A Comparison of the Antibacterial Efficacy of Carbohydrate Lipid-like (Thio)Ether, Sulfone, and Ester Derivatives against Paenibacillus larvae.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), the most serious bacterial disease affecting developing honeybee larvae and pupas. In this study, a library of 24 (thio)glycosides, glycosyl sulfones, 6-O-esters, and ethers derived from d-mannose, d-glucose, and d-galactose having C10 or C12 alkyl chain were evaluated for their antibacterial efficacy against two P. larvae strains. The efficacy of the tested compounds determined as minimal inhibitory concentrations (MICs) varied greatly. Generally, dodecyl derivatives were found to be more potent than their decylated analogs. Thioglycosides were more efficient than glycosides and sulfones. The activity of the 6-O-ether derivatives was higher than that of their ester counterparts. Seven derivatives with dodecyl chain linked (thio)glycosidically or etherically at C-6 showed high efficacy against both P. larvae strains (MICs ranged from 12.5 µM to 50 µM). Their efficacies were similar or much higher than those of selected reference compounds known to be active against P. larvae-lauric acid, monolaurin, and honeybee larval food components, 10-hydroxy-2-decenoic acid, and sebacic acid (MICs ranged from 25 µM to 6400 µM). The high efficacies of these seven derivatives suggest that they could increase the anti-P. larvae activity of larval food and improve the resistance of larvae to AFB disease through their application to honeybee colonies.

Genomic analysis of Paenibacillus larvae isolates from the Czech Republic and the neighbouring regions of Slovakia.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybee larvae. In the Czech Republic, two large infested regions were recognised. This study aimed to analyse P. larvae strains occurring in the Czech Republic in the years 2016-2017 and to characterise the genetic structure of their population with the use of Enterobacterial Repetitive Intergenic Consensus genotyping (ERIC), multilocus sequence typing (MLST) and whole genome sequence (WGS) analysis. The results were complemented by the analysis of isolates collected in the year 2018 in areas of Slovakia located near the Czechia-Slovakia border. ERIC genotyping revealed that 78.9% of tested isolates belonged to the ERIC II genotype and 21.1% to ERIC I genotype. MLST showed six sequence types with ST10 and ST11 being the most frequent among isolates. Within six isolates we found discrepancies in correlations between MLST and ERIC genotypes. The use of MLST and WGS analysis of isolates revealed that each of the large infested geographic regions had its own dominating P. larvae strains. We assume that these strains represented primary sources of infection in the affected areas. In addition, the sporadic presence of strains identified by core genome analysis as genetically related was unveiled in geographically distant regions suggesting possible human-mediated transmission of AFB.

Fructose and Trehalose Selectively Enhance In Vitro Sporulation of Paenibacillus larvae ERIC I and ERIC II Strains.

Paenibacillus larvae is a Gram-positive bacterium, the spores of which are the causative agent of the most destructive brood disease of honeybees, American foulbrood (AFB). Obtaining viable spores of pathogen strains is requisite for different studies concerning AFB. The aim of this work was to investigate the effects of five saccharides that may naturally occur in higher amounts in bee larvae on in vitro sporulation of P. larvae. The effect of individual saccharides at different concentrations on spore yields of P. larvae strains of epidemiologically important ERIC genotypes was examined in Columbia sheep blood agar (CSA) and MYPGP agar media. It was found that fructose in ERIC I and trehalose in ERIC II strains at concentrations in the range of 0.5-2% represent new sporulation factors that significantly enhanced the yields of viable spores in both media, mostly in a concentration-dependent manner. The enhancements in spore yield were mainly caused by improvements of the germination ability of the spores produced. Glucose, maltose and sucrose at 1% or 0.5% concentrations also supported sporulation but to a lower extent and not in all strains and media. Based on the knowledge gained, a novel procedure was proposed for the preparation of viable P. larvae spores with supposed improved quality for AFB research.

Antibacterial Activity of Different Blossom Honeys: New Findings.

