Vasculogenic erectile dysfunction (ED) is an age-related disease partially dependent on vascular growth factors availability and their specific endothelium receptors activation. Among these, angiopoietins-tyrosine kinase with immunoglobulin and EGF homology domain-2 (Tie2) system seems to be particularly dependent on aging, both in rat and in human corpus cavernosum (CC). Angiopoietins (Ang)1 and 2 are produced by pericytes and endothelial cells respectively, and bind with the same affinity to Tie2 receptor. Ang1 was shown to act as an obligatory agonist inducing Tie2 phosphorylation, which promotes cell-cell and cell-extracellular matrix interactions. On the other hand, Ang2 acts in a context-dependent fashion in the increment or, otherwise, destabilization of pre-existent vasculature.

Interestingly, it has been demonstrated that activation of Tie2 receptor could also be modulated by Tie1, an additional member of the Tie family of receptor tyrosine kinases, and that co-expression of Tie2 is required for Tie1 activation. Indeed, Tie1 similarly to Tie2 is involved in the maintenance of the vascular integrity. Nevertheless, its role in Angiopoietins-Tie system regulation remains poorly understood, and the identification of a true ligand has remained elusive. In the present study, we aim to demonstrate the expression of Tie1 in the endothelium of aged CC in the Rat and Human species.

Twenty-five male Wistar rats were divided in five groups (n=5) and sacrificed by decapitation when they reached the ages of study (6, 12, 18, 24 and 36 months). The penises were excised and immediately fixed in formalin solution. Human CC fragments were obtained from organ donors without known risk factors for ED and divided in two groups: young (16–35 years) and aged (59–74 years). Paraffin embedded sections were deparaffinized, rehydrated and submitted to epitope retrieval with heated Tris-EDTA pH 9.0 buffer before dual-immunolabeling of Tie1 (rabbit anti-Tie1 antibody -abcam) and specific markers of endothelium and smooth muscle cell (goat- anti-PECAM-1 and mouse anti-m-actin antibodies, Santa Cruz Biotechnology Inc. and Millipore, respectively). Appropriate secondary antibodies were employed (anti-rabbit labelled with Alexa 488 with anti-mouse, or, anti –goat labelled with Alexa 568). Nuclei counterstaining was achieved with 4´-6-diamino-2-phenylindole. Images were observed in an Apotome microscope (Carl Zeiss System, Göttingen, Germany) using the filter sets, 01 to DAPI, 09 to Alexa 488 and 31 to Alexa 568, and acquired with Axio Vision 3.0 program (Carl Zeiss System).

Tie1 was detected in the endothelium in the CC of Rat, co-localizing with PECAM-1 (Figure 1a) but not with w-actin (Figure 1b). The intensity of Tie1 immunolabeling was higher in the older animals (24 and 36 months). Tie1 was also detected in Human CC endothelium, suggesting an up-regulation in samples obtained from older individuals (Figure 2a and 2b).

The present results demonstrate for the first time the expression of Tie1 in the endothelium of CC in Rat and Human, which appears to exhibit an age-dependent up-regulation. Ongoing research employing molecular studies will clarify this aspect.

Paleopathology, summarily defined as the study of past diseases, has on the differential diagnosis a major challenge. Histological techniques offered the possibility to look inside the microstructure of both normal and abnormal body tissues to diagnose diseases that affected past populations, leading to the development of a new field of research - paleohistology or paleohistopathology. However, and contrary to paleopathology whose journey is well-established, in paleohistopathology there are many gaps that need to be filled. This occurrence is probably the result of a nonsystematic and non-standardized approach to the microscopic study of skeletal abnormalities, especially those of infectious origin involving periosteal new bone formation (PNBF). The aims of this work were: (1) to search for differences in the microstructure of PNBF with regard to the cause of death of the individual; (2) to infer differences between the macroscopic and microscopic proprieties of bone lesions, and (3) to ascertain the impact of diagenetic changes in the bone microstructure. For histological examination under transmitted and polarized light, a total of 34 dry bone specimens: 26 belonging to 23 individuals from the Human Identified Skeletal Collection from the Bocage Museum (Lisbon, Portugal), and eight from archaeological skeletons were prepared. The documented bone samples were collected from individuals who died from tuberculosis-TB (Group 1), non-TB infectious diseases (Group 2), and conditions other than those of TB and non-TB infectious origin (Group 3).

With regard to the diagnosis of pathological conditions, differences in the microstructure of PNBF were seen between Group 1 and Group 2 of cause of death and within groups. Multiple layers of “appositional bone” enclosing numerous primary vascular canals were the pattern most commonly observed (n=4) in the samples from Group 1. This type of PNBF seems to mimic the appositional growth that characterizes the modeling process of the periosteal and endosteal membranes (PEM) during rapid growth periods. An abnormal stimulation of growth factors, especially of the vascular endothelial growth factor (VEGF) may eventually explain the extensive hypervascularization observed (Fig. 1 A-B). Periosteal lesions on ribs are normally associated with pulmonary infection (e.g. TB) disseminated from the lungs (via pleura) to the ribs. However, three samples (one from Group 1, two from Group 2) presented a microstructure compatible with subperiosteal hematomas (Fig. 1 C-D). Repetitive microtrauma (e.g. chest wall vibration) that causes detachment of the periosteum may have leaded to subperiosteal bleeding and hematoma formation. These observations suggest that beyond pulmonary diseases other mechanisms may stimulate PNBF on the visceral surface of ribs. Histological analysis was also fundamental in the description and characterization of bone changes. For example, of the five samples with “consolidated” fracture callus, only two presented a truly mature and remodeled microstructure. This means that the outer surface of a bone lesion may not give a complete picture of the tissues response (Fig. 2 A-B). In spite of the good preservation of some bone samples, massive diagenetic changes due to the action of bacteria and fungi were observed at microscopic level. This clearly suggests that gross inspection is not a good measure of the bone tissue quality. In contrast, microscopy is essential to differentiate between pseudopathology and physiological or pathological signs.

Microscopy revealed surprising results that reinforce the pertinence of applying histological techniques in the description and diagnosis of bone changes in human remains.

This research was supported by Fundação para a Ciência e Tecnologia, Portugal [SFRH/BD/36739/2007, Sandra Assis].

