Honey Bee Species Research Articles

Phylogenetic and recombination analyses of two deformed wing virus strains from different honeybee species in China.

BackgroundDeformed wing virus (DWV) is one of many viruses that infect honeybees and has been extensively studied because of its close association with honeybee colony collapse that is induced by Varroa destructor. However, virus genotypes, sequence characteristics, and genetic variations of DWV remain unknown in China.MethodsTwo DWV strains were isolated from Jinzhou and Qinhuangdao cities in China, and were named China1-2017 (accession number: MF770715) and China2-2018 (accession number: MH165180), respectively, and their complete genome sequences were analyzed. To investigate the phylogenetic relationships of the DWV isolates, a phylogenetic tree of the complete open reading frame (ORF), structural protein VP1, and non-structural protein 3C+RdRp of the DWV sequences was constructed using the MEGA 5.0 software program. Then, the similarity and recombinant events of the DWV isolated strains were analyzed using recombination detection program (RDP4) software and genetic algorithm for recombination detection (GARD).ResultsThe complete genomic analysis showed that the genomes of the China1-2017 and China2-2018 DWV strains consisted of 10,141 base pairs (bp) and 10,105 bp, respectively, and contained a single, large ORF (China1-2017: 1,146–9,827 bp; China2-2018: 1,351–9,816 bp) that encoded 2,894 amino acids. The sequences were compared with 20 previously reported DWV sequences from different countries and with sequences of two closely related viruses, Kakugo virus (KV) and V. destructor virus-1. Multiple sequence comparisons revealed a nucleotide identity of 84.3–96.7%, and identity of 94.7–98.6% in amino acids between the two isolate strains and 20 reference strains. The two novel isolates showed 96.7% nucleotide identity and 98.1% amino acid identity. The phylogenetic analyses showed that the two isolates belonged to DWV Type A and were closely related to the KV-2001 strain from Japan. Based on the RDP4 and GARD analyses, the recombination of the China2-2018 strain was located at the 4,266–7,507 nt region, with Korea I-2012 as an infer unknown parent and China-2017 as a minor parent, which spanned the entire helicase ORF. To the best of our knowledge, this is the first study to the complete sequence of DWV isolated from Apis cerana and the possible DWV recombination events in China. Our findings are important for further research of the phylogenetic relationship of DWVs in China with DWV strains from other countries and also contribute to the understanding of virological properties of these complex DWV recombinants.

Tolerance and response of two honeybee species Apis cerana and Apis mellifera to high temperature and relative humidity.

The ambient temperature and relative humidity affect the metabolic and physiological responses of bees, thus affecting their life activities. However, the physiological changes in bee due to high temperature and high humidity remain poorly understood. In this study, we explored the effects of higher temperature and humidity on the epiphysiology of bees by evaluating the survival, tolerance and body water loss in two bee species (Apis cerana and Apis mellifera). We also evaluated the changes in the activity of antioxidant and detoxification enzymes in their body. We observed that under higher temperature and humidity conditions, the survival rate of A. mellifera was higher than that of A. cerana. On the other hand, a comparison of water loss between the two species revealed that A. mellifera lost more water. However, under extremely high temperature conditions, A. cerana was more tolerant than A. mellifera. Moreover, under higher temperature and humidity conditions, the activity of antioxidant and detoxification enzymes in bees was significantly increased. Overall, these results suggest that high temperatures can adversely affect bees. They not only affect the survival and water loss, but also stimulate oxidative stress in bees. However, unlike our previous understanding, high humidity can also adversely affect bees, although its effects are lower than that of temperature.

Effectiveness of KVK organized training program on knowledge level of Bee keeping

Krishi Vigyan Kendra, Pratapgarh conducted a training program on Bee Keeping. 50 participants from different blocks attended the program. The present study was conducted to assess the change in knowledge level of the trainees regarding various bee keeping technologies after attending the training. The study revealed that there was significant change in knowledge level regarding various aspects of bee keeping technologies. The study revealed that maximum change in knowledge among the trainees was regarding the use of queen cage (70.00%) and lowest change in knowledge level was observed about fertilized and unfertilized eggs within bee hive (12.00%). Moreover, it was also found that 58.00 per cent, 56.00 per cent and 54.00 per cent change in knowledge level regarding ‘Gharchuut’, ‘growth period of honey bee’ and ‘regarding various species of honey bee’. The training helped in overall improvement of knowledge level of trainees regarding various aspects of bee keeping technology.