Antibacterial activity is the most investigated biological property of honey. The goal of this study was to evaluate the antibacterial activity of 57 Slovak blossom honeys against Staphylococcus aureus and Pseudomonas aeruginosa and investigate the role of several bioactive substances in antibacterial action of honeys. Inhibitory and bactericidal activities of honeys were studied to determine the minimum inhibitory and bactericidal concentrations. The contents of glucose oxidase (GOX) enzyme, hydrogen peroxide (H2O2), and total polyphenols (TP) were determined in honeys. We found that honey samples showed different antibacterial efficacy against the tested bacteria as follows: wildflower honeys > acacia honeys > rapeseed honeys. Overall antibacterial activity of the honeys was statistically-significantly correlated with the contents of H2O2 and TP in honeys. A strong correlation was found between the H2O2 and TP content. On the other hand, no correlation was found between the content of GOX and level of H2O2. Antibacterial activity of 12 selected honeys was markedly reduced by treatment with catalase, but it remained relatively stable after inactivation of GOX with proteinase-K digestion. Obtained results suggest that the antibacterial activity of blossom honeys is mainly mediated by H2O2 levels present in honeys which are affected mainly by polyphenolic substances and not directly by GOX content.

Engineering the acceptor substrate specificity in the xyloglucan endotransglycosylase TmXET6.3 from nasturtium seeds (Tropaeolum majus L.).

KEY MESSAGE: The knowledge of substrate specificity of XET enzymes is important for the general understanding of metabolic pathways to challenge the established notion that these enzymes operate uniquely on cellulose-xyloglucan networks. Xyloglucan xyloglucosyl transferases (XETs) (EC 2.4.1.207) play a central role in loosening and re-arranging the cellulose-xyloglucan network, which is assumed to be the primary load-bearing structural component of plant cell walls. The sequence of mature TmXET6.3 from Tropaeolum majus (280 residues) was deduced by the nucleotide sequence analysis of complete cDNA by Rapid Amplification of cDNA Ends, based on tryptic and chymotryptic peptide sequences. Partly purified TmXET6.3, expressed in Pichia occurred in N-glycosylated and unglycosylated forms. The quantification of hetero-transglycosylation activities of TmXET6.3 revealed that (1,3;1,4)-, (1,6)- and (1,4)-ß-D-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-ß-D-xylooligosaccharides, and arabinoxylo- and glucomanno-oligosaccharides were less preferred. The 3D model of TmXET6.3, and bioinformatics analyses of identified and putative plant xyloglucan endotransglycosylases (XETs)/hydrolases (XEHs) of the GH16 family revealed that H94, A104, Q108, K234 and K237 were the key residues that underpinned the acceptor substrate specificity of TmXET6.3. Compared to the wild-type enzyme, the single Q108R and K237T, and double-K234T/K237T and triple-H94Q/A104D/Q108R variants exhibited enhanced hetero-transglycosylation activities with xyloglucan and (1,4)-ß-D-glucooligosaccharides, while those with (1,3;1,4)- and (1,6)-ß-D-glucooligosaccharides were suppressed; the incorporation of xyloglucan to (1,4)-ß-D-glucooligosaccharides by the H94Q variant was influenced most extensively. Structural and biochemical data of non-specific TmXET6.3 presented here extend the classic XET reaction mechanism by which these enzymes operate in plant cell walls. The evaluations of TmXET6.3 transglycosylation activities and the incidence of investigated residues in other members of the GH16 family suggest that a broad acceptor substrate specificity in plant XET enzymes could be more widespread than previously anticipated.

10-HDA, A Major Fatty Acid of Royal Jelly, Exhibits pH Dependent Growth-Inhibitory Activity Against Different Strains of Paenibacillus larvae.

Paenibacillus larvae (P. larvae) is a bacterial pathogen causing American foulbrood (AFB), the most serious disease of honeybee larvae. The food of young larvae could play an important role in the resistance of larvae against AFB. It contains antibacterial substances produced by honeybees that may inhibit the propagation of the pathogen in larval midguts. In this study, we identified and investigated the antibacterial effects of one of these substances, trans-10-hydroxy-2-decenoic acid (10-HDA), against P. larvae strains including all Enterobacterial Repetitive Intergenic Consensus (ERIC) genotypes. Its inhibitory activities were studied by determining the minimum inhibitory concentrations (MICs). It was found that 10-HDA efficacy increases substantially with decreasing pH; up to 12-fold differences in efficacy were observed between pH = 5.5 and pH = 7.2. P. larvae strains showed different susceptibility to 10-HDA; up to 2.97-fold differences existed among various strains with environmentally important ERIC I and ERIC II genotypes. Germinating spores of the pathogen were generally more susceptible to 10-HDA than vegetative cells. Our findings suggest that 10-HDA could play significant role in conferring antipathogenic activity to larval food in the midguts of young larvae and contribute to the resistance of individual larvae to P. larvae.