Membranoproliferative glomerulonephritis (MPGN) encompasses 7 to 10% of all biopsied glomerulonephritis. They are divided in: MPGN type I; MPGN type II and MPGN type III, being primary or secondary. MPGN type I are the most frequent, MPGN types II and III are very rare and difficult to diagnose without clinical and morphologic findings integration. MPGN type II or Dense Deposit Disease has a varied morphologic appearance with a few numbers of cases showing a membranoproliferative pattern by Light microscopy (LM). Electron microscopy (EM) is pivotal to confirm the diagnosis.

We present a case of 35 years old man, with nephrotic proteinuria and mild renal insufficiency since 2 years. The only relevant clinical data is facial lipodystrophy. Complement 3 (C3) was low and C3 nephritic factor negative. There were not other relevant abnormalities. Renal biopsy was fixed in buffered formaldehyde 10% and performed for LM. The frozen fragment, prepared for observation by fluorescence microscopy - immunofluorescence (IMF) -, was prepared to be stained with florescent anti-serums, against immunoglobulines (IgG, IgA and IgM) and complement factors (C3, C4, and C1q). EM was later done on tissue formaldehyde fixed reprocessed from paraffin-embebbed for LM, because there was no tissue fragment fixed in glutaraldehyde.

LM showed variable endocapillary hypercelullarity, with neutrophils infiltration. Capillary walls were thickened due to the deposition of elongate and ribbon-like deposits. Few double contours were visible (Figure 1a). IMF demonstrated the presence of C3 deposits in the capillary walls and mesangium (Figure 1b). EM confirmed the presence of an intramembranous dense deposit along basement membrane which was thickened (Figure 1c). LM and IMF findings favored the diagnosis of MPGN type II with C3 deposits and thickening of basement membrane. Nevertheless EM was essential to confirm intramembranous unequivocally dense deposits.

MPGN type II is a rare glomerulonephritis mediated by complement deregulation. The integration of clinical and morphologic findings is essential to get a correct diagnosis. In this setting EM is highly distinctive and required for a definitive diagnosis.

Hypericum L, the largest genus of Hypericaceae comprising ca. 484 species of shrubs and perennial and annual herbs, is worldwide in a large variety of habitats in subtropical and temperate areas. Hypericum species, namely H. perforatum (St. John’s wort) the most representative species of the genus, have been used in folk medicine thought the centuries for a large number of ailments. Nowadays, it is well known the therapeutic potentialities of their main compounds, hypericin, pseudohypericin and hyperforin, which justify its clinical use. Despite the intense phytochemical and pharmacological research conducted in Hypericum species during the last decades, morpho-anatomical studies on the glands that produce the bioactive compounds are scarce and fragmented, only H. perforatum was studied in detail. As part as an ongoing project on Hypericum glands, the present research aims to provide information about the morphology, anatomy and histochemistry of the secretory structures present on the aerial organs of H. perfoliatum, one of the seventeen species of Hypericum that occur wild in Portugal.

The types of glandular structures and their pattern of distribution on the leaves and flowers were studied by light (MO) and scanning electron microscopy (SEM). Samples for SEM were fixed with glutaraldehyde, dehydrated in a graded acetone series, critical-point dried and coated with gold. For general anatomy samples were fixed in the same fixative and embedded in Leica historesin®. Histochemical tests and standard control procedures were carried out in fresh material to localize in situ the main chemical classes of compounds present in the secretion. Observations were carried out Observations were carried out on a JEOL T220 scanning electron microscope and with a Leica DM-2500 microscope.

The aerial organs of H. perfoliatum present four different types of secretory structures (idioblasts, translucent glands, ducts and black nodules), that can occur exclusively in a specific organ or in more than one organ. Tanniniferous secretory cells are frequent in the epidermis, as well as in the ground parenchyma of all organs, where they are scattered together with crystal idioblasts containing druses of calcium oxalate. Translucent glands are spheroidal subepidermical glandular pockets delimited by two or three cell layers of fattened and densely-stained cells (Fig. 1A). They are typically found in the leaves, giving them a perforated appearance. Two types of secretory ducts, cavities that differ from translucent glands in the length, are present in the vegetative and floral organs. Type A ducts have a narrow lumen delimited by four secretory epithelial cells and occur associated to the phloem in all aerial organs with exception of stamens (Fig. 1B, arrow). Type B ducts have a wider lumen, are generally limited by ten thin-walled secretory cells surrounding by a sheath of thick-walled cells and are located in the parenchyma of sepals, petals and ovary. Black nodules are clusters of cells lacking a central intercellular space (lumen), surrounded by one or two-layers of flat cells of a delimiting sheath (Fig. 1C). The inner cells are large, irregular, tightly packed and filled with a dark red stained content. Spheroidal black nodules are found punctuating the leaf margins and in the connective tissue of the stamen (Fig. 1D), whereas long-shaped black nodules are distributed across the lamina of bracts, sepals and petals. Peculiar glandular emergences, which look like peduncular black nodules, are present along the margin of the bracts and sepals. They consist of a multicellular peduncle and a dark-red multicellular secretory head-a black nodule (Fig. 1E). Histochemical tests showed that translucent glands secreted essential oils rich in phenolic compounds (flavonolic aglycones), ducts produce oleoresins and nodules contain essentially hypericin. In mature organs, the disorganization of the inner cells of the nodules seems to form a large intercellular space, a lumen.

All these secretory structures were also found in H. perforatum with exception of peduncular black nodules, that was only reported in H. elodes, but not studied in detail. The obtained results allow as speculating that nodules may be primitive multicellular structures, relics of an evolutionary process, that give rise to cavities, internal secretory structures that stores secretion material in intercellular spaces.

The authors acknowledge the funding by Fundação para a Ciência e Tecnologia through the project FCT PEst-OE/EQB/LA0023/2011.

Cr(III)-tris(picolinate), [Cr(pic)3], is a very common dietary supplement, recommended for humans, cattle and swine. Chromium is considered an essential trace element, when in oxidation state +3, with some of its compounds seeming to have a beneficial effect on blood sugar regulation mechanisms. However, the safety of the use of a particularly popular Cr(III) compound, ie [Cr(pic)3], remains debatable. Clastogenic, and mutagenic features have been reported by Stearns and co-workers, although surrounded by a controversial and contradictory multitude of publications on this subject. The present work aims to study the effects of [Cr(pic)3] on mice spermatogenesis.

Cr(III)-tris(picolinate) was synthesized and characterized according to the literature. Its composition as a mononuclear complex was tested by ESI-MS and by X-ray powder diffraction followed by single-crystal simulation calculations.

