Polystyrene nanoplastics as an ecotoxicological hazard: cellular and transcriptomic evidences on marine and freshwater in vitro teleost models.

The contamination of marine and freshwater environments by nanoplastics is considered a global threat for aquatic biota. Taking into account the most recent concentration range estimates reported globally and recognizing a knowledge gap in polystyrene nanoplastics (PS-NPs) ecotoxicology, the present work investigated the harmful effects of 20 nm and 80 nm PS-NPs, at increasing biological complexity, on the rainbow trout Oncorhynchus mykiss RTG-2 and gilthead seabream Sparus aurata SAF-1 cell lines. Twenty nm PS-NPs exerted a greater cytotoxicity than 80 nm ones and SAF-1 were approximately 4-fold more vulnerable to PS-NPs than RTG-2. The engagement of PS-NPs with plasma membranes was accompanied by discernible uptake patterns and morphological alterations along with a nuclear translocation already within a 30-min exposure. Cells were structurally damaged only by the 20 nm PS-NPs in a time-dependent manner as indicated by distinctive features of the execution phase of the apoptotic cell death mechanism such as cell shrinkage, plasma membrane blebbing, translocation of phosphatidylserine to the outer leaflet of the cell membrane and DNA fragmentation. At last, functional analyses unveiled marked transcriptional impairment at both sublethal and lethal doses of 20 nm PS-NPs, with the latter impacting the "Steroid biosynthesis", "TGF-beta signaling pathway", "ECM-receptor interaction", "Focal adhesion", "Regulation of actin cytoskeleton" and "Protein processing in endoplasmic reticulum" pathways. Overall, a distinct ecotoxicological hazard of PS-NPs at environmentally relevant concentrations was thoroughly characterized on two piscine cell lines. The effects were demonstrated to depend on size, exposure time and model, emphasizing the need for a comparative evaluation of endpoints between freshwater and marine ecosystems.

Antibacterial and anticancer activity of two NK-lysin-derived peptides from the Antarctic teleost Trematomus bernacchii.

The NK-lysin antimicrobial peptide, first identified in mammals, possesses both antibacterial and cytotoxic activity against cancer cell lines. Homologue peptides isolated from different fish species have been examined for their functional characteristics in the last few years. In this study, a NK-lysin transcript was identified in silico from the head kidney transcriptome of the Antarctic teleost Trematomus bernacchii. The corresponding amino acid sequence, slightly longer than NK-lysins of other fish species, contains six cysteine residues that in mammalian counterparts form three disulphide bridges. Real time-PCR analysis indicated its predominant expression in T. bernacchii immune-related organs and tissues, with greatest mRNA abundance detected in gills and spleen. Instead of focusing on the full T. bernacchii derived NK-lysin mature molecule, we selected a 27 amino acid residue peptide (named NKL-WT), corresponding to the potent antibiotic NK-2 sequence found in human NK-lysin. Moreover, we designed a mutant peptide (named NKL-MUT) in which two alanine residues substitute the two cysteines found in the NKL-WT. The two peptides were obtained by solid phase organic synthesis to investigate their functional features. NKL-WT and NKL-MUT displayed antibacterial activity against the human pathogenic bacterium Enterococcus faecalis and the ESKAPE pathogen Acinetobacter baumannii, respectively. Moreover, at the determined Minimum Inhibitory Concentration and Minimum Bactericidal Concentration values against these pathogens, both peptides showed high selectivity as they did not exhibit any haemolytic activity on erythrocytes or cytotoxic activity against mammalian primary cell lines. Finally, the NKL-MUT selectively triggers the killing of the melanoma cell line B16F10 by means of a pro-apoptotic pathway at a concentration range in which no effects were found in normal mammalian cell lines. In conclusion, the two peptides could be considered as promising candidates in the fight against antibiotic resistance and tumour proliferative action, and also be used as innovative adjuvants, either to decrease chemotherapy side effects or to enhance anticancer drug activity.

The cell biology of the thyroid-disrupting mechanism of dichlorodiphenyltrichloroethane (DDT).

