Morphological and molecular study of Didymodiclinus marginati n. sp. (Trematoda: Didymozoidae) gill parasite of Epinephelus marginatus from the central and western Mediterranean Sea.

The current study provides a morphological and molecular characterization of a new species of Didymodiclinus (Trematoda: Didymozoidae) infecting the dusky grouper, Epinephelus marginatus (Teleostei: Serranidae) from the Mediterranean Sea. A total of 279 dusky grouper specimens were examined for didymozoid gill parasites from the Mediterranean Sea between 1998 and 2020. New species differs from the most similar congeneric species by the rudiments of female reproductive organs in functional male specimens, and the seminal receptacle, Mehlis gland and accessory gland cells in functional female specimens, not observed in Didymodiclinus branchialis (Yamaguti, 1970), Didymodiclinus epinepheli (Abdul-Salam, Sreelatha and Farah, 1990) and Didymodiclinus pacificus (Yamaguti, 1938), respectively. These species are also characterized by their different hosts and location within the host tissues, being from other geographical localities. Moreover, this is the first species reported in E. marginatus from the central and western Mediterranean Sea. Genetic analyses were performed on partial 28S and partial internal transcribed spacer-2 ribosomal RNA regions and the mitochondrial cytochrome oxidase 1 (cox1) gene by polymerase chain reaction. Comparison of genetic sequences of Didymodiclinus marginati n. sp. with the available deposited sequences of 28S revealed that the new isolates cluster with several unidentified didymozoids and groups as a sister clade of the Nematobothrinae subfamily. Moreover, 28S and cox1 phylogenetic trees evidenced that Didymodiclinae is well separated from Didymozoinae and other gonochoric didymozoids. Following both morphological and genetic results, a key of identification for the genus Didymodiclinus is proposed.

Protein Hydrolysates from Anchovy (Engraulis encrasicolus) Waste: In Vitro and In Vivo Biological Activities.

Fish waste utilization to obtain protein hydrolysates has been demonstrated to be a useful strategy to face both environmental and economic impacts while obtaining high-value products with remarkable biological and nutritional properties. In the present study, protein hydrolysates obtained from anchovy Engraulis encrasicolus (APH) by-products were assessed for their potential biological activities in both in vitro and in vivo models. The treatment with APH exerted a significant protection against LPS-induced inflammation in RAW 264.7 cells, decreasing the protein expression of pro-inflammatory mediators (i.e., COX-2) and inhibiting the nuclear translocation of NF-κB through IκB-α. Moreover, APH modulated the expression of iNOS, MnSOD and HO-1, thus decreasing the severity of oxidative stress. The supplementation of APH in the diet of ApoE knockout mice down-regulated the proinflammatory cytokines (i.e., TNF-α, IL-1α, IL-1ß, IL-6) in both aorta and heart tissues, and modulated the expression of oxidative stress-related genes (Cu/ZnSod, MnSod, Cat, Gpx and Ho), indicating that APH can exert a beneficial role, having anti-inflammatory and antioxidant activities. The nutritional properties of APH, together with their biological activities herein reported, highlight the possibility of obtaining bioactive molecules from fish waste and encourage their use as potential nutraceuticals in food and pharmaceutical industries in the next future.

Cytoprotective and Antioxidant Effects of Hydrolysates from Black Soldier Fly (Hermetia illucens).

The black soldier fly (BSF), Hermetia illucens, has been recognized as one of the most promising insect species for its ability to valorize organic waste while producing a valuable larval biomass with a great potential as a sustainable source of nutrients, including proteins and bioactive molecules. In the present study, BSF larvae were used to produce and characterize the protein hydrolysates (BPHs) that were then evaluated for their potential biological activity in vitro. The BPHs obtained from the BSF larvae proteins by enzymatic digestion were characterized by Nuclear Magnetic Resonance (NMR) and polyacrylamide gel electrophoresis and assessed for their antioxidant activity (BPHs in the range of 0.1 to 1.5 mg/mL) in L-929 cells. Our findings show that BPHs can exert a dose-dependent cytoprotective role against H2O2-iduced oxidative stress in cells. This antioxidant activity relies on the reduction of ROS levels in challenged cells as measured by flow cytometry and fluorescence microscopy, together with the induction and nuclear translocation of Nrf2, as evaluated by qPCR and indirect immunofluorescence analysis, respectively. Overall, our findings on the remarkable biological activity of the BPHs obtained in a large-scale process strongly suggest the application of BPHs as ingredients promoting animal health in feed formulations.