Male adult CDI mice from Harlan (Spain) were divided in groups and orally given 25 mg/kg and 50 mg/kg/body weigh/daily of [Cr(pic)3] for two weeks. Controls were also done. Behaviour and body weight were monitored throughout the experiments. After sacrifice, testis were collected, weighed, and fixed in Bouin´s solution. Organs were then prepared for histology using routine techniques. Animal experiments were conducted according to ethics procedures. Histological sections of control group evidenced normal regular features (Fig. 1a). However considerable damage was observed in both experimental groups in a dose dependent manner. In fact, seminiferous tubules showed degenerative changes within epithelium, namely vacuolation and sloughing of immature germ cells into the lumen in the group given the lowest dose (Fig.1 b,c). The high dosed group displayed more conspicuous injury within testis, namely strongly atrophic seminiferous tubules devoid of germs cells and strong vacuolation (Figs.1d-f).

The results of this study have shown an increased risk of adverse events in mice receiving 50 mg/kg/body weight of [Cr(pic)3]. However, little potential for adverse reproductive and developmental effects namely on progeny was recently described for male mice fed a diet containing 200 mg/kg/day [Cr(pic)3]. In conclusion, concerns about using dietary supplements based on [Cr(pic)3] remain to be elucidated in future work.

This work was financed by CICECO, Aveiro University, Portugal.

Plasmodium falciparum causes the most severe/lethal form of malaria, a parasitic infection that affects 500 million people worldwide and leads to the death of nearly one million/year, about 91% being due to P. falciparum. The malaria pigment or hemozoin crystals (HZ) are formed in an enzyme-independent polymerization of heme released during haemoglobin digestion by the parasite during the intra-erythrocytic life cycle. The quinoline and artemisinin based antimalarials (the only available treatment options) appear to act by disrupting the formation of HZ. Therefore, the development of antimalarial agents based on the physicochemical process of heme crystallization appears a good option to drug design.

We have produced β-haematin following an assay adapted from the method described by Basilico et al, with modifications. Briefly, hemin (ferriprotoporphyrin IX chloride; Sigma-Aldrich) was dissolved NaOH and polymerized by the addition of acetic acid and incubation at 37°C. Resulting pellet washed with dimethyl sulfoxide (DMSO) to remove unpolymerized hemin and resuspended in H2O. To extract native HZ (nHZ) from P. falciparum (clone Dd2)-infected erythrocytes, when parasitemia reached >10%, parasites were harvested by saponin lysis, washed with phosphate-buffered saline (PBS) and sonicated in 2% sodium dodecyl sulfate (SDS), further washed in 2% SDS and pellet was resuspended (10 mM Tris-HCl (pH 8.0); 0.5% SDS; 2mg/ml of proteinase K) and incubated at 37°C overnight. The nHZ pellet was washed in 2% SDS and in distilled H2O, then resuspended in distilled H2O water and sonicated again prior to use to minimize aggregation and maintain the nHZ in suspension. β-haematin and native nHZ crystals were analysed by Scanning Electron Microscopy (JEOL 7001FSEM) (Fig.1; A, B.1 and C.1) and Transmission Electron Microscopy (Hitachi H8100) (Fig.1; B.2 and C.2).

Here we report the crystal morphology of nHZ produced by the Plasmodium falciparum clone Dd2 and of β-haematin produced with the above protocol. Chemically, HZ is a stable dimer of iron(III)(protoporphyrin[PP]-IX), also known as β-hematin and consists of heme units dimerized through reciprocal iron-carboxylate bonds. Even though the parasite nHZ and synthetic β-hematin are isostructural, there are large differences in the crystal morphologies of nHZ (Dd2) and β-hematin as evidenced by SEM and TEM images (Fig.1). nHZ crystals are remarkably uniform in size and shape, adopting a parallelipede morphology with high aspect ratio of about 800x200x200 nm (Fig. 1 C1 and C2). Different protocols to prepare β-haematin yield diverse material, which can be poorly crystalline and heterogeneous within the same sample. The present results show that β-haematin is rather homogeneous and constituted by needle-like particles of sizes similar to those presented by nHZ ones (compare B1 with C1). Higher resolution observations evidence differences between the β-haematin and nHZ materials, since the latter seems constituted by uniform flat layers and the former shows flaked ones (Fig. B2).

Morphology seems to be of biological relevance, since structure-function studies revealed that the size and shape of the synthetic crystals influence their ability to activate, for instance, the inflammatory responses in vitro and in vivo. Several protocols have been developed to measure drug inhibition of heme crystallization in vitro although it has been difficult to standardize these assays. In the present study, we report a new method for β-haematin preparation that renders homogenous crystals, both in shape and size, favourable for reproducible drug inhibition of heme crystallization assays.

This work was supported by CMDT through PEst-OE/SAU/LA0018/2011 grant and FCT through PTDC/SAU-FAR/114864/2009 and PEst-OE/CTM-UI0084/2011 grants.

Nanoparticles (NPs), particles with at least one dimension less than 100 nm, are used in many industrial applications and to produce new types of materials with unique physicochemical properties. The aquatic environment is commonly the ultimate recipient for NPs and there is uncertainty of exposure as understanding and data regarding the potential detrimental effects of NPs on aquatic biota are missing. In this study, titanium dioxide (TiO2) was chosen for its potential use in technology and diverse industrial applications. The objective of this work is to evaluate the toxicity of TiO2 NPs on total liver glutathione-S-transferase (GST), lipid peroxidation and tissue structure of the livers of two freshwater fish species (Carassius auratus and Danio rerio).

Stock suspensions of TiO2 NPs, with an average size of 21 nm, were prepared using distilled water and then ultrasonicated (10 min, 35 KHz). The suspensions were added to 10L of tap water in exposure tanks, to obtain nominal concentrations (0.01; 0.1; 1, 10; 100 TiO2 mg/L). The test fish, C. auratus (N=144) and D. rerio (N=80), were randomly distributed by 6 exposure tanks and an additional tank with clean tap water was used as control. Fish were sampled after 7, 14, and 21 days. Six fish from both species were left for depuration in clean tap water during 14 days and then sacrificed. Immediately after sampling the fish were processed for enzymatic determination and histopathology. The GST activity was determined by following the procedure described by Habig et al. and lipid peroxidation was measured based on the Thiobarbituric Acid Reactive Species method. The tissues were processed essentially according to Martoja and Martoja for light microscopy (LM). For transmission electron microscopy (TEM) the samples were fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate, dehydrated in ethanol and embedded in Epon-Araldite according to standard procedures. The histological and ultra-structural observations were carried out using a Leica microscope (Leica-ATC 2000) and a JEOL 100-SX electron microscope respectively.