Characterization of a long-chain α-galactosidase from Papiliotrema flavescens.

α-Galactosidases are assigned to the class of hydrolases and the subclass of glycoside hydrolases (GHs). They belong to six GH families and include the only characterized α-galactosidases from yeasts (GH 27, Saccharomyces cerevisiae). The present study focuses on an investigation of the lactose-inducible α-galactosidase produced by Papiliotrema flavescens. The enzyme was present on the surface of cells and in the cytosol. Its temperature optimum was about 60 °C and the pH optimum was 4.8; the pH stability ranged from 3.2 to 6.6. This α-galactosidase also exhibited transglycosylation activity. The cytosol α-galactosidase with a molecular weight about 110 kDa, was purified using a combination of liquid chromatography techniques. Three intramolecular peptides were determined by the partial structural analysis of the sequences of the protein isolated, using MALDI-TOF/TOF mass spectrometry. The data obtained recognized the first yeast α-galactosidase, which belongs to the GH 36 family. The bioinformatics analysis and homology modeling of a 210 amino acids long C-terminal sequence (derived from cDNA) confirmed the correctness of these findings. The study was also supplemented by the screening of capsular cryptococcal yeasts, which produce the surface lactose-inducible α- and ß-galactosidases. The production of the lactose-inducible α-galactosidases was not found to be a general feature within the yeast strains examined and, therefore, the existing hypothesis on the general function of this enzyme in cryptococcal capsule rearrangement cannot be confirmed.

Crystal structure of cystathionine ß-synthase from honeybee Apis mellifera.

Cystathionine ß-synthase (CBS), the key enzyme in the transsulfuration pathway, links methionine metabolism to the biosynthesis of cellular redox controlling molecules. CBS catalyzes the pyridoxal-5'-phosphate-dependent condensation of serine and homocysteine to form cystathionine, which is subsequently converted into cysteine. Besides maintaining cellular sulfur amino acid homeostasis, CBS also catalyzes multiple hydrogen sulfide-generating reactions using cysteine and homocysteine as substrates. In mammals, CBS is activated by S-adenosylmethionine (AdoMet), where it can adopt two different conformations (basal and activated), but exists as a unique highly active species in fruit fly Drosophila melanogaster. Here we present the crystal structure of CBS from honeybey Apis mellifera, which shows a constitutively active dimeric species and let explain why the enzyme is not allosterically regulated by AdoMet. In addition, comparison of available CBS structures unveils a substrate-induced closure of the catalytic cavity, which in humans is affected by the AdoMet-dependent regulation and likely impaired by the homocystinuria causing mutation T191M.

Bee-derived antibacterial peptide, defensin-1, promotes wound re-epithelialisation in vitro and in vivo.

Royal jelly (RJ) has successfully been used as a remedy in wound healing. RJ has multiple effects, including antibacterial, anti-inflammatory and immunomodulatory activities, in various cell types. However, no component(s) (other than antibacterial) have been identified in RJ-accelerated wound healing. In this study, we demonstrate that keratinocytes are responsible for the elevated production of matrix metalloproteinase-9 (MMP-9) after incubation with a water extract of RJ. Furthermore, the keratinocyte migration and wound closure rates were significantly increased in the presence of RJ extract. MMP-9 production was reduced significantly following proteinase K treatment but remained stable after heat treatment, indicating that active component(s) have a proteinous character. To identify the component responsible for inducing MMP-9 production, RJ extract was fractionated using C18 RP-HPLC. In fractions exhibiting stimulatory activity, we immunochemically detected the bee-derived antibacterial peptide, defensin-1. Defensin-1 was cloned, and recombinant peptide was produced in a baculoviral expression system. Defensin-1 stimulated MMP-9 secretion from keratinocytes and increased keratinocyte migration and wound closure in vitro. In addition, defensin-1 promoted re-epithelisation and wound closure in uninfected excision wounds. These data indisputably demonstrate that defensin-1, a regular but concentration variable factor found in honey and RJ, contributes to cutaneous wound closure by enhancing keratinocyte migration and MMP-9 secretion.

Honeybee glucose oxidase--its expression in honeybee workers and comparative analyses of its content and H2O2-mediated antibacterial activity in natural honeys.

