European Honey Bee Apis Mellifera Research Articles

Honeybees prefer novel insect-pollinated flower shapes over bird-pollinated flower shapes.

Plant–pollinator interactions have a fundamental influence on flower evolution. Flower color signals are frequently tuned to the visual capabilities of important pollinators such as either bees or birds, but far less is known about whether flower shape influences the choices of pollinators. We tested European honeybee Apis mellifera preferences using novel achromatic (gray-scale) images of 12 insect-pollinated and 12 bird-pollinated native Australian flowers in Germany; thus, avoiding influences of color, odor, or prior experience. Independent bees were tested with a number of parameterized images specifically designed to assess preferences for size, shape, brightness, or the number of flower-like shapes present in an image. We show that honeybees have a preference for visiting images of insect-pollinated flowers and such a preference is most-likely mediated by holistic information rather than by individual image parameters. Our results indicate angiosperms have evolved flower shapes which influence the choice behavior of important pollinators, and thus suggest spatial achromatic flower properties are an important part of visual signaling for plant–pollinator interactions.

The Optimal Supply of Crop Pollination and Honey From Wild and Managed Bees: An Analytical Framework for Diverse Socio-Economic and Ecological Settings

Hiring pollination services has become an important strategy to secure fruit set in many pollinator-dependent crops, especially in monocultures such as the Californian almond (Prunus dulcis) groves, where the European honeybee (Apis mellifera) is frequently deployed to compensate deficiencies in the natural provision of insect-mediated pollination. Such beekeeper-farmer cooperation has been the focus of many economic models, although it is only one in a variety of setups under which bees (wild and managed) contribute to agriculture. Based on a thorough literature review and on our own research, we generalize the basic model by Rucker et al. (2012) to describe farmer-beekeeper interactions as determined by diverse agro-ecological, institutional and socioeconomic conditions. The generalized model serves as an analytical framework to classify real world farmer-ecosystem-beekeeper interactions, to identify possible causes for a suboptimal crop pollination and to formulate informed policy recommendations.

Characterisation of the British honey bee metagenome

The European honey bee (Apis mellifera) plays a major role in pollination and food production. Honey bee health is a complex product of the environment, host genetics and associated microbes (commensal, opportunistic and pathogenic). Improved understanding of these factors will help manage modern challenges to bee health. Here we used DNA sequencing to characterise the genomes and metagenomes of 19 honey bee colonies from across Britain. Low heterozygosity was observed in many Scottish colonies which had high similarity to the native dark bee. Colonies exhibited high diversity in composition and relative abundance of individual microbiome taxa. Most non-bee sequences were derived from known honey bee commensal bacteria or pathogens. However, DNA was also detected from additional fungal, protozoan and metazoan species. To classify cobionts lacking genomic information, we developed a novel network analysis approach for clustering orphan DNA contigs. Our analyses shed light on microbial communities associated with honey bees and demonstrate the power of high-throughput, directed metagenomics for identifying novel biological threats in agroecosystems.

Nosema ceranae infection in honeybee samples from Tuscanian Archipelago (Central Italy) investigated by two qPCR methods.

Nosema apis and Nosema ceranae are microsporidian parasite worldwide spread causing an emerging infectious disease of European honeybee Apis mellifera. The Nosema presence was deeply investigated in several countries but low information are presents about islands. In this investigation was evaluated the presence N. ceranae and N. apis in apiaries located in Tuscanian Archipelago islands (Central Italy). For N. ceranae detection, two different Real-Time PCR (qPCR) methods, the 16S rRNA and Hsp70 gene amplification qPCR, were performed on honey bee samples; while, for N. apis only the 16S rRNA qPCR amplification was performed. On all islands, only N. ceranae was present, while N. apis was not found in the samples. The two qPCR showed significant difference (p < 0.040) in N. ceranae spores quantification. The single-copy Hsp70 gene method qPCR assay systematically detected a lower amount of N. ceranae copies compared to the multi-copy 16S rRNA gene method.