The results showed increased activities of the GST in livers with increasing TiO2 NP concentrations after 7 days of exposure, however after 14 days a trend to decrease was observed for both species. The GST results suggest that the increase of activity of these detoxification enzymes can be a response to oxidative stress caused by the generation of reactive oxygen species by the NP. On the other hand, size, chemical composition, surface area, shape, solubility and aggregation may also contribute for NPs toxicity. The results from lipid peroxidation showed an increase according to tested concentrations suggesting that TiO2 NPs is able to cause cell damage and is in agreement with biochemical and histological findings. After 14 days of depuration, GST and lipid peroxidation levels were not significant different from controls suggesting that cells are able to recover in a certain degree. The results from LM (Fig. 1) showed that exposure to TiO2 NPs affected liver structure, with more pronounced changes detected in livers from fish exposed to higher concentrations. Observed changes include tissue degeneration, inflammation and pyknosis among others. The TEM analysis revealed also severe changes in liver cells compatible with oxidative stress. Hepatocytes of treated fish showed glycogen depletion, swollen mitochondria and increased lysosomes, compared to controls. After depuration, some cells recovered nearly normal morphology, but retained the lysosomes, while others underwent necrotic changes (Fig. 2). Differences among the two species studied were of a quantitative nature, and more pronounced in Danio rerio.

The results suggest that potential risk to fish health exist related to the TiO2 NPs release to the aquatic environment and may cause deleterious effects in aquatic organisms. It is evident that the effects of TiO2 NPs on environment is a matter of great concern and the precise mechanisms of toxicity of this and other types of NPs must be clarified.

The authors acknowledge the funding by Fundação para a Ciência e Tecnologia through grant PTDC/CTM/099446/2008 and through project no. PEst-C/EQB/LA0006/2011 granted to Requimte.

Sea anemones species identification of can be difficult, especially in the field. The taxonomic keys currently utilised are based mainly on histological differences and therefore require collection of whole animals. Furthermore, histological analysis of sea anemones requires considerable expertise and worldwide sea anemones histological descriptions are still limited. Here it is reported a detailed histological description of the internal structure of the two most conspicuous species in tidal pool communities in European shores, Actinia equina and Anemonia sulcata. This species description makes them excellent models for studying reproductive strategies that are instrumental in establishing and maintaining new populations. Therefore they are potential indicator species important both for micro and macro scale monitoring programs. The anatomy and histology of main organs of the studied sea anemones were described. The different colour morphs of the species were studied. Fine histological details of their muscular and digestive systems and appendages, down to the cellular level, were studied. They are two-germinal layer animals and possess radial symmetry in body form. Samples of the main organs of sea anemones including epidermis, tentacles, mesentery, gonads and digestive tract (mouth, stomodaeum and gastrovascular cavity) were processed for histological examination according to procedures described elsewhere [1,2]. The epithelia were composed of epithelio-muscular cells, in which many gland cells were distributed. Three types of cnidoblasts (nematooysts) were observed in the epithelioma of the tentacles, mesentery filament and stomodaeum. Cnidoblasts in different places have different functions. The gonads were of the follicular type and were composed of germ cells and nutritional cells. The investigation of the internal organization of invertebrate communities provides important information on the organism’s biology with special interest for the study of ecological processes to evaluate their reproductive modes in order to understand how they may reflect their dispersal strategies [1, 2]. Traits that influence reproduction in animal and plant species have been a central focus of evolutionary biology. Therefore, considering their basal position in evolution, the Anthozoa (Cnidaria) provide important models for further understanding processes affecting reproductive strategies in the eumetazoan (i.e., cnidarian-bilaterian) ancestor and in modern vertebrates.

This work was supported by the Portuguese Foundation for the Science and Technology – Portugal and FEDER funds, through the Projects: PTDC/MAR/464729/2006 and FCT/CNPq (Brazil), Project 6818, Programme 19/ 004.

Microorganisms, that evolve to acquire resistance to environmental amoeba, are likely to become human pathogens due to the physiological similarities of free-living amoebae to human macrophages. In this sense free-living amoebae can be regarded as nurseries of pathogenic microorganisms. Due to the widespread distribution of amoebae in the environment and their resistance to current disinfection procedures, they may constitute important reservoirs of pathogenic microorganisms. Striking examples are Legionella pneumophila and several mycobacteria including Mycobacterium tuberculosis. This work represents the first attempt to detect and characterize amoebae and their associated microorganisms in Portugal.

Detection, isolation and in vitro culture of amoebae from several environmental sources, including lagoon and estuarine water and sediments, were performed according to previously developed methods. Identification of the isolated amoebae was done by optical microscopy based on morphological criteria and by DNA sequence analysis of PCR products amplified with one of the following two sets of primers: EUK and ITS. Transmission electron microscopy (TEM) studies and PCR/sequencing approaches were used to detect and identify amoeba-associated microorganisms (AAMs). Bacterial 16S region was amplified with the primers 616V/630R. TEM studies were performed according to standard procedures. In short, samples were fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate, dehydrated in ethanol and embedded in Epon-Araldite. Thin sections contrasted with uranyl acetate and lead citrate were observed with a JEOL 100-SX electron microscope.

Several species of amoeba (Acanthamoeba lenticulata, A. polyphaga, A. rhyzodes, Platyamoeba oblongata, Saccamoeba limax, Vannella simplex, Vannella sp. and other unidentified amoebae) were found and AAMs were detected both by TEM and PCR/sequencing. The bacteria species Variovorax paradoxus, previously found in association with Saccamoeba and Arcella, were isolated and identified by the PCR/sequencing approach, which also allowed the detection of an unidentified species with about 85% identity with Marivirga tractuosa, a species not yet associated with amoebae. These results point to the existence of AAMs in the environments subjected to this study and the need to evaluate their pathogenic potential. This is an important issue particularly in environments related to human recreational, nutritional and public health activities.