OBJECTIVE: Dichlorodiphenyltrichloroethane (DDT) is an organochlorine known for its pesticide properties and for its negative effects on human health. It was banned in most countries for its toxicity to the endocrine system, but due to its persistence at clinically relevant concentrations in both soil and animal tissues, DDT is still linked to several health and social problems. METHODS: We have previously shown that DDT exposure is causally related to the extracellular release of vesicular organelles such as microvesicles and/or exosomes by using immunocytochemistry with gold-tagged antibodies and various fluorescent membrane markers. RESULTS: It is now well recognized that microvesicles and/or exosomes organelles are implicated in cell-to-cell communication, and that they are fundamental elements for transferring proteins, RNA, DNA, lipids and transcriptional factors among cells. In this short review, we discussed the role of extracellular vesicle formation in the thyroid-disrupting mechanism of DDT. In particular, we described how DDT, by dislodging the thyrotropin hormone (TSH) receptor from the raft containing compartments of the cells, prevents its activation and internalization. CONCLUSION: Based on our earlier finding and on the large body of evidence here reviewed, we propose that DDT-induced formation of extracellular vesicles containing the TSH receptor could be directly involved in the development of autoimmune responses against the TSH receptor and that, therefore, their release could lead to the development of the Graves' disease.

Engineered nanoparticles of titanium dioxide (TIO2): Uptake and biological effects in a sea bass cell line.

With the rapid development of nanotechnology there has been a corresponding increase in the application of titanium dioxide nanoparticles (TiO2-NPs) in various consumer and industrial products, consequently their potential health hazards and environmental effects are considered an aspect of great concern. In the present study, in order to assess the impact of TiO2-NPs in the marine environment, the biological effects of TiO2-NPs on a sea bass cell line (DLEC) were investigated. Cells were exposed for 24 h to different concentrations of TiO2-NPs (1, 8, 40, 200 and 1000 µg/ml) or co-exposed with CdCl2 (Cd). The effects of UV light irradiation were also investigated in cells treated with TiO2-NPs and/or Cd. The internalization of TiO2-NPs and the morphological cell modifications induced by the treatments were examined by transmission and scanning electron microscopy, this latter coupled with energy dispersive X-ray spectroscopy (EDS) for particle element detection. In addition, the effects of controlled exposures were studied evaluating the cytotoxicity, the DNA damage and the expression of inflammatory genes. Our study indicates that TiO2-NPs were localized on the cell surface mainly as agglomerates revealed by EDS analysis and that they were uptaken by the cells inducing morphological changes. Photoactivation of TiO2-NPs and/or co-exposure with Cd affects ATP levels and it contributes to induce acute cellular toxicity in DLEC cells dependent on Ti concentration. The inflammatory potential and the DNA damage, this latter displayed through a caspase-3 independent apoptotic process, were also demonstrated. Overall our data suggest that the interaction of TiO2-NPs with marine water contaminants, such as cadmium, and the UV irradiation, may be an additional threat to marine organisms.

Quantitative immunoenzymatic detection of viral encephalopathy and retinopathy virus (betanodavirus) in sea bass Dicentrarchus labrax.

Viral encephalopathy and retinopathy disease caused by betanodavirus, genus of the family Nodaviridae, affects marine, wild and farmed species including sea bass, one of the most important farmed species in Europe. This work describes a reliable and sensitive indirect ELISA assay to detect betanodavirus in biological samples using a polyclonal antiserum (pAb 283) against the 283/I09 virus strain, the most common red-spotted grouper nervous necrosis virus (RGNNV) genotype in the Mediterranean area, and a capture-based ELISA using a monoclonal antibody (mAb 4C3) specific to a common epitope present on the capsid protein. Using adsorbed, purified VERv preparation, the detection limit of indirect ELISA was 2 µg mL(-1) (3 × 10(5) TCID50 per mL), whereas for capture-based ELISA, the sensitivity for the antigen in solution was 17 µg mL(-1) (35 × 10(5) TCID50 per mL). The capture-based ELISA was employed to detect VERv in brain homogenates of in vivo infected sea bass and resulted positive in 22 of 32 samples, some of these with a high viral load estimates (about 1.1 × 10(8)  TCID50 per mL). The ELISA system we propose may be helpful in investigations where coupling of viral content in fish tissues with the presence of circulating VERv-specific IgM is required, or for use in samples where PCR is difficult to perform.

Disruption of the salivary gland muscle in tsetse, Glossina pallidipes Austen, as a result of salivary gland hypertrophy virus infection.