Medicinal value of sunflower pollen against bee pathogens

Global declines in pollinators, including bees, can have major consequences for ecosystem services. Bees are dominant pollinators, making it imperative to mitigate declines. Pathogens are strongly implicated in the decline of native and honey bees. Diet affects bee immune responses, suggesting the potential for floral resources to provide natural resistance to pathogens. We discovered that sunflower (Helianthus annuus) pollen dramatically and consistently reduced a protozoan pathogen (Crithidia bombi) infection in bumble bees (Bombus impatiens) and also reduced a microsporidian pathogen (Nosema ceranae) of the European honey bee (Apis mellifera), indicating the potential for broad anti-parasitic effects. In a field survey, bumble bees from farms with more sunflower area had lower Crithidia infection rates. Given consistent effects of sunflower in reducing pathogens, planting sunflower in agroecosystems and native habitat may provide a simple solution to reduce disease and improve the health of economically and ecologically important pollinators.

MKast is dispensable for normal development and sexual maturation of the male European honeybee

The European honeybee (Apis mellifera L.) exhibits various social behaviors. The molecular and neural mechanisms underlying these behaviors have long been explored, but causal relations between genes or neurons and behaviors remain to be elucidated because effective gene manipulation methods in the honeybee have not been available until recently. We recently established a basic technology to produce mutant honeybee drones using CRISPR/Cas9. Here we produced mutant drones using CRISPR/Cas9 targeting mKast, which is preferentially expressed in a certain subtype of class I Kenyon cells that comprise the mushroom bodies in the honeybee brain. By immunoblot analysis, we showed that mKast protein expression was completely lost in the mutant drone heads. In addition, during the production process of homozygous mutant workers, we demonstrated that heterozygous mutant workers could be produced by artificial insemination of wild-type queens with the sperm of mutant drones, indicating that mKast mutant drones were sexually mature. These results demonstrate that mKast is dispensable for normal development and sexual maturation in drone honeybees, and allow us to proceed with the production of homozygous mutant workers for the analysis of a particular gene by gene knockout in the future.

Tracing natural and industrial contamination and lead isotopic compositions in an Australian native bee species

This study investigates trace element concentrations (arsenic (As), manganese (Mn), lead (Pb) and zinc (Zn)) and Pb isotopic compositions in an Australian native bee species, Tetragonula carbonaria, and its products of honey and wax. Co-located soil and dust samples were simultaneously analysed with the objective of determining if the bees or their products had potential application as a proxy for monitoring environmental contamination. The most significant relationships were found between Pb concentrations in honey (r = 0.814, p = 0.014) and wax (r = 0.883, p = 0.004) and those in co-located dust samples. In addition, Zn concentrations in honey and soil were significantly associated (r = 0.709, p = 0.049). Lead isotopic compositions of native bee products collected from background sites adjacent to Sydney national parks (206Pb/207Pb = 1.144, 208Pb/207Pb = 2.437) corresponded to local geogenic rock and soil values (206Pb/207Pb = 1.123–1.176, 208Pb/207Pb = 2.413–2.500). By contrast, inner Sydney metropolitan samples, including native bees and wax (206Pb/207Pb = 1.072–1.121, 208Pb/207Pb = 2.348–2.409), co-located soil and dust (206Pb/207Pb = 1.090–1.122, 208Pb/207Pb = 2.368–2.403), corresponded most closely to aerosols collected during the period of leaded petrol use (206Pb/207Pb = 1.067–1.148, 208Pb/207Pb = 2.341–2.410). A large range of Pb isotopic compositions in beehive samples suggests that other legacy sources, such as Pb-based paints and industrials, may have also contributed to Pb contamination in beehive samples. Native bee data were compared to corresponding samples from the more common European honey bee (Apis mellifera). Although Pb isotopic compositions were similar in both species, significant differences in trace element concentrations were evident across the trace element suite, the bees and their products. The statistical association between T. carbonaria and co-located environmental contaminant concentrations were stronger than those in European honey bees, which may be attributable to its smaller foraging distance (0.3–0.7 km versus 5–9 km, respectively). This implies that T. carbonaria may be more suitable for assessing small spatial scale variations of trace element concentrations than European honey bees.

Insight into the substrate specificity change caused by the Y227H mutation of α-glucosidase III from the European honeybee (Apis mellifera) through molecular dynamics simulations.