Foraging preferences of honey bees Apis cerana in Dakshina Kannada, Karnataka, India

Honey bees visit flowers for collecting nectar and pollen. Pollen serves as a source of protein for the survival of honeybees. To understand the dependence and preference of the honey bee species Apis cerana on different pollen resources, we carried out a study to analyze the distribution of different types of pollen in honey in various regions of the coastal plains of the Western Ghats. Fourteen different honey samples from different sites ranging in elevation from 55m to 135m were collected and analyzed. Acetolysis and centrifugation were used for pollen extraction from different honey samples. The extracted pollen was mixed with glycerin jelly and transferred to a glass slide for microscopic analysis. The primary source of pollen as revealed by the current study were found to be Areca catechu, Cocos nucifera, Ixora coccinea, Mimosa pudica, and Psidium guajava. Morphotype analysis revealed 12 different plants to be the source of the pollen. Each honey sample collected from different locations, however, had only three to six types of pollen indicating that honeybees visit a narrow or a small number of pollen sources. Therefore, based on our study, we conclude that lesser types of pollen in each honey sample indicates that their food resources are getting limited. Therefore, it is necessary to conserve pollen resources for the better survival of honey bees and the environment.

Impact of acute oral exposure to thiamethoxam on the homing, flight, learning acquisition and short-term retention of Apis cerana.

Thiamethoxam (TMX) represents the second generation of neonicotinoids that has been widely applied in agricultural activities, while how TMX alters the behavior of Apis cerana, an important native honey bee species in China, is not clear. We carried out three independent experiments to study the impact of acute oral treatment of 20 μL TMX at concentrations of 2.4 ppb (0.048 ng/bee) and 10ppb (0.2ng/bee) on the homing, flight, learning acquisition and short-term retention ability of A. cerana. The homing ability was assessed by the catch-and-release method, the flight ability was assessed by flight mills, and the learning acquisition and short-term retention were evaluated by the proboscis extension response method. When treated with 10ppb of TMX, bees had a significantly higher average homing time, mean flight velocity, flying distance, and flying duration than the control, whereas 2.4ppb concentration did not cause any significant effect on homing or flight ability. Bees treated with either 2.4ppb or 10ppb TMX had significantly lower learning acquisition and short-term retention ability. Results suggest that acute oral exposure to 10ppb of TMX altered the short-distance homing time, flight ability, and learning acquisition and short-term retention ability. Our study also highlights the concern that acute oral exposure to a low concentration of 2.4ppb could have consequences on the behavior of A. creana. Those multiple sublethal alterations on A. cerana's behavior indicate that TMX are likely having complex but negative consequences on bee health in the field. © 2019 Society of Chemical Industry.

Identifying Microbial Resistance of the Apis mellifera Melanization Immune Response

Apis mellifera, one of the most common species of honey bee, has been suffering disease and colony collapse. While there has been effort in identification and treatment of the pathogens, the study of their insufficient immunoresponse has gone under‐researched. Melanization is a common form of immune response in insects and has been seen in Apis mellifera. The goal of this study is to isolate melanin from hemolymph and conduct multiple experiments with known pathogens against it with the purpose of identifying microbial resistance and further inferring the mechanism of immunity. With this knowledge, a method of improving the Apis mellifera immune response may be found.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Longevity, food consumption, and foraging performance of Apis cerana and Apis mellifera in mixed colonies