Assessment of the effectiveness of a novel BioFilm-Membrane BioReactor oil-polluted wastewater treatment technology by applying biomarkers in the mussel Mytilus galloprovincialis.

Petrochemical industries and oil refineries are sources of hazardous chemicals into the aquatic environments, and often a leading cause of reduced oxygen availability, thus resulting in adverse effects in biota. This study is an expansion of our previous work on the assessment of the BioFilm-Membrane Bioreactor (BF-MBR) to mitigate the impact of oil-polluted wastewater on marine environments. Specifically, this study evaluated the reduction of selected chemical constituents (hydrocarbons and trace metals) and toxicity related to hypoxia and DNA damage to mussels Mytilus galloprovincialis, before and after treatment of oil-polluted wastewater with the BF-MBR. The application of a multidisciplinary approach provided evidence of the efficiency of BF-MBR to significantly reducing the pollutants load from oily contaminated seawaters. As result, the health status of mussels was preserved by a hypoxic condition due to oily pollutants, as evidenced by the modulation in the gene expression of HIF-1α and PHD and changes in the level of hypotaurine and taurine. Moreover, ameliorative effects in the energy metabolism were also found in mussel gills showing increased levels of glycogen, glucose and ATP, as well as a mitigated genotoxicity was revealed by the Micronucleus and Comet assays. Overall, findings from this study support the use of the BF-MBR as a promising treatment biotechnology to avoid or limiting the compromise of marine environments from oil pollution.

Black Soldier Fly (Hermetia illucens) Larvae and Prepupae Defatted Meals in Diets for Zebrafish (Danio rerio).

The black soldier fly (BSF) Hermetia illucens is receiving increasing attention as a sustainable fishmeal alternative protein source for aquaculture. To date, no studies have explored the effects of fishmeal replacement with BSF V instar larvae or prepupae meals due to their peculiar nutritional properties on fish performances. This study investigated the effects of 100% replacement of fishmeal (control diet) with defatted BSF meals (V instar larvae and prepupae meals, treatments) on growth performance and welfare of zebrafish (Danio rerio), from larvae to adults, in a 60-day feeding trial. Following the inclusion of BSF meals, the expression of key genes involved in growth (igf1, igf2, mstnb, myod1, myog, myf5), hydrolysis of chitin (chia.2, chia.3, chia.5), immune- (il1b, il6, tnfα), and stress- (hsp70 and nr3c1) responses, as assessed by qPCR, was modulated in all of the molecular pathways, except for the stress response. Overall, our findings showed that both BSF meals can totally replace fishmeal without adverse impacts on adult zebrafish growth parameters (final total and standard length, final body weight, weight gain, daily growth rate, specific growth rate) and welfare, with BSF prepupae meal inducing the most beneficial effects, thus suggesting their potential application to meet fish requirements in aquaculture.

Waste Valorization via Hermetia Illucens to Produce Protein-Rich Biomass for Feed: Insight into the Critical Nutrient Taurine.

Insects have been recognized as sustainable alternative sources of nutrients for food and feed. The Black Soldier Fly (BSF), Hermetia illucens, is a particularly promising species for its great potential in the waste valorization to produce, during the bioconversion process, high-value fat and proteins that currently represent a valuable source for fish feed. The present study aims to evaluate the efficiency to use substrate proteins in two different BSF developmental stages as sustainable biotechnological tools for vegetable waste management. We provide insights into the nutritional values of both V instar larvae and prepupae in terms of valuable amino acids with special focus on taurine, a crucial nutrient for fish. Moreover, we cloned four key genes from BSF involved in the taurine biosynthesis pathway, 2-aminoethanethiol dioxygenase (Hiado), cysteine dioxygenase (Hicdo), cysteine sulfonate decarboxylase (Hicsad), and glutamate decarboxylase (Higad). The gene expression analysis in larvae and prepupae by qPCR showed development-specific profiles suggesting they influence the taurine content during BSF development. These findings showed peculiar phenotypes in larvae and prepupae that can be selected for different biotechnological applications as sustainable source of relevant amino acids and taurine to support the increasing demand for animal feed and aquafeed in the next decades.

Hermetia illucens (Diptera: Stratiomydae) larvae and prepupae: Biomass production, fatty acid profile and expression of key genes involved in lipid metabolism.