The secretory region of the salivary glands in Glossina pallidipes Austen (Diptera: Glossinidae) is characterized by an external muscle layer. Scanning electron microscopy and transmission electron microscopy investigations provide a detailed description of the longitudinal muscle fibres and a comparison of their structure when affected by salivary gland hypertrophy virus. The virus is responsible for hypertrophy of the salivary glands in symptomatic flies, specifically of the muscle fibres, the cytoarchitecture of which is completely altered. Although observations did not reveal viral particles in the muscle cells of either asymptomatic or symptomatic flies, muscle fibres were enlarged and detached from one another and their associated basement membrane only in symptomatic flies. A decrease in type IV collagen labelling in the basement membrane of the muscles in symptomatic flies is reported and is considered a potential cause of the salivary gland muscle alteration and, possibly, myopathy. The maintenance of an organized muscular layer is essential for the normal secretion of saliva and hence its pathology in symptomatic tsetse flies could affect the normal transmission of the trypanosome that develops inside the salivary gland epithelium. Therefore, a better understanding of the possible role of the virus is essential in order to elucidate its impact on salivary deployment in symptomatic flies.

Injectable microcarriers as human mesenchymal stem cell support and their application for cartilage and degenerated intervertebral disc repair.

Degeneration of the intervertebral disc (IVD) is a progressive and chronic process, and the high incidence of discogenic disorders calls for new therapeutic approaches, such as cell-based therapies using three dimensional cultures and mesenchymal stem cells (MSC), which can differentiate to chondrogenic- and IVD-lineages. Here, we investigated the growth and differentiation of human MSC culture on biodegradable collagen scaffolds in order to obtain an injectable suspension. Commercially available wound dressings were downsized to dimensions between 100 and 1500 µm and seeded with freshly isolated or early passages MSC. Proliferation rate and chondrogenic differentiation potential was tested at oxygenation levels of 2%, 5%, 10% and 21% in static and dynamic cultures. Evaluation methods included cell viability test, disc marker genes expression (aggrecan, collagen type I and type II), histological detection of proteoglycans and immunohistochemical analysis. On microcarriers, freshly isolated MSC had lower proliferation rate and chondrogenic differentiation potential compared with early passages MSC. Proliferation of MSC was significantly increased 1.7-fold at 5% oxygen level and in combination with dynamic culture was further increased to 2.3-fold, with respect to normoxia. Chondrogenesis was positively affected by 2% and 5% hypoxia, as shown by increased transcription levels and protein expression of collagen type II and proteoglycan accumulation in static cultures, while it was inhibited in dynamic cultures. Collagen type I and aggrecan expression were not affected by hypoxia. In conclusion, collagen based microcarriers are a suitable support for in vitro MSC growth and chondrogenesis especially when cultured at 5% oxygen level.

Immune modulatory effects of Aloe arborescens extract on the piscine SAF-1 cell line.

The pharmacological potential of Aloe arborescens Miller leaf components was investigated, with special attention deserved to immune modulatory effects on the Sparus aurata fibroblast cell line SAF-1. The cells were treated with Aloe extract at different concentrations (1.2-4.8 mg ml(-1)) for various times (24-72 h). The lowest concentration did not provoke any cellular damage observable by SEM and did not affect ATP amounts after 24 and 48 h, while even induced a significant increase over controls after 72 h. We next examined the transcription kinetics of different immune-related genes (IL-1ß, TGF-ß, TNF-α, COX-2, IFN-I, Mx and MHCI-α) in SAF-1 cells stimulated with LPS or poly I:C. The Aloe extract (1.2 mg ml(-1)) acted as a powerful immune stimulant in LPS- or poly I:C-activated SAF-1 cells, inducing a synergic effect on interconnected genes that are expected to be involved in different aspects of the immune responses. These reports provide a new perspective for the use of A. arborescens to prevent or oppose bacterial and viral fish diseases and to face, as an alternative strategy based on natural plant extracts, the growing unwillingness to rely upon standard solutions involving antibiotics or antimicrobial chemicals.

Binding, uptake, and degradation of internalized thyroglobulin in cultured thyroid and non-thyroid cells.