Since contaminants such as metals, POPs (Persistent Organic Pollutants) and PAHs (Polycyclic aromatic compounds represent a risk to human health and to the environment, it is therefore extremely necessary to study their biological effects. Man-made chemicals endocrine disruptors such as estrogens pose the potential to modulate endocrine function and thus adversely affect humans and other animal’s reproductive development. In this work, sublethal toxicity tests were carried out with the sea anemones Actinia equina and Anemonia sulcata exposed to 17-β-Estradiol. A. equina and A. sulcata are species that present a wide geographic distribution and might possibly be effective pollution indicators. Histological and histochemical techniques were used to detect morphological changes in sea anemones in order to find histological parameters that could be useful as early biomarkers of environmental contamination. The histological and histochemical procedures followed by standard methods to Hematoxylin and eosine (H & E), Periodic acid Shiff reagent (PAS) and Masson`s Tricrome (TMass) stained adaptated to Actiniidae conditions. Such as, the fixation (formalin and alcohol) time is high (96 hours), because this organisms are almost 98% of water body constitution. The slides obtained were observed by light microscopy means. The assemblage of methodologies described permitted the identification of several anomalies/pathologies in different parts of the sea anemones body, with special attention to reproductive structures. Results obtained for A. sulcata showed vitellogenic oocytes with anomalous dimensions, altered cytoplasm or without cellular membrane limits (Figure 1). It can also be observed lipid accumulations and cells membranes not always preserved. In certain areas oocytes presents small reactivity with atypical PAS low basophilic patterns. In the mesoglea the amoebocytes showed more eosinophilic cytoplasm or extracellular bodies suggesting necrosis or protein content.  The effects at 10 µg/L concentrations show a considerable number of oocytes with germinal vesicles membranes and indistinct cytoplasm boundaries (Figure 1). Results obtained for A. equina showed some morphological changes in the spermatocytes of male gonads and in the germinal vesicles the female gonads. The effects observed at higher concentrations shows oocytes and ovarian tissues disintegration. The morphologic alterations observed suggested a delay in spermatogenesis and although there have been no alterations in female vitellogenic granules, there are changes in their maturation. The whole effects lead to verify the role that the estradiol in the Anthozoan reproductive system.

Acknowledgments: This work was supported by the Portuguese Foundation for the Science and Technology – Portugal and FEDER funds, through the Projects: PTDC/MAR/464729/2006 and FCT/CNPq (Brazil), Project 6818, Programme 19/ 004.

Azelaic acid, a saturated dicarboxylic acid, is used in the treatment of acne. However, some side effects and low patient compliance have been associated with several topical formulations of azelaic acid. Thus, nanotechnology presents an innovative strategy that can overcome these problems. Polymeric nanoparticles are generally applied to control drug release and to reduce local drug toxicity. In this study, we used the polymer PLGA (acid poly (DL-lactic-co-glycolic)) which is a biocompatible and biodegradable polymer that provides a slow and controlled drug release. The aim of this study was to evaluate a new topical treatment for acne with azelaic acid-loaded PLGA nanoparticles in terms of size, surface charge, encapsulation efficiency, morphology, drug-polymer interactions, efficacy, toxicity and safety of excipients.

Nanoparticles were produced by a modified spontaneous emulsification/solvent diffusion method and included into a gel of carbopol 940.  Size and zeta potential were determined by photon spectroscopy and electrophoretic mobility, respectively. Efficiency encapsulation was accessed by HPLC analysis using an adaptation of a previously described method. Loaded and blank nanoparticles morphology was observed by SEM. Drug-polymer interactions and thermal characteristics were evaluated by DSC. Staphylococcus epidermidis and Propionibacterium acnes susceptibility to the azelaic acid-loaded nanoparticles were tested by the broth microdilution method (efficacy test) and the minimum inhibitory concentration (MIC) values were determined using vancomycin as a positive control. The cytotoxicity of the nanoparticles was investigated on a Saccharomyces cerevisiae model. Excipients safety was accessed by occluded patch tests in humans (blank nanoparticles). The tests were performed in 12 healthy, female volunteers with skin type II whose ages were in between 22-38 years. All the tests were carried out according to the Declaration of Helsinki and received the approval of local Ethical Committee.

Nanoparticles formed a soft white powder (Figure 1A). The loaded nanoparticles mean size was 378.63±60.86 nm (0.09±0.03, P.I.) (Figure 1B) and zeta potential was -7.82 ± 9.01 mV. The encapsulation efficiency was 76±3.81%. Entrapped nanoparticles were visible in the empty nanoparticles sample (Figure 1C). Monodispersed and spherical loaded nanoparticles were observed by SEM (Figure. 1D). System efficacy test suggested similar results of azelaic acid-loaded nanoparticles and azelaic acid in the free form against S. epidermidis and P. acnes (Table 1). DSC analysis suggested an interaction between the polymer (PLGA) and the drug. Cytotoxicity of azelaic acid-loaded nanoparticles on S. cerevisiae was concentration-dependent although not pronounced. Occluded patch test seemed indicate that the formulation excipients were safe.

Small and anionic PLGA nanoparticles demonstrated to be efficient in the encapsulation of azelaic acid.

Nanoparticles proved to be efficient against the most common bacteria associated with acne. Further studies will include additional bacteria susceptibility tests.

Rheumatoid arthritis is a chronic, systemic inflammatory illness that predominantly affects the joints. Recent works tested inductor agents of arthritis models and their effects when associated to low intensity laser therapy. In this work the effects of the biological agent Infliximab and low intensity laser therapy were investigated in an animal experimental model of Zymosan induced arthritis. This model is widely used in pharmacological studies because it enhances the hypernociception of inflammation, detected throughout the observation of joint articular incapacitation, increase of vascular permeability, release of inflammatory mediators in the joint space and histological changes.

A laser instrument (GaAlAs) DMC brand, model Photon Laser III was used, and the radiation was applied at wavelength 808 nm, power density of 200 mW/cm2, energy density of 2000 mJ/cm2, dose of energy or fluence 2 J/cm2, spot area of 0.02 mm, length of 5 dots per second. Rats with 250g were separated in 5 groups; the control (GI) received intra-articular 50µl of 0.9% saline solution in the right knee. The experimental group GII received intra articular injections of Zymosan (AZy) in the right knee at day zero (1mg, in 50µl of saline solution 0.9%); group GIII received daily doses of the same solution in the medial face of the right knee and 10 applications of laser (904 nm); Group GIV received 3 doses of 1,6ml of endovenous Infliximab in the vein of tail; Group GV received 3 doses of infliximab and daily doses of laser (904 nm). In the 14th day of the experiment the group GII was not treated. The organisms were sacrificed 24 hours after each protocol was completed and submitted to histopathological analysis using standard histological procedures.

