Mushroom body-preferential expression of proteins/genes involved in endoplasmic reticulum Ca(2+)-transport in the worker honeybee (Apis mellifera L.) brain.

To identify the molecular characteristics specific to the mushroom body (MB, a higher processing centre) neurones in the honeybee brain, we previously used proteomics to identify proteins that are preferentially expressed in these MBs. Here we continued our proteomic analysis to show that reticulocalbin, which is involved in endoplasmic reticulum (ER) Ca(2+) transport, is also preferentially expressed in the MBs in the honeybee brain. Gene expression analysis revealed that reticulocalbin is preferentially expressed in the large-type Kenyon cells, which are MB-intrinsic neurones. In addition, the gene for the ryanodine receptor, which is also involved in ER Ca(2+) transport, was also preferentially expressed in the large-type Kenyon cells. In contrast, the expression of three other ER-related genes, protein disulphide isomerase, sec61 and erp60, was not enriched in the MBs. These findings further support the notion that the function of ER Ca(2+)-signalling, but not the mere intracellular density of ER, is specifically enhanced in the large-type Kenyon cells in the honeybee brain.

Evaluation of innate immune stimulating activity of polysaccharides using a silkworm (Bombyx mori) muscle contraction assay.

In silkworm larvae, the mature form of paralytic peptide (PP), an insect cytokine, is produced from pro-PP in association with activation of innate immune responses, resulting in slow muscle contraction. We utilized this reaction, muscle contraction in silkworms coupled with innate immunity stimulation, to quantitatively measure the innate immune stimulating activity of various natural polysaccharides. ß-Glucan of Gyrophora esculenta (GE-3), fucoidan from sporophyll of Undaria pinnatifida, and curldan induced silkworm muscle contraction. We further demonstrated that GE-3 had therapeutic effects on silkworms infected by baculovirus. Based on these findings, we propose that the silkworm muscle contraction assay is useful for screening substances that stimulate innate immunity before evaluating therapeutic effectiveness in mammals.

Evaluation of therapeutic effects and pharmacokinetics of antibacterial chromogenic agents in a silkworm model of Staphylococcus aureus infection.

The therapeutic effect of dye compounds with antibacterial activity was evaluated in a silkworm model of Staphylococcus aureus infection. Among 13 chromogenic agents that show antibacterial activity against S. aureus (MIC = 0.02 to 19 µg/mL), rifampicin had a therapeutic effect. The ED(50) value in the silkworm model was consistent with that in a murine model. Other 12 dyes did not increase survival of the infected silkworms. We examined the reason for the lack of therapeutic efficacy. Amidol, pyronin G, and safranin were toxic to silkworms, which explained the lack of therapeutic effects. Fuchsin basic and methyl green disappeared quickly from the hemolymph after injection, suggesting that they are not stable in the hemolymph. Although coomassie brilliant blue R250/G250, cresyl blue, and nigrosin showed no toxic effects or instability in the hemolymph, they also did not have a therapeutic effect. The in vitro antibacterial actions of these dyes were inhibited by silkworm plasma or bovine serum albumin and filtration experiments demonstrated that cresyl blue bound to plasma proteins in the silkworm, suggesting that plasma protein binding inhibited the therapeutic efficacy of these four dyes. These findings indicate that drug screening using the silkworm infection model is useful for evaluating toxicity and pharmacokinetics of potential antibiotics.

Carbohydrate metabolism genes and pathways in insects: insights from the honey bee genome.

Carbohydrate-metabolizing enzymes may have particularly interesting roles in the honey bee, Apis mellifera, because this social insect has an extremely carbohydrate-rich diet, and nutrition plays important roles in caste determination and socially mediated behavioural plasticity. We annotated a total of 174 genes encoding carbohydrate-metabolizing enzymes and 28 genes encoding lipid-metabolizing enzymes, based on orthology to their counterparts in the fly, Drosophila melanogaster, and the mosquito, Anopheles gambiae. We found that the number of genes for carbohydrate metabolism appears to be more evolutionarily labile than for lipid metabolism. In particular, we identified striking changes in gene number or genomic organization for genes encoding glycolytic enzymes, cellulase, glucose oxidase and glucose dehydrogenases, glucose-methanol-choline (GMC) oxidoreductases, fucosyltransferases, and lysozymes.

Identification of a novel gene, Mblk-1, that encodes a putative transcription factor expressed preferentially in the large-type Kenyon cells of the honeybee brain.

Mushroom bodies (MBs) are considered to be involved in higher-order sensory processing in the insect brain. To identify the genes involved in the intrinsic function of the honeybee MBs, we searched for genes preferentially expressed therein, using the differential display method. Here we report a novel gene encoding a putative transcription factor (Mblk-1) expressed preferentially in one of two types of intrinsic MB neurones, the large-type Kenyon cells, which makes Mblk-1 a candidate gene involved in the advanced behaviours of honeybees. A putative DNA binding motif of Mblk-1 had significant sequence homology with those encoded by genes from various animal species, suggesting that the functions of these proteins in neural cells are conserved among the animal kingdom.