A C-type lectin with dual carbohydrate recognition domains functions in innate immune response in Asian corn borer, Ostrinia furnacalis.

C-type lectins (CTLs) act as pattern recognition receptors (PRRs) to initiate the innate immune response in insects. A CTL with dual carbohydrate recognition domains (CRDs) (named immulectin-4 [IML-4]) was selected from the Ostrinia furnacalis transcriptome dataset for functional studies. We cloned the full-length complementary DNA of O. furnacalis IML-4 (OfIML-4). It encodes a 328-residue protein with a Glu-Pro-Asn (EPN) and Gln-Pro-Asp (QPD) motifs in 2 CRDs, respectively. OfIML-4 messenger RNA levels increased significantly upon the bacterial and fungal infection. Recombinant OfIML-4 (rIML-4) and its individual CRDs (rCRD1 and rCRD2) exhibited the binding ability to various microorganisms including Escherichia coli, Micrococcus luteus, Pichia pastoris, and Beauveria bassiana, and the cell wall components including lipopolysaccharide from E. coli, peptidoglycan from M. luteus or Bacillus subtilis, and curdlan from Alcaligenes faecalis. The binding further induced the agglutination of E. coli, M. luteus, and B. bassiana in the presence of calcium, the phagocytosis of Staphylococcus aureus by the hemocytes, in vitro encapsulation and melanization of nickel-nitrilotriacetic acid beads, and a significant increase in phenoloxidase activity of plasma. In addition, rIML-4 significantly enhanced the phagocytosis, nodulation, and resistance of O. furnacalis to B. bassiana. Taken together, our results suggest that OfIML-4 potentially works as a PRR to recognize the invading microorganisms, and functions in the innate immune response in O. furnacalis.

PLA2 mediates the innate immune response in Asian corn borer, Ostrinia furnacalis.

The eicosanoid signaling pathway mediates insect immune reactions to a wide range of stimuli. This pathway begins with the biosynthesis of arachidonic acid (AA) from the hydrolysis of phospholipids catalyzed by phospholipase A2 (PLA2 ). We report here that the PLA2 inhibitor, dexamethasone (DEX), impaired the innate immune response including nodulation, encapsulation, and melanization in Ostrinia furnacalis larvae, while AA partially reversed these effects of DEX. We cloned a full-length complementary DNA encoding a PLA2 , designated as OfsPLA2 , from O. furnacalis. The open reading frame of OfsPLA2 encodes a 195-amino acid residue protein with a 22-residue signal peptide. Sequence alignment analyses indicated that O. furnacalis PLA2 might be a Group III secretory PLA2 . The highest transcript levels of OfsPLA2 were detected in the fat body, and its transcript levels increased dramatically after infection with Escherichia coli, Micrococcus luteus, or Beauveria bassiana. Recombinant OfsPLA2 significantly induced prophenoloxidase (PPO) activation in larval hemolymph in the presence of Ca2+ and encapsulation of agarose beads. Injection of recombinant OfsPLA2 into larvae resulted in increased transcript levels of attacin, defencin, and moricin-3 genes. Our results demonstrate the involvement of the eicosanoid signaling pathway in the innate immune response of O. furnacalis larvae and provide new information about the roles of O. furnacalis secretory PLA2 in activating PPO and antimicrobial peptide production.

Determination of low-level ink photoinitiator residues in packaged milk by solid-phase extraction and LC-ESI/MS/MS using triple-quadrupole mass analyzer.

A confirmatory and quantitative method based on liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) has been developed for simultaneous determination of seven photoinitiator residues: benzophenone, (1-hydroxycyclohexyl)phenylketone (Irgacure 184), isopropylthioxanthone (ITX), 2-ethylhexyl-(4-dimethylamino)benzoate (EHA or EHDAB), 2-methyl-1-[4-(methylthio)phenyl]-2-(4-morpholinyl)-1-propanone (Irgacure 907), (2,4,6-trimethylbenzoyl)diphenylphosphine oxide (TPO) and 2-benzyl-2-(dimethylamino)-1-(4-morpholinophenyl)-1-butanone (Irgacure 369) in packaged milk and related packaging materials. Residues of photoinitiators were extracted from milk using acetonitrile, and further enriched and purified on HLB solid-phase extraction cartridges prior to being analyzed by LC-ESI/MS/MS with selected reaction monitoring mode, while photoinitiators in packaging materials were extracted using the same solvent. Satisfactory recovery (from 80 to 111%), intra- and inter-day precision (below 12%), and low limits of quantification (from 0.1 to 5.0 microg kg(-1)) were evaluated from spiked samples at three concentration levels (5.0, 10.0 and 25.0 microg kg(-1) for Irgacure 184 and 2.5, 5.0 and 25.0 microg kg(-1) for others). These excellent validation data suggested the possibility of using the LC-ESI/MS/MS method for simultaneous determination of low-level photoinitiator residues migrating from printed food-packaging materials into milk. The method has been successfully applied to the analysis of real samples of different fat contents ranging from 8 to 30 g L(-1). The photoinitiator residues were revealed to be higher in milk with higher fat content and the most important contaminations were benzophenone and ITX in concentration ranges of 2.84-18.35 and 0.83-8.87 microg kg(-1), respectively.