Honey from the European honeybee, Apis mellifera, is produced by α-glucosidases (HBGases) and is widely used in food, pharmaceutical, and cosmetic industries. Categorized by their substrate specificities, HBGases have three isoforms: HBGase I, II and III. Previous experimental investigations showed that wild-type HBGase III from Apis mellifera (WT) preferred sucrose to maltose as a substrate, while the Y227H mutant (MT) preferred maltose to sucrose. This mutant can potentially be used for malt hydrolysis because it can efficiently hydrolyze maltose. In this work, to elucidate important factors contributing to substrate specificity of this enzyme and gain insight into how the Y227H mutation causes substrate specificity change, WT and MT homology models were constructed, and sucrose/maltose was docked into active sites of the WT and MT. AMBER14 was employed to perform three independent molecular dynamics runs for these four complexes. Based on the relative binding free energies calculated by the MM-GBSA method, sucrose is better than maltose for WT binding, while maltose is better than sucrose for MT binding. These rankings support the experimentally observed substrate specificity that WT preferred sucrose to maltose as a substrate, while MT preferred maltose to sucrose, suggesting the importance of binding affinity for substrate specificity. We also found that the Y227H mutation caused changes in the proximities between the atoms necessary for sucrose/maltose hydrolysis that may affect enzyme efficiency in the hydrolysis of sucrose/maltose. Moreover, the per-residue binding free energy decomposition results show that Y227/H227 may be a key residue for preference binding of sucrose/maltose in the WT/MT active site. Our study provides important and novel insight into the binding of sucrose/maltose in the active site of Apis mellifera HBGase III and into how the Y227H mutation leads to the substrate specificity change at the molecular level. This knowledge could be beneficial in the design of this enzyme for increased production of desired products.

Introgression in native populations of Apis mellifera mellifera L: implications for conservation

Hybridisation and introgression can have negative impacts on regional biodiversity through the potential erosion of locally adapted lineages. The honey bee (Apis mellifera L.) occurs in twenty-seven subspecies across Europe, is an extremely economically important insect, yet threatened by multifarious impacts. Transhumance of the most commercially appealing varieties threatens native honey bee diversity by introgression and subsequent loss of locally adapted traits, or even by complete removal of some subspecies from parts of the range. Here levels of admixture and introgression are examined in UK honey bees suspected to be from hives of the dark European honey bee (Apis mellifera mellifera). Microsatellite DNA and STRUCTURE analyses reveal that the studied populations are generally admixed, and discriminant analysis of principal components shows them to be intermediate between A. m. mellifera and Apis mellifera carnica populations. However, examining mitochondrial haplotype data (COI-COII intergenic spacer region) and nuclear DNA reveal that some hives are relatively pure (from 4 to 15 hives, depending on the Q-value threshold). Genetic diversity is relatively high in comparison with other European populations. Implications for conservation and management are discussed.

A significant pure population of the dark European honey bee (Apis mellifera mellifera) remains in Ireland

The natural range of the dark European honey bee, Apis mellifera mellifera has been significantly reduced in recent years as a result of importation and replacement of queens with those of other Apis subspecies. Previous studies have indicated that a substantial amount of A. m. mellifera populations throughout Europe are heavily hybridized but that pockets of pure populations do still exist and need to be protected as this subspecies is a highly valuable gene pool and is of considerable conservational interest. Small numbers of Irish bees have been included in previous studies, but scientific information is limited and questions remain about the genetic diversity of bees in Ireland and the extent of introgression into apparent black bees from introduced races and hybrids. The objective of this study was to investigate the genetic composition of the A. m. mellifera population on the island of Ireland with both nuclear (microsatellites) and mitochondrial markers. Molecular data was generated from 412 bees sampled from 24 counties across the island. Mitochondrial data identified 34 different haplotypes, with 63% of bees having sequences identical to three European haplotypes but all other haplotypes being novel. Population structure analysis using microsatellite markers indicates that the Irish population is genetically diverse and that 97.8% of sampled bees were determined to be pure A. m. mellifera. Results from cluster analysis using a Bayesian model approach, and the presence of novel alleles, shows evidence of distinctiveness within the Irish population.

Molecular, physiological and behavioral responses of honey bee (Apis mellifera) drones to infection with microsporidian parasites.

