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.

Transcriptome profiling of microdissected cortex and medulla unravels functional regionalization in the European sea bass Dicentrarchus labrax thymus.

The thymus is a sophisticated primary lymphoid organ in jawed vertebrates, but knowledge on teleost thymus remains scarce. In this study, for the first time in the European sea bass, laser capture microdissection was leveraged to collect two thymic regions based on histological features, namely the cortex and the medulla. The two regions were then processed by RNAseq and in-depth functional transcriptome analyses with the aim of revealing differential gene expression patterns and gene sets enrichments, ultimately unraveling unique microenvironments imperative for the development of functional T cells. The sea bass cortex emerged as a hub of T cell commitment, somatic recombination, chromatin remodeling, cell cycle regulation, and presentation of self antigens from autophagy-, proteasome- or proteases-processed proteins. The cortex therefore accommodated extensive thymocyte proliferation and differentiation up to the checkpoint of positive selection. The medulla instead appeared as the center stage in autoimmune regulation by negative selection and deletion of autoreactive T cells, central tolerance mechanisms and extracellular matrix organization. Region-specific canonical markers of T and non-T lineage cells as well as signals for migration to/from, and trafficking within, the thymus were identified, shedding light on the highly coordinated and exquisitely complex bi-directional interactions among thymocytes and stromal components. Markers ascribable to thymic nurse cells and poorly characterized post-aire mTEC populations were found in the cortex and medulla, respectively. An in-depth data mining also exposed previously un-annotated genomic resources with differential signatures. Overall, our findings contribute to a broader understanding of the relationship between regional organization and function in the European sea bass thymus, and provide essential insights into the molecular mechanisms underlying T-cell mediated adaptive immune responses in teleosts.

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 Effect of the Hypertrophy Virus (MdSGHV) on the Ultrastructure of the Salivary Glands of Musca domestica (Diptera: Muscidae).

The salivary glands of insects play a key role in the replication cycle and vectoring of viral pathogens. Consequently, Musca domestica (L.) (Diptera: Muscidae) and the Salivary Gland Hypertrophy Virus (MdSGHV) serve as a model to study insect vectoring of viruses. A better understanding of the structural changes of the salivary glands by the virus will help obtain a better picture of the pathological impact the virus has on adult flies. The salivary glands are a primary route for viruses to enter a new host. As such, studying the viral effect on the salivary glands is particularly important and can provide insights for the development of strategies to control the transmission of vector-borne diseases, such as dengue, malaria, Zika, and chikungunya virus. Using scanning and transmission electron microscopic techniques, researchers have shown the effects of infection by MdSGHV on the salivary glands; however, the exact location where the infection was found is unclear. For this reason, this study did a close examination of the effects of the hypertrophy virus on the salivary glands to locate the specific sites of infection. Here, we report that hypertrophy is present mainly in the secretory region, while other regions appeared unaffected. Moreover, there is a disruption of the cuticular, chitinous lining that separates the secretory cells from the lumen of the internal duct, and the disturbance of this lining makes it possible for the virus to enter the lumen. Thus, we report that the chitinous lining acts as an exit barrier of the salivary gland.

Gut immunity in European sea bass (Dicentrarchus labrax): a review.

In this review, we summarize and discuss the trends and supporting findings in scientific literature on the gut mucosa immune role in European sea bass (Dicentrarchus labrax L.). Overall, the purpose is to provide an updated overview of the gastrointestinal tract functional regionalization and defence barriers. A description of the available information regarding immune cells found in two immunologically-relevant intestinal compartments, namely epithelium and lamina propria, is provided. Attention has been also paid to mucosal immunoglobulins and to the latest research investigating gut microbiota and dietary manipulation impacts. Finally, we review oral vaccination strategies, as a safe method for sea bass vaccine delivery.

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.

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.

MHC II-ß chain gene expression studies define the regional organization of the thymus in the developing bony fish Dicentrarchus labrax (L.).

MHC II-ß chain gene transcripts were quantified by real-time PCR and localised by in situ hybridization in the developing thymus of the teleost Dicentrarchus labrax, regarding the specialization of the thymic compartments. MHC II-ß expression significantly rose when the first lymphoid colonization of the thymus occurred, thereafter increased further when the organ progressively developed cortex and medulla regions. The evolving patterns of MHC II-ß expression provided anatomical insights into some mechanisms of thymocyte selection. Among the stromal cells transcribing MHC II-ß, scattered cortical epithelial cells appeared likely involved in the positive selection, while those abundant in the cortico-medullary border and medulla in the negative selection. These latter most represent dendritic cells, based on typical localization and phenotype. These findings provide further proofs that efficient mechanisms leading to maturation of naïve T cells are operative in teleosts, strongly reminiscent of the models conserved in more evolved gnathostomes.

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.

Impact of environmental changes and human-related factors on the potential malaria vector, Anopheles labranchiae (Diptera: Culicidae), in Maremma, Central Italy.

