A molluscan IRF interacts with IKKα/ß family protein and modulates NF-κB and MAPK activity.

Interferon regulatory factor (IRF) family proteins are key transcription factors involved in vital physiological processes such as immune defense. However, the function of IRF in invertebrates, especially in marine shellfish is not clear. In this study, a new IRF gene (CfIRF2) was identified in the Zhikong scallop, Chlamys farreri, and its immune function was analyzed. CfIRF2 has an open reading frame of 1107 bp encoding 368 amino acids. The N-terminus of CfIRF2 consists of a typical IRF domain, with conserved amino acid sequences. Phylogenetic analysis suggested close evolutionary relationship with shellfish IRF1 subfamily proteins. Expression pattern analysis showed that CfIRF2 mRNA was expressed in all tissues, with the highest expression in the hepatopancreas and gills. CfIRF2 gene expression was substantially enhanced by a pathogenic virus (such as acute viral necrosis virus) and poly(I:C) challenge. Co-immunoprecipitation assay identified CfIRF2 interaction with the IKKα/ß family protein CfIKK1 of C. farreri, demonstrating a unique signal transduction mechanism in marine mollusks. Moreover, CfIRF2 interacted with itself to form homologous dimers. Overexpression of CfIRF2 in HEK293T cells activated reporter genes containing interferon stimulated response elements and NF-κB genes in a dose-dependent manner and promoted the phosphorylation of protein kinases (JNK, Erk1/2, and P38). Our results provide insights into the functions of IRF in mollusks innate immunity and also provide valuable information for enriching comparative immunological theory for the prevention of diseases in scallop farming.

Molecular characterization and functional analysis of a mollusk Rel homologous gene.

Members of the nuclear factor-kappa B (NF-κB) family are crucial regulators of physiological processes such as apoptosis, inflammation, and the immune response, acting as vital transcription factors to perform their function. In this study, we identified a NF-κB homologous gene (CfRel1) in Zhikong scallops. The 3006-bp-long open reading frame encodes 1001 amino acids. The N-terminus of the CfRel1 protein harbors a conserved Rel homology domain (RHD) that contains a DNA-binding domain and a dimerization domain. According to the multiple sequence alignment results, both the DNA-binding and dimerization domains are highly conserved. Phylogenetic analysis indicated that CfRel1 is closely related to both the Dorsal protein of Pinctada fucata and the Rel2 protein of Crassostrea gigas. CfRel1 mRNA was expressed in all tissues tested in the quantitative reverse transcription PCR experiments, with hepatopancreatic tissue expressing the highest levels. Furthermore, after stimulation with lipopolysaccharide, peptidoglycan, or polyinosinic:polycytidylic acid, the mRNA expression level of CfRel1 was markedly increased. The co-immunoprecipitation test results showed that CfRel1 interacted with scallop IκB protein through its RHD DNA-binding domain, suggesting that IκB may regulate the activity of Rel1 by binding to this domain. Dual-luciferase reporter gene assays revealed that CfRel1 overexpression in HEK293T cells activated the activator protein 1 (AP-1), NF-κB, interferon (IFN)α, IFNß, and IFNγ reporter genes, indicating the diverse functions of the protein. In summary, CfRel1 is capable of responding to attacks from pathogen-associated molecular patterns, participating in immune signaling, and activating NF-κB and IFN reporter genes. Our findings contribute to the advancement of invertebrate innate immunity theory, enrich the theory of comparative immunology, and serve as a reference for the future screening of disease-resistant strains in scallops.

Performance effects of feed-borne Fusarium mycotoxins on broiler chickens: Influences of timing and duration of exposure.

In commercial practice, broiler chickens may be exposed to Fusarium mycotoxins either during specific growth stages or throughout the entire production cycle. A 34-day feeding trial was conducted to identify sensitive periods for mycotoxin effects during the growth cycle of broiler chickens. A total of 420 newly-hatched Ross 308 male broilers were randomly assigned to 60 cages with 7 birds/cage. Sources of clean wheat (<0.5 mg/kg deoxynivalenol [DON]) and Fusarium-contaminated wheat (11.4 mg/kg DON) were used to formulate the starter diets (0.41 and 6.62 mg/kg DON) provided from 1 to 21 d of age and the grower diets (0.54 and 7.90 mg/kg DON) provided from 22 to 34 d. Control and DON diets were provided to broilers according to treatments (control, DON 1 to 14 d, DON 15 to 21 d, DON 22 to 34 d and DON 1 to 34 d). Birds were monitored daily for morbidity or mortality. Broiler growth performance (body weight, average daily gain, average daily feed intake and feed to gain ratio) was measured weekly. Segments of duodenum, jejunum and ileum were collected at 21 and 34 d and morphometric parameters (villus height, crypt depth, villus width, muscularis thickness and villi:crypt ratio) were measured. Birds fed the DON starter diet during the first 14 d did not exhibit any changes in growth performance; however, growth performance was suppressed in birds fed DON-contaminated diets during the grower period (22 to 34 d). At 34 d, birds that received the DON grower diet (DON 22 to 34 d and DON 1 to 34 d) were lighter (1,433 vs. 1,695 g) than birds fed the control diet. Feed to gain ratio was higher in birds fed the DON grower diet from 22 to 28 d (1.77 vs. 1.56) and 28 to 34 d (2.24 vs. 1.85) compared with corresponding controls. These results suggest that providing older broiler chicks (22 to 34 d) feed contaminated with Fusarium mycotoxins (specifically DON) may result in production losses. Histopathological analysis of the ileum region revealed that birds provided the DON diets throughout the entire trial (1 to 34 d) had shorter villi (506 vs. 680 µm) and shallower crypt (85 vs. 115 µm) than control birds. Taken together, these results indicate that DON-induced growth suppression may be a result of adverse effects on intestinal morphology during later growth phases of broilers.