Antibacterial properties of honey largely depend on the accumulation of hydrogen peroxide (H2O2), which is generated by glucose oxidase (GOX)-mediated conversion of glucose in diluted honey. However, honeys exhibit considerable variation in their antibacterial activity. Therefore, the aim of the study was to identify the mechanism behind the variation in this activity and in the H2O2 content in honeys associated with the role of GOX in this process. Immunoblots and in situ hybridization analyses demonstrated that gox is solely expressed in the hypopharyngeal glands of worker bees performing various tasks and not in other glands or tissues. Real-time PCR with reference genes selected for worker heads shows that the gox expression progressively increases with ageing of the youngest bees and nurses and reached the highest values in processor bees. Immunoblot analysis of honey samples revealed that GOX is a regular honey component but its content significantly varied among honeys. Neither botanical source nor geographical origin of honeys affected the level of GOX suggesting that some other factors such as honeybee nutrition and/or genetic/epigenetic factors may take part in the observed variation. A strong correlation was found between the content of GOX and the level of generated H2O2 in honeys except honeydew honeys. Total antibacterial activity of most honey samples against Pseudomonas aeruginosa isolate significantly correlated with the H2O2 content. These results demonstrate that the level of GOX can significantly affect the total antibacterial activity of honey. They also support an idea that breeding of novel honeybee lines expressing higher amounts of GOX could help to increase the antibacterial efficacy of the hypopharyngeal gland secretion that could have positive influence on a resistance of colonies against bacterial pathogens.

Methylglyoxal may affect hydrogen peroxide accumulation in manuka honey through the inhibition of glucose oxidase.

Although hydrogen peroxide (H2O2) is one of the major antibacterial factors in most honeys, it does not accumulate in medical-grade manuka honey. The goal of this study was to investigate the effect of artificially added methylglyoxal (MGO) on H2O2 accumulation in natural non-manuka honeys. H2O2 concentrations in the honey solutions were determined using a fluorimetric assay. Two, the most potent H2O2 producers honeydew honeys were mixed with MGO at final concentrations of 250, 500, and 1000 mg/kg, and incubated for 4 days at 37°C. Subsequently, H2O2 concentrations were determined in 50% (wt/vol) MGO supplemented honey solutions. In vitro crosslinking of the enzyme glucose oxidase (GOX) after incubation with MGO was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tested honeys at a concentration of 50% (wt/vol) accumulated up to 495.8±9.1 µM H2O2 in 24 h. The most potent producers were the two honeydew honeys, whose 50% solutions accumulated 306.9±6.8 and 495.8±9.1 µM H2O2, respectively. Levels of H2O2 increased significantly over time in both honey solutions. Contrary to this, the MGO-treated honeys generated significantly lower amounts of H2O2 (P<.001), and this reduction was dose dependent. In addition, MGO-treated GOX formed high molecular weight adducts with increasing time of incubation accompanied by loss of its enzymatic activity. High levels of MGO in manuka honey, by modifying the enzyme GOX, might be responsible for suppressing H2O2 generation. These data highlight the detrimental effect of MGO on significant proteinaceous components of manuka honey.

Anti-biofilm effects of honey against wound pathogens Proteus mirabilis and Enterobacter cloacae.

Biofilm growth and its persistence within wounds have recently been suggested as contributing factors to impaired healing. The goal of this study was to investigate the anti-biofilm effects of several honey samples of different botanical origin, including manuka honey against Proteus mirabilis and Enterobacter cloacae wound isolates. Quantification of biofilm formation was carried out using a microtiter plate assay. All honeys at a sub-inhibitory concentration of 10% (w/v) significantly reduced the biofilm development of both isolates. Similarly, at a concentration of 50% (w/v), each of the honeys caused significant partial detachment of Pr. mirabilis biofilm after 24 h. On the other hand, no honey was able to significantly detach Ent. cloacae biofilm. In addition, treatment of Ent. cloacae and Pr. mirabilis biofilms with all honeys resulted in a significant decrease in colony-forming units per well values in a range of 0.35-1.16 and 1.2-7.5 log units, respectively. Of the tested honeys, manuka honey possessed the most potent anti-biofilm properties. Furthermore, methylglyoxal, an antibacterial compound of manuka honey, was shown to be responsible for killing biofilm-embedded wound bacteria. These findings suggest that manuka honey could be used as a potential therapy for the treatment of wounds containing Pr. mirabilis or Ent. cloacae.

Fir honeydew honey flavonoids inhibit TNF-α-induced MMP-9 expression in human keratinocytes: a new action of honey in wound healing.

