided by a wide range of affected species and environments. The present scale of use, combined with the properties of these compounds, has resulted in widespread contamination of agricultural soils, freshwater resources, wetlands, non-target vegetation and estuarine and coastal marine systems, which means that many organisms inhabiting these habitats are being repeatedly and chronically expose...

Fatal powdering of bees in flight with particulates of neonicotinoids seed coating and humidity implication

Losses of honeybees have been reported in Italy concurrent with the sowing of corn coated with neonicotinoids using a pneumatic drilling machine. Being unconvinced that solid particles containing systemic insecticide, falling on the vegetation surrounding the sown area, could poison bees foraging on contaminated nectar and pollen, the effect of direct aerial powdering was tested on foragers in free flight near the drilling machine. Bees were conditioned to visit a dispenser of sugar solution whilst a drilling machine was sowing corn along the flight path. Samples of bees were captured on the dispenser, caged and held in the laboratory. Chemical analysis showed some hundred nanograms of insecticide per bee. Nevertheless, caged bees, previously contaminated in flight, died only if kept in conditions of high humidity. After the sowing, an increase in bee mortality in front of the hives was also observed. Spring bee losses, which corresponded with the sowing of corn‐coated seed, seemed to be related to the casual encountering of drilling machine during foraging flight across the ploughed fields.

Standard methods for toxicology research in Apis mellifera

Summary Modern agriculture often involves the use of pesticides to protect crops. These substances are harmful to target organisms (pests and pathogens). Nevertheless, they can also damage non-target animals, such as pollinators and entomophagous arthropods. It is obvious that the undesirable side effects of pesticides on the environment should be reduced to a minimum. Western honey bees (Apis mellifera) are very important organisms from an agricultural perspective and are vulnerable to pesticide-induced impacts. They contribute actively to the pollination of cultivated crops and wild vegetation, making food production possible. Of course, since Apis mellifera occupies the same ecological niche as many other species of pollinators, the loss of honey bees caused by environmental pollutants suggests that other insects may experience a similar outcome. Because pesticides can harm honey bees and other pollinators, it is important to register pesticides that are as selective as possible. In this manuscript, we describe a selection of methods used for studying pesticide toxicity/selectiveness towards Apis mellifera. These methods may be used in risk assessment schemes and in scientific research aimed to explain acute and chronic effects of any target compound on Apis mellifera.

OVIPOSITIONAL DETERRENTS INDACUS OLEAE

The deterrent substances diverting D. oleae females from ovipositing on already attacked olives are contained, at least partly, in the juice which trickles from the oviposition wounds. Surprisingly, the water fraction of the olive juice had limited deterrent activity. The principal deterrent stimuli are present in the oil fraction. Acetophenone and benzaldehyde are likely to be involved.

Reduced fitness of the leafhopper vector Scaphoideus titanus exposed to Flavescence dorée phytoplasma

Scaphoideus titanus Ball (Homoptera: Cicadellidae), a specialist and univoltine leafhopper on grapevine (Vitis vinifera L.) (Vitaceae), is a vector of Flavescence dorée phytoplasma (FDP) in vineyards of European temperate areas. Males and females of the leafhopper were exposed to FDP by feeding on infected broad bean (Vicia faba L.) (Fabaceae). Detection of FDP by the amplification of phytoplasma DNA with polymerase chain reaction assays of individual insects revealed an acquisition rate of 91.4% (96/105) after an acquisition access period of 13 days. The adult life span of FD‐exposed males and females was much less than that of leafhoppers fed on healthy broad bean, as revealed by ANOVA on the quartiles of survival distribution and Weibull scale parameter. The progeny of exposed females (number of nymphs emerging from eggs deposited on woody cane segments) was significantly less than the progeny of unexposed females. Eggs produced by FD‐exposed females were slightly but significantly delayed in hatching. Reduced fecundity was confirmed by dissecting FD‐exposed and non‐exposed 42‐day‐old females and counting the number of fully sized eggs in each leafhopper. There was no evidence of transovarial passage of FDP in the offspring of infected females after 72 nymphs were reared on a healthy grapevine until the fifth instar or adult appearance and then confined on broad bean seedlings.

