In this study, we investigated if industrial high-density polyethylene (HDPE) particles, a model microplastic free of additives, ranging > 0-80 μm are ingested and taken up into the cells and tissue of the blue mussel Mytilus edulis L. The effects of exposure (up to 96 h) and plastic ingestion were observed at the cellular and subcellular level. Microplastic uptake into the gills and digestive gland was analyzed by a new method using polarized light microscopy. Mussel health status was investigated incorporating histological assessment and cytochemical biomarkers of toxic effects and early warning. In addition to being drawn into the gills, HDPE particles were taken up into the stomach and transported into the digestive gland where they accumulated in the lysosomal system after 3 h of exposure. Our results show notable histological changes upon uptake and a strong inflammatory response demonstrated by the formation of granulocytomas after 6 h and lysosomal membrane destabilization, which significantly increased with longer exposure times. We provide proof of principle that microplastics are taken up into cells and cause significant effects on the tissue and cellular level, which can be assessed with standard cytochemical biomarkers and polarized light microscopy for microplastic tracking in tissue.

SummaryA variety of methods are used in honey bee research and differ depending on the level at which the research is conducted. On an individual level, the handling of individual honey bees, including the queen, larvae and pupae are required. There are different methods for the immobilising, killing and storing as well as determining individual weight of bees. The precise timing of developmental stages is also an important aspect of sampling individuals for experiments. In order to investigate and manipulate functional processes in honey bees, e.g. memory formation and retrieval and gene expression, microinjection is often used. A method that is used by both researchers and beekeepers is the marking of queens that serves not only to help to locate her during her life, but also enables the dating of queens. Creating multiple queen colonies allows the beekeeper to maintain spare queens, increase brood production or ask questions related to reproduction. On colony level, very useful techniques are the measurement of intra hive mortality using dead bee traps, weighing of full hives, collecting pollen and nectar, and digital monitoring of brood development via location recognition. At the population level, estimation of population density is essential to evaluate the health status and using beelines help to locate wild colonies. These methods, described in this paper, are especially valuable when investigating the effects of pesticide applications, environmental pollution and diseases on colony survival. Métodos estándar diversos para la investigación en Apis mellifera ResumenEn la investigación de la abeja de la miel, se han usado una variedad de métodos que se diferencian en función del nivel en el que se realiza la investigación. Al nivel individual, el manejo de las abejas individuales es necesario, incluyendo a la reina, las larvas y las pupas. Existen diferentes métodos para la inmovilización, mortandad y almacenamiento, así como para la determinación del peso individual de las abejas. La precisión en la sincronización de las etapas de desarrollo es también un aspecto importante de los experimentos con muestreos individuales.La microinyección se utiliza a menudo con el fin de investigar y manipular los procesos funcionales de las abejas melíferas, como por ejemplo, la formación y recuperación de la memoria y la expresión génica. Un método utilizado tanto por investigadores como apicultores es el marcado de las reinas, que sirve no sólo para ayudar a localizarlas durante su vida, sino que también permite su datación. La creación de varias colmenas a partir de reinas permite al apicultor mantener reinas de repuesto, aumentar la producción de cría o hacer preguntas relacionadas con la reproducción. Al nivel de colmena, la medición de la mortalidad intra colmena utilizando trampas de abejas muertas, el pesaje de las colmenas completas, la recolección de polen y néctar, y el seguimiento digital del desarrollo de la cría a través del reconocimiento de su ubicación, son algunas de las técnicas más útiles. Al nivel p...

Plastic pollution is an emerging global threat for marine wildlife. Many species of birds, reptiles, and fishes are directly impaired by plastics as they can get entangled in ropes and drown or they can ingest plastic fragments which, in turn, may clog their stomachs and guts. Microplastics of less than 1 mm can be ingested by small invertebrates, but their fate in the digestive organs and their effects on the animals are yet not well understood. We embedded fluorescent microplastics in artificial agarose-based food and offered the food to marine isopods, Idotea emarginata. The isopods did not distinguish between food with and food without microplastics. Upon ingestion, the microplastics were present in the stomach and in the gut but not in the tubules of the midgut gland which is the principal organ of enzyme-secretion and nutrient resorption. The feces contained the same concentration of microplastics as the food which indicates that no accumulation of microplastics happens during the gut passage. Long-term bioassays of 6 weeks showed no distinct effects of continuous microplastic consumption on mortality, growth, and intermolt duration. I. emarginata are able to prevent intrusion of particles even smaller than 1 μm into the midgut gland which is facilitated by the complex structure of the stomach including a fine filter system. It separates the midgut gland tubules from the stomach and allows only the passage of fluids and chyme. Our results indicate that microplastics, as administered in the experiments, do not clog the digestive organs of isopods and do not have adverse effects on their life history parameters.