Matrix metalloproteinase-9 (MMP-9) appears to be a major protease responsible for the degradation of matrix and growth-promoting agents in chronic wounds. Honey has been successfully used for treating non-healing wounds associated with infections. However, the mechanisms of its action at the cellular level have remained poorly understood. The aim of this study was to investigate the effect of fir honeydew honey on TNF-α-induced MMP-9 expression and secretion from human keratinocytes (HaCaT) and to identify the honey component(s) responsible for a discovered effect. A C18 solid-phase column was used for preparation of honey aqueous extract (HAE). Expression and production of MMP-9 by HaCaT cells were determined by reverse transcription-PCR, gelatine zymography and Western blot analysis using a polyclonal antibody against MMP-9. We found that HAE inhibited TNF-α-induced production of MMP-9 in keratinocytes in a dose-dependent manner at both the mRNA and protein levels. Apigenin and kaempferol, identified flavonoids in HAE, markedly inhibited MMP-9 production from HaCaT and epidermal keratinocytes. Taken together, fir honeydew honey, which contains certain flavonoids, prevents TNF-α-induced proteolytic activity in cutaneous inflammation. Thus, our findings provide clear evidence that honey may serve as a natural treatment for dermatological problems associated with a persistent inflammation.

Purification, crystallization and preliminary crystallographic analysis of the full-length cystathionine ß-synthase from Apis mellifera.

Cystathionine ß-synthase (CBS) is a pyridoxal-5'-phosphate-dependent enzyme that catalyzes the first step of the transsulfuration pathway, namely the condensation of serine with homocysteine to form cystathionine. Mutations in the CBS gene are the single most common cause of hereditary homocystinuria, a multisystemic disease affecting to various extents the vasculature, connective tissues and central nervous system. At present, the crystal structure of CBS from Drosophila melanogaster is the only available structure of the full-length enzyme. Here we describe a cloning, overexpression, purification and preliminary crystallographic analysis of a full-length CBS from Apis mellifera (AmCBS) which maintains 51 and 46% sequence identity with its Drosophila and human homologs, respectively. The AmCBS yielded crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a=85.90, b=95.87, c=180.33 Å. Diffraction data were collected to a resolution of 3.0 Å. The crystal structure contained two molecules in the asymmetric unit which presumably correspond to the dimeric species observed in solution.

Methylglyoxal-induced modifications of significant honeybee proteinous components in manuka honey: Possible therapeutic implications.

Methylglyoxal (MGO) is a major antibacterial component of manuka honey. Another antibacterial component found in Revamil honey, peptide defensin1, was not identified in manuka honey. The primary aim of the study was to evaluate the content of defensin1 in honeys of different botanical origins and to investigate a presumed effect of reactive MGO on defensin1 and a dominant protein of honey MRJP1 in manuka honey. Immunoblotting of honey samples showed that defensin1 was a regular but quantitatively variable component of honeys. One of the reasons of varying contents of defensin1 in different honeys seems to be constitutive but varying defensin1 expression in individual honeybees in bee populations that we documented on samples of nurse and forager bees by RT-PCR. Comparative analyses of honeys revealed a size modification of defensin1, MRJP1 and probably also α-glucosidase in manuka honey. We further showed that (i) the treatment of purified defensin1 in solution containing high amount of MGO caused a time-dependent loss of its antibacterial activity and (ii) increasing MGO concentrations in a non-manuka honey were connected with a gradual increase in the molecular weight of MRJP1. Obtained results demonstrate that MGO abrogates the antibacterial activity of defensin1 and modifies MRJP1 in manuka honey. We assume that MGO could also have negative effects on the structure and function of other proteins/peptides in manuka honey, including glucose oxidase, generating hydrogen peroxide.

Effect of honey and its major royal jelly protein 1 on cytokine and MMP-9 mRNA transcripts in human keratinocytes.

Honey has been used since ancient times as a remedy in wound healing. However, even though the results from randomized clinical trials document that honey accelerates wound healing, no study dealing with its influence on human skin cells (epidermal keratinocytes and dermal fibroblast) has been performed. We demonstrate that keratinocytes, which are known to be involved in wound healing, are responsible for elevated production of mediators including cytokines (TNF-alpha, IL-1beta and TGF-beta) and matrix metalloproteinase-9 (MMP-9) after incubation with honey. Real-time PCR was performed for the quantification of mRNA level of selected cytokines and MMP-9. Furthermore, we show that the increased level of MMP-9 in the epidermis following incubation with honey leads to degradation of type IV collagen in the basement membrane. These data indisputably demonstrate that honey activates keratinocytes and support the findings that honey may accelerate wound healing process.