The Black Soldier Fly (BSF) Hermetia illucens provides a promising strategy in the waste valorisation process and a sustainable alternative source of valuable nutrients, including lipids for food and feed. In the present study, the differences in growth performances and nutritional values of BSF V instar larvae and prepupae reared on vegetable waste were analyzed and compared focusing on fat content. V instar larvae showed higher capacity to bioconvert the substrate into biomass than prepupae. The nutritional composition and the fatty acid profiles were dependent on the developmental stage. The expression levels of acetyl-CoA carboxylase (acc), fatty acid synthase (fas), lipase (lip) and acyl-CoA dehydrogenase (acd) genes involved in the lipid metabolism pathway and herein characterized for the first time, were evaluated in order to understand the molecular basis underlying the observed differences in fatty acid profiles. Our results suggest that the different fatty acid profiles of BSF V instar larvae and prepupae may be related to the modulation of the lipid metabolism-related genes expression during larval development. Our study highlights substantial differences between H. illucens V instar larvae and prepupae giving important features regarding the opportunity to modulate the preferable fatty acid profile to meet the industrial requirements.

Two anion transporters AtClCa and AtClCe fulfil interconnecting but not redundant roles in nitrate assimilation pathways.

* In plants, the knowledge of the molecular identity and functions of anion channels are still very limited, and are almost restricted to the large ChLoride Channel (CLC) family. In Arabidopsis thaliana, some genetic evidence has suggested a role for certain AtCLC protein members in the control of plant nitrate levels. In this context, AtClCa has been demonstrated to be involved in nitrate transport into the vacuole, thereby participating in cell nitrate homeostasis. * In this study, analyses of T-DNA insertion mutants within the AtClCa and AtClCe genes revealed common phenotypic traits: a lower endogenous nitrate content; a higher nitrite content; a reduced nitrate influx into the root; and a decreased expression of several genes encoding nitrate transporters. * This set of nitrate-related phenotypes, displayed by clca and clce mutant plants, showed interconnecting roles of AtClCa and AtClCe in nitrate homeostasis involving two different endocellular membranes. * In addition, it revealed cross-talk between two nitrate transporter families participating in nitrate assimilation pathways. The contribution to nitrate homeostasis at the cellular level of members of these different families is discussed.

Responses of marine mussel Mytilus galloprovincialis (Bivalvia: Mytilidae) after infection with the pathogen Vibrio splendidus.

Bivalve molluscs possess effective cellular and humoral defence mechanisms against bacterial infection. Although the immune responses of mussels to challenge with pathogenic vibrios have been largely investigated, the effects at the site of injection at the tissue level have not been so far evaluated. To this aim, mussels Mytilus galloprovincialis were herein in vivo challenged with Vibrio splendidus to assess the responses induced in hemolymph and posterior adductor muscle (PAM), being the site of bacterial infection. The number of living intra-hemocyte bacteria increased after the first hour post-injection (p.i.), suggesting the occurrence of an intense phagocytosis, while clearance was observed within 24 h p.i. A recruitment of hemocytes at the injection site was found in mussel PAM, together with marked morphological changes in the volume of muscular fibers, with a recovery of muscle tissue organization after 48 h p.i. A concomitant impairment in the osmoregulatory processes were observed in PAM by an initial inhibition of aquaporins and increased immunopositivity of Na+/K+ ATPase ionic pump, strictly related to the histological alterations and hemocyte infiltration detected in PAM. Accordingly, an intense cell turnover activity was also recorded following the infection event. Overall, results indicated the hemolymph as the system responsible for the physiological adaptations in mussels to stressful factors, such as pathogenicity, for the maintenance of homeostasis and immune defence. Also, the osmotic balance and cell turnover can be used as objective diagnostic criteria to evaluate the physiological state of mussels following bacterial infection, which may be relevant in aquaculture and biomonitoring studies.

Baseline levels of metabolites in different tissues of mussel Mytilus galloprovincialis (Bivalvia: Mytilidae).