Thyroid hormone release requires degradation of thyroglobulin (Tg) by thyroid epithelial cells, which occurs mainly in the lysosomal pathway following Tg endocytosis. Non-specific fluid-phase endocytosis is thought to be the main route of Tg uptake leading to degradation, whereas receptor- mediated endocytosis is believed to lead to post-endocytic pathways other than degradation. To gain more insights into these issues, we investigated handling of Tg by various cell types. Tg bound similarly to thyroid (FRTL-5, FRT) and non-thyroid (COS-7, IRPT) cells, indicating the presence of membrane-binding sites, presumably receptors, in both cell types. Tg was internalized and degraded by all cells and degradation paralleled uptake, with the exception of FRTL- 5 cells, in which a lower proportion of Tg was degraded, suggesting that in FRTL-5 cells mechanisms that target Tg to the various post-endocytic pathways (either receptors or postreceptorial factors) are differently represented. Immunoelectronmicroscopy showed a common path of endocytosis in FRTL-5, COS-7, and IRPT cells, namely the formation of pseudopods engulfing Tg, followed by internalization and accumulation of Tg in cytoplasmic vesicles and lysosomes. The fastest rate was observed in COS-7 cells, probably reflecting a lower impact of endocytic receptors. Our findings suggest that Tg uptake and degradation are not thyroid-specific, that Tg binding sites exist in different cell types, and that uptake and/or degradation are differently regulated in differentiated thyroid cells, presumably because of a different impact of endocytic receptors or post-endocytic mechanisms, which are probably responsible for the regulation of hormone release.

Thyroid disruptor 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) prevents internalization of TSH receptor.

The thyroid-stimulating hormone (TSH) receptor (TSHr) was made specifically fluorescent by insertion of a tetracysteine motif (TSHr-FlAsH) into the C-terminal end and transiently transfected into COS-7 and HeLa cells. The observation that TSH administration caused the intracellular level of cAMP to increase in both TSHr-FlAsH-transfected cell types indicated that the FlAsH binding motif did not alter normal TSHr functioning. When transfected into HeLa cells and stimulated with TSH, the TSHr-FlAsH receptor exhibited a pronounced perinuclear labelling pattern, whereas labelling remained on the cell surface following pre-incubation with 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT). Chinese hamster ovary (CHO)-TSHr cells probed with anti-TSHr antibodies were fluorescent mainly in the proximity of the plasma membrane, with fluorescence being primarily restricted to a juxta-nuclear position when exposed to 10 mU/ml TSH for 1 or 5 min. However, in the presence of DDT, the anti-TSHr fluorescence maintained a peripheral location along the cell plasma membrane, even if CHO-TSHr cells were stimulated with TSH for 1 and 5 min. To verify that DDT acted specifically on the TSHr, CHO cells transfected with the A(2)a receptor were used as controls. Following a 1-min stimulation with 5'-(N-ethyl-carboxamido)-adenosine, A(2)a receptors were gradually internalized regardless of the presence of DDT in the culture medium. Finally, immunoelectron microscopy of CHO-TSHr cells showed that a 1-min exposure to TSH sufficed to displace anti-TSHr antibodies tagged with 10-nm gold particles into coated pits and vesicles but that their superficial location was retained along the plasma membrane in the presence of DDT.

The cell wall of kiwifruit pollen tubes is a target for chromium toxicity: alterations to morphology, callose pattern and arabinogalactan protein distribution.

Trivalent chromium has previously been found to effectively inhibit kiwifruit pollen tube emergence and elongation in vitro. In the present study, a photometric measure of increases in tube wall production during germination showed that 25 and 50 mum CrCl(3) treatment induced a substantial reduction in levels of polysaccharides in walls over those in controls. Moreover, chromium-treated kiwifruit pollen tubes had irregular and indented cell walls. Callose, the major tube wall polysaccharide, was deposited in an anomalous punctuate pattern. Arabinogalactan proteins (AGPs), which are integral in maintaining correct tube growth and shape in kiwifruit pollen, were found to be strongly altered in their distribution after CrCl(3) treatment compared to control tube walls. Transmission electron microscopy-immunogold analysis using four monoclonal antibodies (JIM8, JIM13, JIM14 and MAC207) revealed discontinuous AGP distribution within the treated tube walls. Such clearly discernable alterations in the molecular and morphological architecture of pollen tube walls may be detrimental in vivo for the male gametophyte to accomplish its vital role in the fertilisation process.

In vitro toxicity towards kiwifruit pollen of the antimicrobial peptides magainins 1 and 2.