Susceptibility to pathogens and parasites often varies between sexes due to differences in life history traits and selective pressures. Nosema apis and Nosema ceranae are damaging intestinal pathogens of European honey bees (Apis mellifera). Nosema pathology has primarily been characterized in female workers where infection is energetically costly and accelerates worker behavioral maturation. Few studies, however, have examined infection costs in male honey bees (drones) to determine if Nosema similarly affects male energetic status and sexual maturation. We infected newly emerged adult drones with Nosema spores and conducted a series of molecular, physiological, and behavioral assays to characterize Nosema etiology in drones. We found that infected drones starved faster than controls and exhibited altered patterns of flight activity in the field, consistent with energetic distress or altered rates of sexual maturation. Moreover, expression of candidate genes with metabolic and/or hormonal functions, including members of the insulin signaling pathway, differed by infection status. Of note, while drone molecular responses generally tracked predictions based on worker studies, several aspects of infected drone flight behavior contrasted with previous observations of infected workers. While Nosema infection clearly imposed energetic costs in males, infection had no impact on drone sperm numbers and had only limited effects on antennal responsiveness to a major queen sex pheromone component (9-ODA). We compare Nosema pathology in drones with previous studies describing symptoms in workers and discuss ramifications for drone and colony fitness.

Antioxidant activity and irritation property of venoms from Apis species

Pharmacological effects of bee venom has been reported, however, it has been restricted to the bee venom collected from European honey bee (Apis mellifera). The aim of the present study was to compare the antioxidant activities and irritation properties of venoms collected from four different Apis species in Thailand, which includes Apis cerena (Asian cavity nesting honeybee), Apis florea (dwarf honeybee), Apis dorsata (giant honeybee), and A. mellifera. Melittin content of each bee venom extracts was investigated by using high-performance liquid chromatography. Ferric reducing antioxidant power, 2, 2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid), and 1, 1-diphenyl-2-picrylhydrazyl assay were used to determine the antioxidant activity, whereas, hen's egg test chorioallantoic membrane assay was used to determine the irritation property of each bee venom extracts. Melittin was the major constituent in all bee venom extracts. The melittin content in A. dorsata, A. mellifera, A. florea, and A. cerena were 95.8 ± 3.2%, 76.5 ± 1.9%, 66.3 ± 8.6%, and 56.8 ± 1.8%, respectively. Bee venom extract from A. dorsata possessed the highest antioxidant activity with the inhibition of 41.1 ± 2.2% against DPPH, Trolox equivalent antioxidant capacity of 10.21 ± 0.74 mM Trolox/mg and equivalent concentration (EC1) of 0.35 ± 0.02 mM FeSO4/mg. Bee venom extract from A. mellifera exhibited the highest irritation, followed by A. cerena, A. dorsata, and A. florea, respectively. Melittin was the compound responsible for the irritation property of bee venom extracts since it could induce severe irritation (irritation score was 13.7 ± 0.5, at the concentration of 2 mg/ml). The extract from A. dorsata which possessed the highest antioxidant activity showed no irritation up to the concentration of 0.1 mg/ml. Therefore, bee venom extract from A. dorsata at the concentration not more than 0.1 mg/ml would be suggested for using as cosmetic ingredients since it possessed the highest antioxidant activity with no irritation. This study is the first report to compare the bee venom extracts from different Apis species and display their potential application of bee venom extracts in cosmetic products.

Survival and health improvement of Nosema infected Apis florea (Hymenoptera: Apidae) bees after treatment with propolis extract

Nosema ceranae is now considered to be an emerging infectious disease of the European honey bee Apis mellifera. Only one antibiotic, Fumagillin, is commercially available to combat Nosema infections. This antibiotic treatment is banned from use in Europe and elsewhere there is a high probability for antibiotic resistance to develop. We are therefore interested in investigating the effects of a natural propolis extract on its ability to reduce N. ceranae infection loads in the dwarf honey bee, Apis florea, a native honey bee with a range that overlaps with Apis cerana and Apis mellifera that is at risk of infection. Experimentally infected caged bees were fed a treatment consisting of 0%, 50%, or 70% propolis extract. All 50% and 70% propolis treated bees had significantly lower infection loads, and the 50% treated bees had higher survival in comparison to untreated bees. In addition, propolis treated bees had significantly higher haemolymph trehalose levels and hypopharyngeal gland protein content similar to levels of uninfected bees. Propolis ethanolic extract treatment could therefore be considered as a possible viable alternative to Fumagillin to improve bee health. This natural treatment deserves further exploration to develop it as a possible alternative to combat N. ceranae infections distributed around the world.