Results showed that, with 14 days of AZy, the groups presented a moderate to intense inflammation, presence of giant cell granulomas and synovial hyperplasia. When treatment was associated with laser therapy the inflammation was substantially reduced. The results suggest the efficiency of laser therapy associated or not with the biological agent application, to the treatment of induced chronical synovitis.

Arbuscular mycorrhizal fungi (AMF) are ubiquitous underground symbiotic associations between most plants and fungi from the phylum Glomeromycota. From this symbiosis plants generally improve their capacity to obtain less mobile soil nutrients (like phosphorus) and increase resistance against biotic and abiotic stresses. Concomitantly the fungus has access to host carbon photosynthates. As obligate symbionts these fungi require always a host to survive, hence dual cultures is an obligatory laboratory requisite to maintain and multiply live Glomeromycota fungi. These cultures are mainly of three sorts: 1) Open pot cultures, 2) in vitro cultures with Ti DNA transformed root systems, 3) and in vitro cultures with L. cruciata, presented here. The internal mycelium of AMF usually assumes one or both of the morph-types named Arum (first described on Arum maculatum) and Paris (observed in Paris quadrifolia). The Paris-type morphology is characterized by hyphae with an intra-cellular growth, from cell-to-cell, forming coils and arbusculate coils. The Arum-type shows mainly intercellular hyphae growth, longitudinally between cells, with the arbuscules mounting upright on short intra-cellular branches.

This study presents the first comparative morphologic characterization of 4 species of AMF grown in vitro with Lunularia cruciata (L.) Dumortier ex. Lindberg. Glomus clarum Nicol. & Schenck (CNPAB 005), G. intraradices Schenck & Smith (MUCL 43204), G. proliferum Dalpé & Declerck (MUCL 41827); and Gigaspora margarita Becker & Hall (CNPAB 001) internal hyphae share the same morph-type of L. cruciata colonization: the Paris-type. Stable and viable monoxenic cultures of AMF with L. cruciata were first presented by Fonseca and co-workers in 2006 for G. intraradices and G. proliferum on Petri dish (Fig. 1.1). In this work we bring two more species (G. clarum and Gi. margarita) cultured in vitro with L. cruciata, on bi-layer medium in flask containers (Fig. 1.2). The production of spores occurred mainly, among the rhizoids, between overlapping thallus and over the thallus (Fig. 1.3). Only the Glomus species produced spores within the medium.

L. cruciata belongs to a group of complex thalloid liverworts with an internally differentiated anatomy: an upper epidermis forming air chambers where the chlorophyllous cells are located, a conspicuous vacuolated parenchyma, and the lower epidermis with its scales and rhizoids (Fig. 1.4). All four species show highly similar architecture of colonization with most fungi distributed in the thallus’ midrib parenchyma, and with internal mycelium architecture compatible with the Paris-type colonization (Figures 1.5 to 1.8). Although consistent the occurrence of Paris-type colonization among these four species, more species needed to be examined before a generalization can be given. The availability of AMF isolates for research at international fungi collections and laboratories is limited, and from these axenic isolates for in vitro culture are very few, which hinders the prove of the hypotheses that Paris-type colonization is the main pattern of hyphae colonization in L. cruciata mycothallus.

Several approaches for cell image segmentation have been proposed and applied with success in the biomedical sciences field, but cell content estimation is still a not solved problem. This paper presents a novel formed classifier that combine appropriately “intelligent scissors” (livewire), object’s pixel information (RGB values of colors) and probabilistic artificial neural networks (ANN) techniques for segmenting and estimating precisely the cytoplasm composition in relation to protein, lipid, and carbohydrates. Based on this it is possible to estimate the gonad’s state of development and the maturation stage of oocytes in adult ovigerous females gonads.

The ovigerous mature females (identifiable by their brood pouch – empty, with eggs, with eyeless or eyed embryos) were selected under a dissecting microscope in the laboratory. A total of 170 gonadic masses from females containing oocytes in different developmental stages were analyzed. A total of 100 sections were observed by light microscopy in order to record the arrangement of the ovaries and determine the maturity stage of the various germ cells.

With the ANN network (Fig. 1) trained is possible to carry out the classification step. Classification aims to associate each cytoplasm pixel to one of the selected classes. In this process each cytoplasm pixel is simulated (Figure 2 A). As result a new image containing the classified cytoplasm is produced, where each class is represented by a different colour level (Figure 2 B). Figure 2 C show from left to right the full “classification flow process” including ROI selection, cytoplasm and nucleus contour extraction, cytoplasm segmentation and cytoplasm content classification. The proposed classifier was tested successfully in a representative image dataset, demonstrating the effectiveness and robustness of this new method facilitating the histochemical analysis of cell contents, based on a gonads model.

A fully automatic approach to locate polyomavirus particles in images of transmission electronic microscopy is presented, that can localize intact particles, many damaged capsids and an acceptable percentage of superposed ones; the performance is quantified in 25 electron micrographs containing nearly 390 particles, compared to the interpretation of two independent electron microscopy experts. As these particles often does not have a well-defined edge, in highly textured backgrounds (Figure 1) the initial guess of candidate points from local entropy proportion measures retains a large set of points, in order to avoid false negatives.

Based in human criteria - focused on the pattern generated by the capsomeres typical of polyomavirus surface, this approach uses indicators calculated from the local co-occurrence matrix of grey levels to assess the textured pattern and prune the initial set of candidates; in some more complicated backgrounds about 2-10% of the elements will survive. A restricted set of the points accepted (Figure 2) is used to evaluate typical average and variance and reduce the set of survivors accordingly. These intermediate points are locally evaluated using i) a statistical index concerning the radiometric distribution of a circular neighborhood around the centroid of each candidate, and ii) a structural index resuming the expected morphological characteristics of eight radial intensity profiles encompassing the area of the possible particle. All the parameterization is based on the particle expected dimensions.

This hierarchical approach attains 95% efficiency in the detection of intact virus particles, tolerating a certain lack of differentiation in the borders and a certain amount of shape alterations. Only 5 to 7% (according to the reference used) of the intact particles are missed by the algorithm – cases of particles with abnormal radial structures, particles with different degrees of agglutination or particles showing empty (or with different degrees of permeation) capsids. Superposed particles are detected with a success rate of 71% to 77%. In the case of distorted and/or doubtful particles, a correct detection of 72% is attained; these particles are the main source of differences between experts (a difference of 48% between both interpretations).