In vitro toxicity of the antimicrobial peptides (AMPs) magainin 1 and 2 to a higher plant organism, i.e., the bicellular male gametophyte of Actinidia Deliciosa (kiwifruit), is investigated. Heavy damage to the plasma membrane, the primary cellular target of the peptides, was rapidly induced: in as few as 15 min, from 70 to nearly 100 % of pollen grains were rendered unviable by 20 microM magainin 1 or 2, respectively. Therefore, kiwifruit pollen sensitivity to natural magainins seemed to be higher if compared to the sensitivity of other pollen species towards magainin 2 amide or synthetic magainin analogues. Strong dose-dependent inhibitory effects on kiwifruit pollen performance were registered: as for magainin 1, the EC (50) at 120 min varied from 14.0 (germination) to 15.8 microM (tube elongation). The inhibitory effect was much greater when administering magainin 1 to elongating tubes rather than to ungerminated pollen grains. The two peptides differentially affected kiwifruit pollen, in line with the previously documented greater activity of magainin 2 in other cell systems. Furthermore, 20 microM magainin 1-treated pollen grains took on a shrivelled shape within 30 min of incubation, an increasingly widespread effect with higher peptide concentration. At the ultrastructural level, both protoplast shrinkage and striking organelle alterations were evident, including chromatin condensation, swelling and loss of mitochondrial cristae, dilation of rough endoplasmic reticulum cisternae, and vacuolization of cytoplasm. To our knowledge, similar alterations in animal or plant cells treated with AMPs have not been described yet.

Viral particles of the endogenous retrovirus ZAM from Drosophila melanogaster use a pre-existing endosome/exosome pathway for transfer to the oocyte.

BACKGROUND: Retroviruses have evolved various mechanisms to optimize their transfer to new target cells via late endosomes. Here, we analyzed the transfer of ZAM, a retroelement from Drosophila melanogaster, from ovarian follicle cells to the oocyte at stage 9-10 of oogenesis, when an active yolk transfer is occurring between these two cell types. RESULTS: Combining genetic and microscopic approaches, we show that a functional secretory apparatus is required to tether ZAM to endosomal vesicles and to direct its transport to the apical side of follicle cells. There, ZAM egress requires an intact follicular epithelium communicating with the oocyte. When gap junctions are inhibited or yolk receptors mutated, ZAM particles fail to sort out the follicle cells. CONCLUSION: Overall, our results indicate that retrotransposons do not exclusively perform intracellular replication cycles but may usurp exosomal/endosomal traffic to be routed from one cell to another.

A pneumococcal pilus influences virulence and host inflammatory responses.

Streptococcus pneumoniae (pneumococcus) is a major cause of morbidity and mortality world-wide. The initial event in invasive pneumococcal disease is the attachment of encapsulated pneumococci to epithelial cells in the upper respiratory tract. This work provides evidence that initial bacterial adhesion and subsequent ability to cause invasive disease is enhanced by pili, long organelles able to extend beyond the polysaccharide capsule, previously unknown to exist in pneumococci. These adhesive pili-like appendages are encoded by the pneumococcal rlrA islet, present in some, but not all, clinical isolates. Introduction of the rlrA islet into an encapsulated rlrA-negative isolate allowed pilus expression, enhanced adherence to lung epithelial cells, and provided a competitive advantage upon mixed intranasal challenge of mice. Furthermore, a pilus-expressing rlrA islet-positive clinical isolate was more virulent than a nonpiliated deletion mutant, and it out-competed the mutant in murine models of colonization, pneumonia, and bacteremia. Additionally, piliated pneumococci evoked a higher TNF response during systemic infection, compared with nonpiliated derivatives, suggesting that pneumococcal pili not only contribute to adherence and virulence but also stimulate the host inflammatory response.

Formation of the egg envelope of a teleost, Dicentrarchus labrax (L.): immunochemical and cytochemical detection of multiple components.

The formation of the egg envelope in a teleost, Dicentrarchus labrax (L.), was analysed at histological and ultrastructural level. The sequential deposition of three main layers (Z1, Z2 and Z3) constitutes the extracellular matrix throughout oocyte development. Various findings indicate that these subunits are biochemically distinct: (1) periodate- and phosphotungstic acid-reactive carbohydrates are obviously detected only in the Z1, that constitutes the initial deposit of the egg envelope in early lipidic oocytes; (2) a monoclonal antibody (DLE7) against egg envelope polypeptides did not immunostain the Z1 and the underlying Z2; (3) the antigenic determinants recognised by DLE7, thought to be exogenous in origin (synthesised in the liver), are incorporated in the inner layer (Z3). In addition, DLE7 immunostained a thin layer, assembled together with Z3. This line has not yet been described in teleost eggs and was named Z1a. This study first describes at fine cytological level the contribution of exogenous proteins to formation of the different egg envelope layers. Results obtained with conventional, immunochemical and cytochemical techniques suggest multiple synthetic sources (exogenous and follicular) of egg envelope proteins.

Is cryopreservation a homogeneous process? Ultrastructure and motility of untreated, prefreezing, and postthawed spermatozoa of Diplodus puntazzo (Cetti).