Chronic Nosema ceranae infection inflicts comprehensive and persistent immunosuppression and accelerated lipid loss in host Apis mellifera honey bees

Nosema ceranae is an intracellular microsporidian parasite of the Asian honey bee Apis cerana and the European honey bee Apis mellifera. Until relatively recently, A. mellifera honey bees were naïve to N. ceranae infection. Symptoms of nosemosis, or Nosema disease, in the infected hosts include immunosuppression, damage to gut epithelium, nutrient and energetic stress, precocious foraging and reduced longevity of infected bees. Links remain unclear between immunosuppression, the symptoms of nutrient and energetic stress, and precocious foraging behavior of hosts. To clarify physiological connections, we inoculated newly emerged A. mellifera adult workers with N. ceranae spores, and over 21 days post inoculation (21 days pi), gauged infection intensity and quantified expression of genes representing two innate immune pathways, Toll and Imd. Additionally, we measured each host’s whole-body protein, lipids, carbohydrates and quantified respirometric and activity levels. Results show sustained suppression of genes of both humorally regulated immune response pathways after 6 days pi. At 7 days pi, elevated protein levels of infected bees may reflect synthesis of antimicrobial peptides from an initial immune response, but the lack of protein gain compared with uninfected bees at 14 days pi may represent low de novo protein synthesis. Carbohydrate data do not indicate that hosts experience severe metabolic stress related to this nutrient. At 14 days pi infected honey bees show high respirometric and activity levels, and corresponding lipid loss, suggesting lipids may be used as fuel for increased metabolic demands resulting from infection. Accelerated lipid loss during nurse honey bee behavioral development can have cascading effects on downstream physiology that may lead to precocious foraging, which is a major factor driving colony collapse.

Honey Bee (Apis mellifera) Pollen Foraging Reflects Benefits Dependent on Individual Infection Status.

Parasites often modify host foraging behavior, for example, by spurring changes to nutrient intake ratios or triggering self-medication. The gut parasite, Nosema ceranae, increases energy needs of the European or Western honey bee (Apis mellifera), but little is known about how infection affects foraging behavior. We used a combination of experiments and observations of caged and free-flying individual bees and hives to determine how N. ceranae affects honey bee foraging behavior. In an experiment with caged bees, we found that infected bees with access to a high-quality pollen were more likely to survive than infected bees with access to a lower quality pollen or no pollen. Non-infected bees showed no difference in survival with pollen quality. We then tested free-flying bees in an arena of artificial flowers and found that pollen foraging bees chose pollen commensurate with their infection status; twice as many infected bees selected the higher quality pollen than the lower quality pollen, while healthy bees showed no preference between pollen types. However, healthy and infected bees visited sucrose and pollen flowers in the same proportions. Among hive-level observations, we found no significant correlations between N. ceranae infection intensity in the hive and the proportion of bees returning with pollen. Our results indicate that N. ceranae-infected bees benefit from increased pollen quality and will selectively forage for higher quality while foraging for pollen, but infection status does not lead to increased pollen foraging at either the individual or hive levels.

A real-time PCR method for quantification of the total and major variant strains of the deformed wing virus.

European honey bees (Apis mellifera) are critically important to global food production by virtue of their pollination services but are severely threatened by deformed wing virus (DWV) especially in the presence of the external parasite Varroa destructor. DWV exists as many viral strains with the two major variants (DWV-A and DWV-B) varying in virulence. A single plasmid standard was constructed containing three sections for the specific determination of DWV-A (VP2 capsid region), DWV-B (IRES) and a conserved region suitable for total DWV (helicase region). The assays were confirmed as specific and discriminatory with limits of detections of 25, 25 and 50 genome equivalents for DWV-A, DWV-B and total-DWV, respectively. The methods were successfully tested on Apis mellifera and V. destructor samples with varying DWV profiles. The new method determined a more accurate total DWV titre in samples with substantial DWV-B than the method currently described in the COLOSS Beebook. The proposed assays could be utilized for the screening of large quantities of bee material for both a total DWV load overview along with more detailed investigations into DWV-A and DWV-B profiles.