This kind of algorithm is "tailored" for one virus family particle, in this case, the polyomavirus; it can be parameterized for different working magnifications, but cannot be applied to different virus, that has different texture, structure and typical radiometry characteristics. We believe that similar results can be obtained in other virus families with the necessary adaptations.

Zinc is a trace metal that is regularly found at pollution sites, amongst other metals and pollutants, contributing to the toxicity of marine ecosystems. In this study zinc acute toxicity tests were conducted on the sea anemone Actinia equina. This sea anemone presents a wide geographic distribution on rocky shore marine habitats, is quite resistant to some degree of various forms of contaminants and plays an important ecological key role in the marine environments.

After a period of acclimatising on a flow through system, organisms of the same average size were exposed to different concentrations of zinc solution during a period of 96 hours (0 µg/l, 10 µg/l, 50 µg/l, 100 µg/l, and 200µg/l). The histological processing of the specimens was performed using standard procedures, slightly modified due to the peculiar nature of the biological material.

The general effects exhibited necrosis of tentacles, mesenterial filaments, and in the gastrodermal regions (Figures 1, 2 and 3). It were observed tentacular cysts, black particulate accumulations in the gastrodermis, hypertrophied cells in the lobes of the mesenteries filaments, and spirocyst and associated necrosis (Fig. 2-3). All anemones exhibited extensive areas of tissue necrosis, degeneration of the epidermis in various stages, with loss of mucous cells, vacuolation, necrosis and ptychocysts from the epidermis, necrosis of retractor muscles and ducts (Figures 2-3). Neoplasms of the gastrointestinal tract and gonads were observed in internal structures. In the former, modifications of the mesenteries such as intensive fragmentation, vacuolization and epithelial thinning and in the latter abnormal gonad development and necrosis were noted (Figures 2-3).

The data suggests that Actinia equina may be adversely affected by acute exposures to high levels of zinc as seen during laboratory exposures of other invertebrates and is also probably sensitive to acute exposures to metal’s toxic sediments in the marine environment. The loss of mucous secretory cells and ptychocysts from the epidermis suggest that energy reserves for cell replenishment were adversely affected. Mucus production in benthic cnidarians is necessary to prevent burial by sedimentation and to protect against toxins or invading microorganisms. The serious effects on the gastrodermis integrity indicate that sea water in the coelenteron is no longer circulated. The observed modifications could be considered as a general cnidarians response to stress.

Further investigations of bioaccumulation, physiology, and histopathology in this anemone and other anthozoans from polluted environments should prove to be valuable in pollution monitoring studies, as they have for other benthic invertebrates

Acknowledgments

This work was supported by the Portuguese Foundation for the Science and Technology – Portugal and FEDER funds, through the Projects: PTDC/MAR/464729/2006 and FCT/CNPq (Brazil), Project 6818, Programme 19/ 004.

Engineered nanomaterials such as nanoparticles (NPs) are increasingly being used for commercial purposes in products within medicine, electronics, sporting goods, tires, textiles and cosmetics. They comprise diverse types of materials from metals, polymers, ceramic to biomaterials and have been defined as particles with at least one dimension in the order up to 100 nm1. The higher toxicological potential of NPs is mostly due to their small size, wide surface, increase of their chemical reactivity and biological activity and the capacity to generate free radicals. NPs also can have the ability to penetrate trough the biological barriers and to move easily through the biological systems. Therefore, is mandatory to assess the toxicity of these nanomaterials, since their industrial production and uses will also result in releases to the environment with unclear consequences.

The aim of the present work is to evaluate the toxicity of titanium dioxide (TiO2) NPs on gill gluthatione-S-transferase activity (GST), lipid peroxidation and on structure of the gills of two freshwater fish species (Carassius auratus and Danio rerio). Suspensions of TiO2 NPs, with an average size of 21 nm, were prepared using distillate water and then ultrasonicated (10 min, 35 KHz). The suspensions were added to 10L of tap water in exposure tanks, to obtain nominal concentrations (0.01; 0.1; 1, 10; 100 TiO2 mg/L). The test fish, C. auratus (N=144) and D. rerio (N=80), were randomly distributed by 6 exposure tanks and an additional tank with clean tap water was used as control. Fish were sampled after 7, 14, and 21 days. Six fish from both species were left for depuration in clean tap water during 14 days and then sacrificed. The GST activity was determined by following the procedure described by Habig et al. and lipid peroxidation was measured based on the Thiobarbituric Acid Reactive Species method. The tissues were processed essentially according to Martoja and Martoja for light microscopy (LM). For transmission electron microscopy (TEM) the samples were fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate, dehydrated in ethanol and embedded in Epon-Araldite according to standard procedures. The histological and ultrastructural observations were performed using a Leica-ATC 2000 microscope and a JEOL 100-SX electron microscope respectively.

The results show a significant increase of GST in gills tissues for C.auratus exposed to 10 and 100 mg/L TiO2 NPs and a decrease following the depuration period. With respect to D. rerio a significant increase was observed in fish exposed to 1, 10 and 100 mg/L TiO2 NPs. Lipid peroxidation are in agreement with GST results but showing a significant increase for fish (C.auratus and D. rerio) exposed to concentrations of 0.1 TiO2 mg/L NPs and higher. Usually, the oxidative stress caused by exposure to TiO2 NPs is attributed to hydroxyl radicals (OH) generated by photochemical (UV/vis) processes but it may be also related to specific properties of TiO2 NPs such as size, surface area and solubility that can influence the degree of toxicity. The results from LM observations (Fig. 1) showed that exposure to TiO2 NPs affected gill tissues, with changes being detected in both fish species exposed to 0.1 TiO2 mg/L NPs and higher which is in accordance with biochemical results. Changes include different degrees of hyperplasia (from low to complete fusion of lamellae). The TEM analysis revealed that TiO2 NPs were internalized by gills epithelial cells accumulating in vacuoles inside these cells (Fig. 2). After the depuration period it was observed that the capability for gills to recover was not complete. The results show a strong response to oxidative stress caused by exposure to TiO2 NPs, possibly because they are in direct contact with the exposure medium and function as a first barrier against external aggression. However, the gills changes observed following exposure and a partial recover after depuration suggest that TiO2 NPs may cause deleterious effects in fish gills compromising fish homeostasis.