The Maremma Plain (central Italy) was hyper-endemic for malaria until the mid-20th century, when a national campaign for malaria elimination drastically reduced the presence of the main vector Anopheles labranchiae Falleroni. However, the introduction of rice cultivation over 30 yr ago has led to an increase in the An. labranchiae population and concern over possible malaria reemergence. We studied the impact of anthropogenic environmental changes on the abundance and distribution of An. labranchiae in Maremma, focusing on rice fields, the main breeding sites. Adults and larvae were collected in three main areas with diverse ecological characteristics. Data were collected on human activity, land use, and seasonal climatic and demographic variations. We also interviewed residents and tourists regarding their knowledge of malaria. Our findings showed that the most important environmental changes have occurred along the coast; An. labranchiae foci are present throughout the area, with massive reproduction strictly related to rice cultivation in coastal areas. Although the abundance of this species has drastically decreased over the past 30 yr, it remains high and, together with climatic conditions and the potential introduction of gametocyte carriers, it may represent a threat for the occurrence of autochthonous malaria cases. Our findings suggest the need for the continuous monitoring of An. labranchiae in the study area. In addition to entomological surveillance, more detailed knowledge of human-induced environmental changes is needed, so as to have a more complete database that can be used for vector-control plans and for properly managing emergencies related to autochthonous introduced cases.

Transcription of T cell-related genes in teleost fish, and the European sea bass (Dicentrarchus labrax) as a model.

In recent years the cloning of genes coding for immuno-regulatory peptides, as well as the sequencing of genomes, provided fish immunologists with a growing amount of information on nucleotide sequences. Research is now also addressed in investigating the functional immunology counterpart of nucleotide sequence transcripts in various fish species. In this respect, studies on functional immunology of T cell activities are still at their beginning, and much work is needed to investigate T cell responses in teleost fish species. In this review we summarise the current knowledge on the group of genes coding for main T cell-related peptides in fish, and the expression levels of these genes in organs and tissues. Particular attention is paid to European sea bass (Dicentrarchus labrax), a marine species in which some information on functional immunology has been obtained, and we reassume here the expression of some T cell-related genes in basal conditions. In addition, we provide original data showing that T cells purified from the intestinal mucosa of sea bass with a specific mAb, express transcripts for TRß, TRγ, CD8α, and RAG-1, thus showing similarities with intra-epithelial leucocytes of mammals.

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.

Intestinal T cells of Dicentrarchus labrax (L.): gene expression and functional studies.

Cellular and molecular data have evidenced a gut-associated lymphoid tissue in a variety of teleost species, abundantly containing T cells, whose origin, selection and functions are still unclear. This study reports CD4, CD8-α, MHCI-α, MHCII-ß, rag-1 and TCR-ß gene transcription along the intestine (anterior, middle and posterior segments) and in the thymus of one year-old Dicentrarchus labrax (L.). Real-time PCR findings depicted a main role of the thymus in T-cell development, but also rag-1 and CD8-α transcripts are detected in the intestine, having significant expression in the posterior segment. In the whole intestine TCR-ß and CD8-α exceeded CD4 transcripts. RNA ISH confirmed these data and detailed that mucosal CD8-α+ cells were especially numerous in the epithelium and in aggregates in the lamina propria. Regional differences in T-cell-specific gene expressions are first described in the intestine of a bony fish. High non-specific cytotoxic activity against xenogeneic and allogeneic cells was found in lymphocytes purified from the intestinal mucosa, providing further insight into their local defence roles.

Results preliminary attempts to control the larvae of Leptoconops (Holoconops) kerteszi Kieffer, 1908 (Diptera, Ceratopogonidae) in the coastal wetlands of Tuscany, Italy.

Leptoconops spp. are small midges, members of the family Ceratopogonidae, that are relatively widespread in wetlands with sandy or silty-clay soils, including many tourist sites. Although very few of the species are proven vectors of pathogens, the blood-feeding females attack mammals, including humans, in large swarms and their painful bites may cause severe reactions, especially in children. Although Leptoconops spp. may limit the socio-economic development of an area, there are currently no effective methods for the control of their natural populations, and the repellents and netting used against mosquitoes are ineffective against these midges. The diurnal control of the adults with pesticides may have unacceptable impacts on non-target species, including humans, and be ineffective because the adult females can easily be carried, from untreated areas to treated, on the wind. In the present study, the use of a diflubenzuron-based insecticide against the larvae of Leptoconops (Holoconops) kerteszi Kieffer, 1908 - a sand-reproducing species that is widespread in certain coastal areas of the Italian province of Grosseto - was explored. In Grosseto, in summer, attacks by swarms of adult L. kerteszi create problems for the local people and the many tourists. The encouraging results of preliminary tests are discussed in relation to the potential use of diflubenzuron for the integrated control of L. kerteszi populations.

Do all stoneflies nymphs have respiratory proteins? Further data on the presence of hemocyanin in the larval stages of plecoptera species.