Acute bee paralysis virus occurs in the Asian honey bee Apis cerana and parasitic mite Tropilaelaps mercedesae

Viruses, and especially RNA viruses, constantly change and adapt to new host species and vectors, posing a potential threat of new and reemerging infectious diseases. Honey bee Acute bee paralysis virus (ABPV) and Deformed wing virus (DWV) are two of the most common honey bee viruses found in European honey bees Apis mellifera and have been implicated in worldwide Varroa-associated bee colony losses. Previous studies have shown that DWV has jumped hosts several times in history causing infection in multiple host species. In the present study, we show that DWV infection could be detected in the Asian honey bee, A. cerana, and the parasitic mite Tropilaelaps mercedesae, confirming previous findings that DWV is a multi-host pathogen and supporting the notion that the high prevalence of DWV in honey bee host populations could be attributed to the high adaptability of this virus. Furthermore, our study provides the first evidence that ABPV occurs in both A. cerana and T. mercedesae in northern Thailand. The geographical proximity of host species likely played an important role in the initial exposure and the subsequent cross-species transmission of these viruses. Phylogenetic analyses suggest that ABPV might have moved from T. mercedesae to A. mellifera and to A. cerana while DWV might have moved in the opposite direction from A. cerana to A. mellifera and T. mercedesae. This result may reflect the differences in virus life history and virus-host interactions, warranting further investigation of virus transmission, epidemiology, and impacts of virus infections in the new hosts. The results from this study indicate that viral populations will continue to evolve and likely continue to expand host range, increasing the need for effective surveillance and control of virus infections in honey bee populations.

Neonicotinoid insecticides in pollen, honey and adult bees in colonies of the European honey bee (Apis mellifera L.) in Egypt.

Honeybee losses have been attributed to multiple stressors and factors including the neonicotinoid insecticides (NIs). Much of the study of hive contamination has been focused upon temperate regions such as Europe, Canada and the United States. This study looks for the first time at honey, pollen and bees collected from across the Nile Delta in Egypt in both the spring and summer planting season of 2013. There is limited information upon the frequency of use of NIs in Egypt but the ratio of positive identification and concentrations of NIs are comparable to other regions. Metabolites of NIs were also monitored but given the low detection frequency, no link between matrices was possible in the study. Using a simple hazard assessment based upon published LD50 values for individual neonicotinoids upon the foraging and brood workers it was found that there was a potential risk to brood workers if the lowest reported LD50 was compared to the sum of the maximum NI concentrations. For non-lethal exposure there was significant risk at the worst case to brood bees but actual exposure effects are dependant upon the genetics and conditions of the Egyptian honeybee subspecies that remain to be determined.

Human dietary intake and hazard characterization for residues of neonicotinoides and organophosphorus pesticides in Egyptian honey

ABSTRACTIn two recently published reports, hazards posed by dietary exposure to organophosphate and neonicotinoid plant protection products on the European honey bee (Apis mellifera L.) in Egypt were investigated. Using concentrations reported in those studies, an assessment of hazards posed by these two classes of insecticides to humans due to consumption of Egyptian honey from the Nile Delta during both spring and summer was performed. Twenty-eight compounds including metabolites were assessed for exposure of adult Egyptians based on the best- and worst-case scenarios. Even for the worst-case scenario, exposure to these two classes of pesticides in honey was 15-fold less than hazard index value of 1.0 for adverse effects on humans. Based upon this analysis, people exposed to these insecticides through consumption of honey products would be unlikely to exhibit adverse health outcomes.

The anti-hepatocellular carcinoma activity of Mel-P15 is mediated by natural killer cells.

Mel-P15 is a peptide derived from melittin, the main toxic component in the venom of the European honeybee Apis mellifera. In the present study, the antitumor effects of Mel-P15 and the underlying molecular mechanisms of these effects in vivo were investigated. Mel-P15 directly stimulated natural killer (NK) cell cytotoxicity in vitro, which was increased to 55.45% at a 4 µg/ml dose of Mel-P15. In the mouse liver cancer (H22) xenograft mice model, Mel-P15 suppressed tumor growth in vivo; the tumor inhibitory rate was 61.15% following treatment with 2 mg/kg Mel-P15. In addition, the immune response was activated following Mel-P15 treatment. Mel-P15 treatment increased the spleen and thymus indices, promoted splenocyte proliferation, stimulated NK cytotoxicity and upregulated the secretion of cytokines, including interleukin-2, interferon-γ and tumor necrosis factor-α. In addition, the tumor inhibitory effect of Mel-P15 on BEL-7402-bearing nude mice was abrogated by the selective depletion of NK cells via the intraperitoneal injection of an anti-asialo GM-1 antibody. The results suggest that Mel-P15 inhibits tumor growth in vivo by promoting NK cell cytotoxicity. Mel-P15 may therefore be a potential immunotherapy candidate for the treatment of hepatocellular carcinoma.