The authors acknowledge the funding by Fundação para a Ciência e Tecnologia through grant PTDC/CTM/099446/2008 and through project no. PEst-C/EQB/LA0006/2011 granted to Requimte.

A large decrease in the side effects of a drug can be obtained if it is efficiently delivered in a timely manner and in the needed location only. By combining therapeutic and diagnostic (theranostic) functionalities with targeting capabilities and large surface areas, nanoparticles provide an ideal vehicle for personalized medicine.

The main objective of our work is to develop hybrid Mesoporous Silica Nanoparticles (MSNs) for theranostics, carrying fluorescent beacons for traceability and imaging, featuring a smart release control mechanism, able to accommodate large drug loads and to deliver their cargo on demand to a desired location. This communication focus on the preparation of the fluorescent MSNs, and characterization by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and laser scanning fluorescence confocal microscopy (LSFCM).

Mesoporous Silica Nanoparticles (MSNs) with well-defined and controllable particle morphology are exceptional supports/nanocontainers for molecules and polymers.This class of materials are characterized by an ordered pore system of 2-8nm diameter, pore volumes above 1mL/g and particle size from 40nm to several hundred nanometers. The preparation of fluorescent hybrid MSNs involves the presence of a fluorescent molecule during particle synthesis, which becomes aligned with the pores, thus impervious to aggregation and self-quenching effects. The MSNs external surface can be selectively functionalized to immobilize polymers or (bio)molecules for possible targeting or sensing, and the pore is available for solvent diffusion, allowing the incorporation of different molecules.

We prepared monodispersed hybrid MSNs incorporating a fluorescent perylenediimide (PDI) derivative in the wall structure. The MSN-PDI were characterized by TEM (Figure 1), SEM and fluorescence emission spectroscopy. LSFCM images of the MSN-PDI after incubation in HEK293 cells show the internalization of the nanoparticles (Figure 2). These new hybrid nanoparticles, after surface-functionalization with stimuli-responsive gate systems, open possibilities for the development of traceable drug delivery systems.

This work was partially supported by Fundação para a Ciência e a Tecnologia (FCT-Portugal) and COMPETE (FEDER) within projects PTDC/CTM/101627/2008 and PEst-OE/CTM/LA0024/2011. T.R. and F.F. also thank FCT for Ph.D. (SFRH/BD/64702/2009) and Pos-Doc (SFRH/BPD/64320/2009) grants.

Mathematical morphology is a novel geometry-based technique for image processing and analysis, originally developed to process binary images, based on the use of simple concepts from set theory and geometry such as set inclusion, intersection, union, complementation, and translation Geometry is used to represent object shape, physics inflict constraints on how the shape may vary over space and time, and optimal approximation theory make available the formal underpinnings of mechanisms for fitting the models to measured data. Deformable curve, surface, and solid models gained popularity after they were proposed by Terzopoulos for use in computer vision and computer graphics in the mid 1980’s introducing the theory of continuous (multidimensional) deformable models in a Lagrangian dynamics setting, based on deformation energies in the form of generalized splines. The deformable model that has attracted the most attention to date is popularly known as “snakes”, planar deformable contours that are useful in several image analysis tasks. They are often used to approximate the locations and shapes of object boundaries in images based on the reasonable assumption that boundaries are piecewise continuous or smooth.

This work presents a novel approach since it joins (and, indeed, reinforces) the index framework with the evaluation of the same biological samples by a suitable combination of deformable models. Nucleus contour is identified through Active Shape Models techniques, and cytoplasm contour’s detected through parametric Snakes, with prior image preprocessing based on statistical and mathematical morphology techniques. Morphometric parameters such as nucleus and cytoplasm area and ratio between them are then easily computed. Biometry was performed using an ocular micrometer and nucleus/cytoplasm ratios were obtained characterizing each of the three identified stages: Immature, Vitellogenic and Mature. This resulted in a collection of tools, called morphological operators, which are eminently suited for the analysis of shape and structure in binary images. Acartia tonsa was used as model to establish an index for oocyte maturity determination based in citometry and histochemical evaluation of gonadic masses. It was validated the application of a methodology with a realistic background and a new, more accurate and ecologically realistic index for oocyte staging emerged.

Species of Aspergillus from section Nigri, also known as black aspergilli, are distributed worldwide and have been widely used for purposes of various types such as: biotechnological, industrial, medical, among others. They have been extensively studied for being the causing agents of biodeterioration of commodities and food.

Within this section, new species have been recently described and among them Aspergillus ibericus and Aspergillus uvarum were both isolated from grapes. The polyphasic approach used in the analysis of these species, either by morphological techniques as well as by molecular methods, allowed not only their characterisation but also their consequent separation from all others in the section. Microbial taxonomy has the identification of species as one of its fundamental goals. Data such as: morphological characteristics description, physiological and biochemical properties, ecological roles, and societal risks or benefits, are key elements in any fungal identification process. But, this process is subjected to periodic changes due to frequent revisions of the taxonomic schemes, therefore becoming time consuming and more demanding and difficult, even for skilled specialists. Furthermore, each taxonomic group has specialised literature, terminology and characters. This takes place since identifications have difficulties of consensual naming, depending on the criteria used and the amount of information available when producing all data. It is increasingly becoming clear that, to better achieve a more accurate concept of species, microbial identification and authentication require a polyphasic approach to produce consistent, useful and quality data.

Characterisation of morphologic and structural aspects of spores from Aspergillus strains, section Nigri, has been carried out using scanning electron microscopy (SEM) and intends to contribute to the associated data of the strains from this section.

Colonies from 13 Aspergillus from section Nigri, from the Micoteca da Universidade do Minho (MUM), were grown at 25 ºC for 3 or 4 days in malt extract agar directly mounted in a sterilised SEM stub. The samples were covered with a mixture of gold and platinum (80/20%) and then examined in the SEM [NanoSEM - FEI NovaTM 200 (FEG/SEM); EDAX - Pegasus X4M (EDS/EBSD)]. A stereomicroscope (Leica MZ12.5) was used to have a clear image comparison of the size of the conidial head.

Although the analysed strains presented dimensional, morphological and structural diversity, common or typical features could be inferred and related to each taxon like those represented in Fig 1.

From the SEM analysis we were able to conclude that in the section Nigri of genus Aspergillus the spore wall ornamentation, and its size and shape continue to present themselves as important primary diagnostic traits for species differentiation.