The Mediterranean mussel Mytilus galloprovincialis (Lamarck 1819) is a popular shellfish commonly included in human diet and is routinely used as bioindicator in environmental monitoring programmes worldwide. Recently, metabolomics has emerged as a powerful tool both in food research and ecotoxicology for monitoring mussels' freshness and assessing the effects of environmental changes. However, there is still a paucity of data on complete metabolic baseline of mussel tissues. To mitigate this knowledge gap, similarities and differences in metabolite profile of digestive gland (DG), gills (G), and posterior adductor muscle (PAM) of aquaculture-farmed M. galloprovincialis were herein investigated by a proton nuclear magnetic resonance (1H NMR)-based metabolomic approach and discussed considering their physiological role. A total of 44 metabolites were identified in mussel tissues and grouped in amino acids, energy metabolites, osmolytes, neurotransmitters, nucleotides, alkaloids, and miscellaneous metabolites. A PCA showed that mussel tissues clustered separately from each other, suggesting a clear differentiation in their metabolic profiles. A Venn diagram revealed that mussel DG, G and PAM shared 27 (61.36%) common metabolites, though with different concentrations. Osmolytes were found to dominate the metabolome of all tissues. The DG exhibited higher level of glutathione and carbohydrates. The G showed greater level of osmolytes and the exclusive presence of neurotransmitters, namely acetylcholine and serotonin. In PAM higher levels of energetics-related metabolites were found. Overall, findings from this study are helpful for a better understanding of mussel tissue-specific physiological functions as well as for future NMR-based metabolomic investigations of marine mussel health and safety.

Copper oxide nanoparticles induce the transcriptional modulation of oxidative stress-related genes in Arbacia lixula embryos.

Copper oxide nanoparticles (CuO NPs) are widely used in various industrial applications, i.e. semiconductor devices, batteries, solar energy converter, gas sensor, microelectronics, heat transfer fluids, and have been recently recognized as emerging pollutants of increasing concern for human and marine environmental health. Therefore, the toxicity of CuO NPs needs to be thoroughly understood. In this study, we evaluated the potential role of oxidative stress in CuO NP toxicity by exploring the molecular response of Arbacia lixula embryos to three CuO NP concentrations (0.7, 10, 20 ppb) by investigating the transcriptional patterns of oxidative stress-related genes (catalase and superoxide dismutase) and metallothionein, here cloned and characterized for the first time. Time- and concentration-dependent changes in gene expression were detected in A. lixula embryos exposed to CuO NPs, up to pluteus stage (72 h post-fertilization, hpf), indicating that oxidative stress is one of the toxicity mechanisms for CuO NPs. These findings provide new insights into the comprehension of the molecular mechanisms underlying copper nanoparticle toxicity in A. lixula sea urchin and give new tools for monitoring of aquatic areas, thus corroborating the suitability of this embryotoxicity assay for future evaluation of impacted sites.

Molecular characterization and expression analysis of heat shock protein 70 and 90 from Hermetia illucens reared in a food waste bioconversion pilot plant.

Two full-length cDNAs of heat shock protein (HSP) genes (Hihsp70 and Hihsp90) were cloned from the black soldier fly (BSF) Hermetia illucens larvae reared in a food waste bioconversion pilot plant. The Hihsp70 and Hihsp90 transcripts were 2243 and 2507bp long, contained 1923 and 2166bp open reading frames encoding proteins of 640 and 721 amino acids with a molecular mass of 69.8 and 83kDa, respectively. Comparative analysis of protein sequences revealed the presence of the conserved HSP motifs in both proteins, showing high homology to their counterparts in other insect species from six different orders. Hihsp70 and Hihsp90 transcriptional expression profiles during two key developmental stages in the bioconversion process were evaluated by quantitative real time PCR showing that both genes were modulated during larval development. HiHsp70 mRNA expression levels during the II instar larvae was higher in respect to the V instar larvae. A similar difference in mRNA expression levels, but in a less extent, was found for the Hihsp90. Moreover, a diverse transcript level between the two genes at the V larval stage was observed where Hihsp90 was up-regulated compared to Hihsp70. These results suggested the involvement of Hsp70 and Hsp90 in H. illucens development and provide further evidences on the ecological and evolutionary importance of HSPs in the insect developmental processes together with valuable information on molecular features of adaptability to peculiar rearing conditions during food waste bioconversion.

Effects of Oxygen Availability on Oxidative Stress Biomarkers in the Mediterranean Mussel Mytilus galloprovincialis.