Towards abolition of immunogenic structures in insect cells: characterization of a honey-bee (Apis mellifera) multi-gene family reveals both an allergy-related core alpha1,3-fucosyltransferase and the first insect Lewis-histo-blood-group-related antigen-synthesizing enzyme.

Glycoproteins from honey-bee (Apis mellifera), such as phospholipase A2 and hyaluronidase, are well-known major bee-venom allergens. They carry N-linked oligosaccharide structures with two types of alpha1,3-fucosylation: the modification by alpha1,3-fucose of the innermost core GlcNAc, which constitutes an epitope recognized by IgE from some bee-venom-allergic patients, and an antennal Lewis-like GalNAcbeta1,4(Fucalpha1,3)GlcNAc moiety. We now report the cloning and expression of two cDNAs encoding the relevant active alpha1,3-FucTs (alpha1,3-fucosyltransferases). The first sequence, closest to that of fruitfly (Drosophila melanogaster) FucTA, was found to be a core alpha1,3-FucT (EC 2.4.1.214), as judged by several enzyme and biochemical assays. The second cDNA encoded an enzyme, most related to Drosophila FucTC, that was shown to be capable of generating the Le(x) [Galbeta1-4(Fucalpha1-3)GlcNAc] epitope in vitro and is the first Lewis-type alpha1,3-FucT (EC 2.4.1.152) to be described in insects. The transcription levels of these two genes in various tissues were examined: FucTA was found to be predominantly expressed in the brain tissue and venom glands, whereas FucTC transcripts were detected at highest levels in venom and hypopharyngeal glands. Very low expression of a third homologue of unknown function, FucTB, was also observed in various tissues. The characterization of these honey-bee gene products not only accounts for the observed alpha1,3-fucosylation of bee-venom glycoproteins, but is expected to aid the identification and subsequent down-regulation of the FucTs in insect cell lines of biotechnological importance.

Two structurally different defensin genes, one of them encoding a novel defensin isoform, are expressed in honeybee Apis mellifera.

Two defensins showing high mutual similarity have previously been characterized in honeybee Apis mellifera: royalisin, a peptide isolated from the royal jelly, and defensin, found in the hemolymph of bacterially infected bees. Here we show that both these peptides are encoded by the same polymorphic gene, which we termed defensin1. Besides this gene, we identified an additional defensin gene coding for a novel honeybee defensin designated defensin2. The pre-pro-peptide sequence of defensin 2 was inferred from its cDNA. Mature defensin 2 peptide shows 55.8% identity with defensin 1. Sequences of genomic loci of the two defensin genes revealed their different structure. Defensin1 possesses an exon-intron structure unique among arthropoda defensin genes. Its second intron splits exactly the common structural module of defensins from a short amidated C-terminal extension found only in hymenopteran defensins. Transcription of defensin genes in some nurse honeybees tissues was studied by RT-PCR. Both defensins are expressed in heads and thoraces. Defensin1 but not defensin2 mRNA was detected in hyphopharyngeal, mandibular and thoracic salivary glands. Immune response elements were identified by computer analysis of the promoter regions of defensin genes. Their different representation in these genes reflects presumably observed tissue-specific expression of defensins.

The MRJP/YELLOW protein family of Apis mellifera: identification of new members in the EST library.

Major royal jelly proteins (named MRJP1-5) of honeybee (Apis mellifera), yellow proteins of Drosophila, together with putative proteins found in several bacteria, form a protein family termed the MRJP/yellow family. Members of the family exert diverse physiological functions and amongst eukaryotes appear to be restricted to the order Insecta. MRJPs constitute about 90% of total protein of royal jelly, which is secreted by nurse bees to feed the queen and growing larvae. We looked for mrjp and yellow homologues in a honeybee brain expressed sequence tags (EST) library. In addition to the five mrjp cDNAs previously characterized, we found three additional cDNAs encoding novel MRJPs and importantly, two cDNAs coding for orthologues of Drosophila yellow proteins. One yellow cDNA and all three cDNAs coding for the novel MRJPs were assembled completely, the sequence of the other yellow homologue was partially assembled. The data we present here supports the view that repeated duplications and functional divergence occurred during the evolution of MRJPs in honeybees, with even closely related MRJPs appearing to perform diverse physiological functions. Conversely, yellow protein orthologues appear to be conserved and thus candidates for maintaining the former function(s) of yellow proteins.