The induced production of secondary metabolites in herbivore-attacked plants varies in space and time. However, the consequences of these spatiotemporal patterns for herbivore performance are not well understood. This is particularly true for 1,4-benzoxazin-3-ones (BXs), the major induced defensive metabolites of maize. Here we report on the spatiotemporal dynamics of BX induction and its consequences for the leaf feeder Spodoptera littoralis. Defence-related phytohormones and transcript levels of BX biosynthetic genes were upregulated locally at the wound site within 12 h of herbivory. Within another 12 h, the insecticidal BX HDMBOA-Glc started to accumulate in a highly localized manner at the feeding site. Changes in BX metabolism away from the feeding site within the same leaf were much weaker and were undetected in systemic leaves. Following the removal of the caterpillars, local HDMBOA-Glc levels remained elevated for 7 days. Caterpillars that were forced to feed directly on locally induced leaf parts, but not on adjacent leaf parts, suffered from reduced growth. This effect was abolished in the BX-deficient bx1 mutant. We did not find any evidence that BXs regulate defensive phytohormones or their own accumulation. In summary, this study shows that induced herbivore resistance in maize is highly localized and dependent on BXs.

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Plant defences vary in space and time, which may translate into specific herbivore-foraging patterns and feeding niche differentiation. To date, little is known about the effect of secondary metabolite patterning on within-plant herbivore foraging. We investigated how variation in the major maize secondary metabolites, 1,4-benzoxazin-3-one derivatives (BXDs), affects the foraging behaviour of two leaf-chewing herbivores. BXD levels varied substantially within plants. Older leaves had higher levels of constitutive BXDs while younger leaves were consistently more inducible. These differences were observed independently of plant age, even though the concentrations of most BXDs declined markedly in older plants. Larvae of the well-adapted maize pest Spodoptera frugiperda preferred and grew better on young inducible leaves irrespective of plant age, while larvae of the generalist Spodoptera littoralis preferred and tended to grow better on old leaves. In BXD-free mutants, the differences in herbivore weight gain between old and young leaves were absent for both species, and leaf preferences of S. frugiperda were attenuated. In contrast, S. littoralis foraging patterns were not affected. In summary, our study shows that plant secondary metabolites differentially affect performance and foraging of adapted and non-adapted herbivores and thereby likely contribute to feeding niche differentiation.

Secondary metabolites produced by plants for herbivore defence are often found in floral nectar, but their effect on the foraging behaviour and physiological performance of pollinators is largely unknown. Nicotine is highly toxic to most herbivores, and nicotine-based insecticides may contribute to current pollinator declines. We examined the effects of nectar nicotine on honeybee foraging choices and worker longevity. Free-flying honeybee (Apis mellifera scutellata) workers from six colonies were given a choice between multiple nicotine concentrations (0-1000 µM) in artificial nectar (0.15-0.63 M sucrose). The dose-dependent deterrent effect of nicotine was stronger in lower sugar concentrations, but even the highest nicotine concentrations did not completely repel honeybees, i.e. bees did not stop feeding on these diets. Nicotine in nectar acts as a partial repellent, which may keep pollinators moving between plants and enhance crosspollination. In the second part of the study, newly emerged workers from 12 colonies were caged and fed one of four nicotine concentrations (0-300 µM) in 0.63 M sucrose for 21 days. Moderate (≤30 µM) nicotine concentrations had no significant detrimental effect, but high nicotine concentrations reduced the survival of caged workers and their nectar storage in the honey comb.In contrast, worker groups that survived poorly on sugar-only diets demonstrated increased survival on all nicotine diets. In the absence of alternative nectar sources, honeybees tolerate naturally occurring nectar nicotine concentrations; and low concentrations can even be beneficial to honeybees. However, high nicotine concentrations may have a detrimental effect on colony fitness.

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