Contrary to what was assumed regarding the presence of respiratory proteins in insects, a functional hemocyanin was recently found in larvae and adults of the stoneflies species Perla marginata, whereas in the close species Perla grandis, hemocyanin functionality was deduced from sequence data. In order to verify if the presence of this ancient trait is widespread within the order and to investigate why stoneflies have maintained it, we have extended the search for hemocyanin to species of other Plecoptera families. In particular, we assessed the presence of hemocyanin in the larval stage of nine Plecoptera species, belonging to six of the seven families of the European stonefly-fauna, and analyzed its potential functionality as deduced by sequence data. We cloned and sequenced the corresponding cDNAs and studied their expression with RT-PCR technique. Moreover, we performed homology studies using the deduced amino acid sequences. On the basis of our analysis, we hypothesized a functional role of the hemocyanin only for two species: Dinocras cephalotes and Isoperla grammatica (Perloidea). In all the investigated Nemouroidea and in Siphonoperla torrentium (Perloidea), this protein may have been lost. Larval size, life-cycle length, trophic role and environmental induction are discussed as possible explanations of these different physiological requirements.

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.

Immunolocalization of a lipase-like protein in the reproductive apparatus of female Phlebotomus papatasi, at various stages of the gonotrophic cycle.

In female phlebotomine sandflies, little is known about the reproductive accessory glands that presumably contribute to egg production and/or oviposition. The main protein secreted in the accessory glands of female Phlebotomus papatasi was recently characterised as a lipase-like protein, the first to be found in the female accessory glands of any insect. This protein, named PhpaLIP (for Phlebotomus papatasi lipase), has now been detected and localized in the reproductive tissues of female P. papatasi, at different stages of the gonotrophic cycle, using a polyclonal anti-PhpaLIP serum and both confocal scanning laser and immuno-electron microscopy. PhpaLIP appears to be always present in the accessory glands (with a secretory peak shortly before oviposition) but was also detected in the follicle cells of the ovarioles, within the developing vitelline envelope, and in the oviducts. The results are discussed in relation to the functions that PhpaLIP could have during the gonotrophic cycle, in the various reproductive structures of female P. papatasi.

Differential tissue expression of a calpastatin isoform in Xenopus embryos.

This study is aimed at demonstrating the role played by a calpastatin isoform (Xcalp3) in Xenopus embryos. A specific monoclonal antibody (mAb) was raised against a glutathione S-transferase (GST)-Xcalp3 fusion protein and characterized by immunoblotting and confocal fluorescence microscopy on stage 20-36 embryos. Under these conditions, calpastatin reactivity is associated with a major 110kDa protein fraction and preferentially expressed by notochord and somitic cells. In notochord cells, anti-calpastatin reactive sites were initially restricted to the luminal space of the vacuoles and later became diffused throughout the cytoplasm. In contrast, anti-calpastatin reactive sites in somitic cells were initially diffused throughout the cytoplasm and became restricted to a few intracellular granules in the later developmental stages. At the ultrastructural level, notochord cells appeared as flattened discs containing several vacuoles and numerous electron-dense granules. During transition from stages 26 to 32, electron-dense granules were gradually reduced in number as vacuoles enlarged in size and losed their calpastatin reactivity. Electron-dense granules were also present in myoblast cells and their number gradually reduced during development. To determine whether these observations bear any causal relationship to the calpain/calpastatin system, a number of Xenopus embryos were examined both ultrastructurally and histochemically following exposure to a specific calpain inhibitor (CI3). Under these conditions, Xenopus embryos exhibited an altered right-left symmetry and an abnormal axial shortening. In CI3-treated stage 32 embryos, notochord cells had a reduced vacuolar extension and exhibited at the same time an increase in granular content. The overall morphology of the somites was also distorted and myoblasts were altered both in shape and granular content. Based on these findings, it is concluded that the calpain/calpastatin may play an important role in the control of notochord elongation and somite differentiation during Xenopus embryogenesis.

High levels of human chitotriosidase hinder the formation of peritrophic membrane in anopheline vectors.

In the Anopheles midgut, Plasmodium falciparum produces a specific chitinase able to penetrate the blood meal surrounding the chitin-containing peritrophic membrane (PM). High levels of an analogous chitinase, chitotriosidase (CHIT), may be found in human blood, being the markers of macrophage activation. To verify the hypothesis that CHIT present in malaria patient blood could help parasite to overcome PM, we carried out a bioassay by feeding Anopheles stephensi females on an artificial apparatus that contained human blood from four different sources and with different chitinase concentrations: (1) healthy donors, as negative controls; (2) patients with malaria; (3) patients with Gaucher disease; and (4) whole blood enriched with commercial P. falciparum chitinase, as positive controls. After 16, 20 and 24 h of bloodfeeding, mosquitoes were dissected to extract the midgut and assess the effect of the different chitinases on membrane structure. Optical microscopy showed that formation of PM was clearly complete after 16 h in the posterior midgut from Anopheles already fed with healthy donor bloods. By contrast, PM formation was visible after 16 h in the posterior midgut of mosquitoes fed with malaria and Gaucher patient bloods but appeared clearly damaged at 20 and 24 h. At the same time, the PM formation was almost completely inhibited in the midgut of Anopheles fed with P. falciparum chitinase-enriched bloods. These alterations were clearly confirmed by transmission electronic microscopy. In the present paper, we demonstrate that human CHIT from different sources is active on anophelines' PM.