Apis mellifera ligustica, Spinola 1806 as bioindicator for detecting environmental contamination: a preliminary study of heavy metal pollution in Trieste, Italy.

Honeybees have become important tools for the ecotoxicological assessment of soil, water and air metal contamination due to their extraordinary capacity to bioaccumulate toxic metals from the environment. The level of heavy metal pollution in the Trieste city was monitored using foraging bees of Apis mellifera ligustica from hives owned by beekeepers in two sites strategically located in the suburban industrial area and urban ones chosen as control. The metal concentration in foraging bees was determined by inductively coupled plasma-mass spectrometry. The chemical analysis has identified and quantified 11 trace elements accumulated in two different rank orders: Zn> Cu > Sr > Bi > Ni > Cr > Pb = Co > V > Cd > As in foraging bees from the suburban site and Zn > Cu > Sr > Cr > Ni > Bi > Co = V > Pb > As > Cd in bees from urban site. Data revealed concentrations of Cr and Cu significantly higher and concentration of Cd significantly lower in bees from urban sites. The spatial difference and magnitude order in heavy metal accumulation along the urban-suburban gradient are mainly related to the different anthropogenic activity within sampled sites and represent a risk for the human health of people living in the city. We discussed and compared results with the range of values reported in literature.

Immune challenges trigger cellular and humoral responses in adults of Pterostichus melas italicus (Coleoptera, Carabidae).

The present study focuses on the ability of Pterostichus melas italicus Dejean to mount cellular and humoral immune responses against invading pathogens. Ultrastructural analyses revealed the presence of five morphologically distinct types of hemocytes: prohemocytes, plasmatocytes, granulocytes, oenocytoids and macrophage-like cells. Differential hemocyte counts showed that plasmatocytes and granulocytes were the most abundant circulating cell types and plasmatocytes exhibited phagocytic activity following the latex bead immune challenge. Macrophage-like cells were recruited after the immune challenge to remove exhausted phagocytizing cells, apoptotic cells and melanotic capsules formed to immobilize the latex beads. Total hemocyte counts showed a significant reduction of hemocytes after latex bead treatment. Phenoloxidase (PO) assays revealed an increase of total PO in hemolymph after immune system activation with lipopolysaccharide (LPS). Moreover, the LPS-stimulated hemocytes showed increased protein expression of inducible nitric oxide synthase, indicating that the cytotoxic action of nitric oxide was engaged in this antimicrobial collaborative response. These results provide a knowledge base for further studies on the sensitivity of the P. melas italicus immune system to the environmental perturbation in order to evaluate the effect of chemicals on non-target species in agroecosystems.

Mediterranean mussel gene expression profile induced by okadaic acid exposure.

Seasonal seawater temperature increases define optimal growth conditions for Dinoflagellate species which can reach high concentrations in water column and also in filter-feeding organisms like Mytilus galloprovincialis. Commonly produced by Dinophysis and Prorocentrum spp., okadaic acid (OA) and its analogues are responsible for the Diarrheic Shellfish Poisoning (DSP) syndrome in humans. Closure of shellfishing grounds is therefore recommended by the EU when DSP toxin levels in shellfish exceed 16 µg OA 100 g(-1) flesh. Despite not being responsible for casualties either in humans or mussels, DSP outbreaks are considered natural events causing health and economic issues due to the frequency of their occurrence. Since gene expression studies offer a wide range of different solutions, we used a mussel cDNA microarray to evaluate gene expression changes in the digestive gland of mussels fed for five weeks with OA-contaminated nutrient. Among the differentially expressed genes we observed a general up-regulation of transcripts coding for stress proteins, proteins involved in cellular synthesis, and a few not annotated proteins. Overall, at the first time point analyzed we identified 58 candidate transcripts for OA-induced stress in mussels, half of which have unknown function. In this paper we present the first gene expression analysis performed on Mediterranean mussels exposed to okadaic acid. The characterization of these transcripts could be useful for the identification of an early physiological response to OA exposure.

Circulating hemocytes from larvae and adults of Carabus (Chaetocarabus) lefebvrei Dejean 1826 (Coleoptera, Carabidae): cell types and their role in phagocytosis after in vivo artificial non-self-challenge.

Carabus lefebvrei Dejean 1826 is an helicophagous Italian endemic ground beetle that lives in central and south Apennines mountain forests, from lower altitudes to about 1500 m. In ground beetles, no morphofunctional data about immune system is available, even though they are well known both taxonomically and ecologically and they have been often used as indicators of the habitat quality due to their specificity to certain habitat types. In the current investigation the cellular population in the hemolymph of adult and third instar larvae of C. lefebvrei has been characterized by means of light and electron microscopy analysis and phagocytosis assays were performed in vivo by injection of 0.9 microm carboxylate-modified polystyrene latex beads in order to identify the hemocyte types involved in phagocytosis. Four morphotypes of circulating hemocytes were found both in larvae and in adults: prohemocytes, granulocytes, oenocytoids and plasmatocytes. After in vivo artificial non-self-challenge treatments, C. lefebvrei showed a non-specific immune response involving phagocytosis performed by plasmatocytes, both in adults and in larvae and by oenocitoids in larvae. In untreated animals, the hemocyte type presenting a firm phagocytic activity, the plasmatocytes, presented a percentage significantly higher in larvae than in adults, and after latex beads injections in larvae there was a tendency of significant difference in plasmatocyte percentage compared to controls injected with phosphate saline buffer. We think that these differences could be correlated with the peculiar morphology (less chitinization) and ecology of larval stages that are more sensitive to pathogens than adults.

Ultrastructural and functional characterization of circulating hemocytes from the freshwater crayfish Astacus leptodactylus: cell types and their role after in vivo artificial non-self challenge.

The freshwater crayfish Astacus leptodactylus (Eschscholtz, 1823) is an important aquacultured decapod species as well as an invasive species in some European countries. In the current investigation we characterized the different classes of circulating blood cells in A. leptodactylus by means of light and electron microscopy analysis and we explored their reaction to different latex beads particles in vivo by total and differential cell counts at 0.5, 1, 2 and 4h after injections. We identified hemocytes by granule size morphometry as hyaline hemocytes with no or rare tiny granules, small granule hemocytes, unimodal medium diameter granule hemocytes and both small and large granule containing hemocytes. The latter granular hemocytes showed the strongest phenoloxidase l-DOPA reactivity both in granules and cytoplasm. A. leptodactylus respond to foreign particles with strong cellular immune responses. All treatments elicited a total hemocyte increase with a conspicuous recruitment of large granule containing hemocytes. All hemocyte types mounted some phagocytic response but the small granule hemocytes were the only ones involved in phagocytic response to all foreign particles with the highest percentages. These results (1) depict the variability in decapod hemocyte functional morphology; (2) identify the small granule hemocyte as the major phagocytic cell; (3) suggest that the rather rapid recruitment of large granule hemocyte in all treatments plays a relevant role by this hemocyte type in defense against foreign particles, probably in nodule formation.