In aquatic environments, hypoxia and oxygen-deficient areas are increasing worldwide. Transitions in oxygen levels can influence the production of reactive oxygen species (ROS), eventually leading to oxidative stress. The transcriptional response of oxidative stress biomarkers was evaluated by qPCR in gill tissue from Mytilus galloprovincialis experimentally subjected to 48-h air exposure followed by 48-h re-oxygenation, as compared to normoxic control mussels. Superoxide dismutases (CuZnsod and Mnsod), catalase (cat), and glutathione S-transferase (gst) were over-expressed early after 8-h air exposure and returned to normoxic levels during re-oxygenation. Moreover, the mRNAs and protein expression patterns of heat shock proteins (HSP70 and HSP90) and metallothioneins (MT-10 and MT-20) were modulated by oxygen availability with increased levels during re-oxygenation suggesting the participation of these cytoprotective mechanisms in the physiological oxidative stress response when oxygen concentration was restored. Overall, the observed modulation of the oxidative stress-related and general stress genes indicates that M. galloprovincialis responds to changes in oxygen availability enhancing the antioxidant potential under low oxygen conditions for dealing with the oxidative burst during future re-oxygenation. The present investigation brings further insights in understanding how intertidal molluscs cope with short-term oxygen variations and gives useful biomarkers for environmental monitoring of hypoxic areas that are predicted to occur in the next future.

Alteration of neurotransmission and skeletogenesis in sea urchin Arbacia lixula embryos exposed to copper oxide nanoparticles.

The extensive use of copper oxide nanoparticles (CuO NPs) in many applications has raised concerns over their toxicity on environment and human health. Herein, the embryotoxicity of CuO NPs was assessed in the black sea urchin Arbacia lixula, an intertidal species commonly present in the Mediterranean. Fertilized eggs were exposed to 0.7, 10 and 20ppb of CuO NPs, until pluteus stage. Interferences with the normal neurotransmission pathways were observed in sea urchin embryos. In detail, evidence of cholinergic and serotoninergic systems affection was revealed by dose-dependent decreased levels of choline and N-acetyl serotonin, respectively, measured by nuclear magnetic resonance (NMR)-based metabolomics, applied for the first time to our knowledge on sea urchin embryos. The metabolic profile also highlighted a significant CuO NP dose-dependent increase of glycine, a component of matrix proteins involved in the biomineralization process, suggesting perturbed skeletogenesis accordingly to skeletal defects in spicule patterning observed previously in the same sea urchin embryos. However, the expression of skeletogenic genes, i.e. SM30 and msp130, did not differ among groups, and therefore altered primary mesenchyme cell (PMC) migration was hypothesized. Other unknown metabolites were detected from the NMR spectra, and their concentrations found to be reflective of the CuO NP exposure levels. Overall, these findings demonstrate the toxic potential of CuO NPs to interfere with neurotransmission and skeletogenesis of sea urchin embryos. The integrated use of embryotoxicity tests and metabolomics represents a highly sensitive and effective tool for assessing the impact of NPs on aquatic biota.

Effects of petrochemical contamination on caged marine mussels using a multi-biomarker approach: Histological changes, neurotoxicity and hypoxic stress.

This work was designed to evaluate the biological effects of petrochemical contamination on marine mussels. Mytilus galloprovincialis, widely used as sentinel organisms in biomonitoring studies, were caged at the "Augusta-Melilli-Priolo" industrial site (eastern Sicily, Italy), chosen as one of the largest petrochemical areas in Europe, and Brucoli, chosen as reference site. Chemical analyses of sediments at the polluted site revealed high levels of PAHs and mercury, exceeding the national and international guideline limits. In mussels from the polluted site, severe morphological alterations were observed in gills, mainly involved in nutrient uptake and gas exchange. Changes in serotonergic and cholinergic systems, investigated through immunohistochemical, metabolomics and enzymatic approaches, were highlighted in gills, as well as onset of hypoxic adaptive responses with up-regulation of hypoxia-inducible factor transcript. Overall, the application of a multi-biomarker panel results effective in assessing the biological effects of petrochemical contamination on the health of aquatic organisms.

PCB and OCP accumulation and evidence of hepatic alteration in the Atlantic bluefin tuna, T. thynnus, from the Mediterranean Sea.

Persistent organic pollutants (POPs) are known to act as "obesogens", being fat-soluble and affecting lipid metabolism. The Atlantic bluefin tuna, Thunnus thynnus, are top pelagic predators prone to bioaccumulate and biomagnify environmental contaminants. This study aimed at evaluating POPs-induced ectopic lipid accumulation in liver of adult tuna from the Mediterranean Sea. PCBs and organochlorine pesticides were measured in tuna liver, and marked morphological changes observed, namely poorly compacted tissues, intense vacuolization, erythrocyte infiltration and presence of melanomacrophages. The expression of perilipin, a lipid-droplet marker, positively correlated with the gene expression of PPARγ, a master regulator of adipogenesis, and its heterodimeric partner, RXRα. Changes in metabolites involved in fatty acid biosynthesis and ketogenesis were also observed. Although male bluefin tuna appeared to be more sensitive than females to the adverse effects of environmental obesogens, the alterations observed in tuna liver of both sexes suggest a potential onset of hepatic steatosis.