This study subdivides the cryopreservation procedure for Diplodus puntazzo spermatozoa into three key phases, fresh, prefreezing (samples equilibrated in cryosolutions), and postthawed stages, and examines the ultrastructural anomalies and motility profiles of spermatozoa in each stage, with different cryodiluents. Two simple cryosolutions were evaluated: 0.17 M sodium chloride containing a final concentration of 15% dimethyl sulfoxide (Me(2)SO) (cryosolution A) and 0.1 M sodium citrate containing a final concentration of 10% Me(2)SO (cryosolution B). Ultrastructural anomalies of the plasmatic and nuclear membranes of the sperm head were common and the severity of the cryoinjury differed significantly between the pre- and the postfreezing phases and between the two cryosolutions. In spermatozoa diluted with cryosolution A, during the prefreezing phase, the plasmalemma of 61% of the cells was absent or damaged compared with 24% in the fresh sample (P < 0.001). In spermatozoa diluted with cryosolution B, there was a pronounced increase in the number of cells lacking the head plasmatic membrane from the prefreezing to the postthawed stages (from 32 to 52%, P < 0.01). In both cryosolutions, damages to nuclear membrane were significantly higher after freezing (cryosolution A: 8 to 23%, P < 0.01; cryosolution B: 5 to 38%, P < 0.001). With cryosolution A, the after-activation motility profile confirmed a consistent drop from fresh at the prefreezing stage, whereas freezing and thawing did not affect the motility much further and 50% of the cells were immotile by 60-90 s after activation. With cryosolution B, only the postthawing stage showed a sharp drop of motility profile. This study suggests that the different phases of the cryoprocess should be investigated to better understand the process of sperm damage.

Sperm quality and reproductive traits in male offspring of female rabbits exposed to lindane (gamma-HCH) during pregnancy and lactation.

Fifteen Grimaud female hybrid rabbits, 135 days old and weighting an average of 3.74+/-0.01 kg each, were administered an oral dose of 1 mg x kg(-1) body weight of Lindane during gestation and lactation period. Fertility rate, libido, volume of ejaculate, concentration and morphology of spermatozoa were investigated to test the effects of the treatment on reproductive traits of first generation male rabbits. Ultrastructure of abnormal spermatozoa was described by Transmission Electron Microscopy and the different abnormalities were quantified. The results obtained indicate that low dose exposure of Lindane has effects on spermatozoa ultrastructure that proved to be susceptible to the treatment with the pesticide (cytoplasmic droplets: 5.3% in control group and 10.3% in Lindane group, P < or = 0.05; coiled tails: 1.3% in control group and 4.3% in Lindane group, P < or = 0.05) and could be utilised as a good marker of toxicity.

Herbicides and the microtubular apparatus of Nicotiana tabacum pollen tube: immunofluorescence and immunogold labelling studies.

Herbicides are chemical compounds widely used in agriculture. As their intensive application is becoming a cause of environmental pollution, detailed and more sophisticated investigations are needed to understand better their consequences at the biological level. After herbicides are dispersed in the fields, they establish chemical interactions with both target and non-target plants. In both cases, herbicides can interact with the plant reproductive apparatus; consequently they could play a role during the fertilisation process in higher plants. Using an antibody to the alpha-tubulin subunit in immunofluorescence and immunoelectron microscopy techniques, we investigated the distribution of microtubules in Nicotiana tabacum pollen tubes grown under in vitro conditions in the presence of five different herbicides selected among those used frequently in central Italy. Herbicides have a specific effect on the microtubular apparatus of both pollen tube and generative cell. In addition to other tests and assays, these results suggest that the microtubule cytoskeleton of pollen tubes can be used as a bioindicator for studying the toxicity effects induced by herbicides.

Ultrastructure of the seminal vesicle of Phlebotomus perniciosus Newstead (Diptera, Psychodidae).

The seminal vesicles of Phlebotomus perniciosus were investigated by light microscopy, confocal scanning laser microscopy and by scanning and transmission electron microscopy. They have a complex structure, and three different morphological compartments called A, B and C are distinguished on the basis of their position and fine structure. Compartment A is continuous with the vasa deferentia and consists of a cylindrical wall limiting a lumen in which the spermatozoa are stored. Compartment B is hemispherical and surrounds compartment A like a muff. Compartment C constitutes an external coat surrounding A and B. The epithelial cells of each compartment are characterized by morphologically different secretory granules. The ultrastructural features of these cells are described and their role in sandfly reproductive biology is discussed.