Aerial powdering of bees inside mobile cages and the extent of neonicotinoid cloud surrounding corn drillers

Sudden losses of bees have been observed in spring during maize sowing. The death of bees has been correlated with the use of neonicotinoid‐coated seed and the toxic particulates emitted by pneumatic drilling machines. The contamination of foragers in flight over the ploughed fields has been hypothesized. The airborne contamination has been proven, both with bees inside fixed cages around the field and in free flight near the driller. A new trial involving mobile cages has been established and consists of making rapid passes with single bees inside cages fixed to an aluminium bar. The bar was moved by two operators at different distances from the working drilling machine. A single pass was shown as sufficient to kill all the bees exposed to exhaust air on the emission side of the drill, when bees were subsequently held in high relative humidity. The extent of toxic cloud around driller was evaluated at the height of 0.5, 1.8 and 3.5 m and proved to be about 20 m in diameter, with an ellipsoidal shape. The shape may be influenced by working speed of the drill and environmental parameters, and is easily shown by adding talc powder to the seed in the machine hopper. A new driller equipment was evaluated consisting of two tubes inclined towards the soil that direct the exhaust air towards the ground. The survival rate of the bees was not substantially increased using the modified drill and was lower than 50%. Chemical analyses show up to 4000 ng of insecticide in single bees with an average content around 300 ng. Similar quantities were observed at increased distances from the modified or unmodified drillers. This new evaluation of bee mortality in the field is an innovative biological test to verify the hypothetical efficiency (or not) of driller modifications.

UHPLC-DAD method for the determination of neonicotinoid insecticides in single bees and its relevance in honeybee colony loss investigations.

In the understanding of colony loss phenomena, a worldwide crisis of honeybee colonies which has serious consequences for both apiculture and bee-pollination-dependent farm production, analytical chemistry can play an important role. For instance, rapid and accurate analytical procedures are currently required to better assess the effects of neonicotinoid insecticides on honeybee health. Since their introduction in agriculture, neonicotinoid insecticides have been blamed for being highly toxic to honeybees, possibly at the nanogram per bee level or lower. As a consequence, most of the analytical methods recently optimized have focused on the analysis of ultratraces of neonicotinoids using liquid chromatography–mass spectrometry techniques to study the effects of sublethal doses. However, recent evidences on two novel routes—seedling guttations and seed coating particulate, both associated with corn crops—that may expose honeybees to huge amounts of neonicotinoids in the field, with instantly lethal effects, suggest that selected procedures need optimizing. In the present work, a simplified ultra-high-performance liquid chromatography–diode-array detection method for the determination of neonicotinoids in single bees has been optimized and validated. The method ensures good selectivity, good accuracy, and adequate detection limits, which make it suitable for the purpose, while maintaining its ability to evaluate exposure variability of individual bees. It has been successfully applied to the analysis of bees in free flight over an experimental sowing field, with the bees therefore being exposed to seed coating particulate released by the pneumatic drilling machine.

Second generation of grape berry moths, Lobesia botrana (Den. & Schiff.) (Lep., Tortricidae) and Eupoecilia ambiguella (Hb.) (Lep., Cochylidae): spatial and frequency distributions of larvae, weight loss and economic injury level

Abstract: Spatial and frequency distributions of grape berry moth larvae were studied in vineyards of northeastern Italy. The larval density varied in relation to position of grape clusters within vines. For density values below 0.5 larval nests per cluster, the larvae were almost randomly distributed and fit the Poisson distribution, while above 0.5 larval nests per cluster were slightly aggregated and fit the negative binomial distribution better. A new method to estimate cluster weight loss cause by second generation of grape berry moths, based on weighing and counting of berries injured, was proposed. Therefore an estimate of economic injury level based on this method was put forward.