Hypoxia-Inducible Factor α and Hif-prolyl Hydroxylase Characterization and Gene Expression in Short-Time Air-Exposed Mytilus galloprovincialis.

Aquatic organisms experience environmental hypoxia as a result of eutrophication and naturally occurring tidal cycles. Mytilus galloprovincialis, being an anoxic/hypoxic-tolerant bivalve, provides an excellent model to investigate the molecular mechanisms regulating oxygen sensing. Across the animal kingdom, inadequacy in oxygen supply is signalled predominantly by hypoxia-inducible factors (HIF) and Hif-prolyl hydroxylases (PHD). In this study, hif-α 5'-end and partial phd mRNA sequences from M. galloprovincialis were obtained. Phylogenetic and molecular characterization of both HIF-α and PHD putative proteins showed shared key features with the respective orthologues from animals strongly suggesting their crucial involvement in the highly conserved oxygen sensing pathway. Both transcripts displayed a tissue-specific distribution with prominent expression in gills. Quantitative gene expression analysis of hif-α and phd mRNAs from gills of M. galloprovincialis demonstrated that both these key sensors are transcriptionally modulated by oxygen availability during the short-time air exposure and subsequent re-oxygenation treatments proving that they are critical players of oxygen-sensing mechanisms in mussels. Remarkably, hif-α gene expression showed a prompt and transient response suggesting the precocious implication of this transcription factor in the early phase of the adaptive response to hypoxia in Mytilus. HIF-α and PHD proteins were modulated in a time-dependent manner with trends comparable to mRNA expression patterns, thus suggesting a central role of their transcriptional regulation in the hypoxia tolerance strategies in marine bivalves. These results provide molecular information about the effects of oxygen deficiency and identify hypoxia-responsive biomarker genes in mussels applicable in ecotoxicological studies of natural marine areas.

Influence of continuous light treatment on expression of stress biomarkers in Atlantic cod.

Continuous light treatment during early juvenile stages in Gadus morhua is a common farming management practice but the effects of these unnatural light conditions on fish stress have received scant attention. In the present study we investigated how continuous illumination affects transcription levels of key stress-related and antimicrobial peptide genes in juvenile Atlantic cod. Gene expression quantification by real-time PCR revealed higher levels of transcripts coding for antioxidant enzymes, namely superoxide dismutase, catalase and glutathione reductase in liver of fish reared under continuous illumination, concomitantly with a 43% decrease in glutathione content. Transcription of antimicrobial peptides such as piscidins, hepcidin and cathelicidin was also affected by constant illumination. Overall, the significant changes in liver transcript levels of these biomarkers in response to continuous light may be an adaptation to light stress.

Pseudomonas aeruginosa induces interleukin-8 (IL-8) gene expression in human conjunctiva through the recruitment of both RelA and CCAAT/enhancer-binding protein beta to the IL-8 promoter.

The purpose of this study was to identify the Pseudomonas aeruginosa-activated signaling pathway leading to interleukin (IL)-8 gene expression and protein synthesis by human conjunctival epithelium. IL-8 protein and mRNA were determined by enzyme-linked immunosorbent assay and reverse transcription-PCR, respectively. Activation of MAPKs and NF-kappaB was analyzed by Western blotting using phosphospecific antibodies. We used transfection with wild-type or mutated IL-8 promoters and cotransfection with transcription factor overexpressing plasmids or small interfering RNAs. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) were performed for in vitro and in vivo protein-DNA binding studies, respectively. P. aeruginosa increased IL-8 expression at the transcriptional level by phosphorylating CCAAT/enhancer-binding protein beta (C/EBPbeta) via p38MAPK and activating NF-kappaB. The simultaneous involvement of RelA and C/EBPbeta and the integrity of the corresponding consensus sites were required, whereas c-Jun was involved only in basal IL-8 expression. Re-ChIP experiments showed that RelA and C/EBPbeta act together at the IL-8 promoter level upon P. aeruginosa infection. Taken together, our results suggest that P. aeruginosa induces IL-8 promoter expression and protein production in conjunctival epithelial cells by activating RelA and C/EBPbeta and by promoting the cooperative binding of these transcription factors to the IL-8 promoter that in turn activates transcription.