Apis cerana is an endemic species of honey bee in Asia. A. cerana is believed to have better adaptation to scattered nectar sources than Apis mellifera, which implies that honey can be harvested from A. cerana colonies, but A. mellifera will starve at the same location. We hypothesize that there are differences in foraging performance, worker longevity, and daily sugar consumption between A. cerana and A. mellifera workers. These parameters in both species were determined. A. cerana had longer longevity than A. mellifera if longevity was measured in caged bees without pollen or in mixed species colonies with A. mellifera queens, but A. mellifera had much longer longevity when measured inside cages provided with pollen. A. cerana also had more foraging trips and higher proportion of foragers than A. mellifera in mixed colonies. The daily sugar consumption of A. cerana workers was 32% of that of A. mellifera, perhaps mainly due to the smaller biomass of A. cerana workers. In addition, A. cerana workers showed significantly lower variance in daily sugar consumption. In conclusion, A. cerana has more foraging trips, higher forager proportion, less sugar consumption, and longer survival than those of A. mellifera, when measured inside the same colonies. These differences might explain why during a dearth or in scattered resource areas, A. cerana can store or produce more honey than A. mellifera.

Learning of monochromatic stimuli in Apis cerana and Apis mellifera by means of PER conditioning

Honey bees are globally distributed and have received increased attention due to their high economic and ecological value for pollination, their exceptional eusocial lifestyle and complex behavioral repertoire. Interestingly, most research on learning and memory in honey bees has been performed in the Western honey bee, Apis mellifera L., and other honey bee species were largely neglected. In the current study, we thus compared visual learning performance of A. mellifera and the Eastern honey bee, A. cerana Fabr., using the proboscis extension response (PER) paradigm. Workers of A. mellifera and A. cerana were differentially conditioned to two monochromatic light stimuli, with peak maxima at 435 and 528 nm. Both honey bee species were able to form an association between the color stimulus and a sugar reward and significantly distinguished between the two color stimuli in a differential discrimination test. However, besides similar performance levels during visual learning, A. cerana showed a reduced mid-term memory (tested after 2 h) compared to A. mellifera. Finally, performance of the visual PER conditioning in our study reached similar levels as found in olfactory PER conditioning, and we thus recommend the visual PER conditioning approach in addition to olfactory conditioning as a useful tool for studying species-specific learning and memory capabilities in honey bees under controlled laboratory conditions.

Apis florea workers show a prolonged period of nursing behavior

Studies on behavioral maturation and division of labor in open-nesting honey bee species are scarce as the bee curtain inhibits direct examination of intranidal behaviors. We observed and studied nursing behavior in Apis florea by attaching a foreign comb with open brood to a host colony. Several of the workers that explored the attached comb visited the cells with brood more often and spent more time in cells compared to empty cells. Workers seen inspecting and feeding the brood had well-developed hypopharyngeal glands compared to foragers of their own colony indicating that they are nurses performing nursing behavior. Further, introducing marked 1-day-old workers into the host colony and repeatedly attaching combs with open brood allowed us to estimate the age range of nursing behavior. In our experiments, A. florea workers started to perform significant nursing behavior 8 days after eclosion and continued to show nursing behavior until the age of 28 days, the end of our observation period. Thus, nursing behavior in A. florea appears to be substantially extended relative to A. mellifera.

Double-stranded RNA reduces growth rates of the gut parasite Crithidia mellificae.

Parasites of managed bees can disrupt the colony success of the host, but also influence local bee-parasite dynamics, which is regarded as a threat for wild bees. Therapeutic measures have been suggested to improve the health of managed bees, for instance, exploiting the bees' RNA interference (RNAi) pathway to treat against viral pathogens. Gut trypanosomes are an important group of bee parasites in at least two common managed bee species, i.e., managed Apis mellifera and reared Bombus terrestris. In several trypanosomes, RNAi activity is present, while in other associated genes of RNAi, such as Dicer-like (DCL) and Argonaute (AGO), it is lost. Up to date, the ability to exploit the RNAi of gut trypanosomes of bees has remained unexplored. Here, we screened parasite genomes of two honey bee protozoa (Crithidia mellificae and Lotmaria passim) and two bumble bee protozoa (Crithidia bombi and Crithidia expoeki) for the presence of DCL and AGO proteins. For C. mellificae, we constructed a double-stranded RNA (dsRNA) targeting kinetoplastid membrane protein-11 (KMP-11) to test the RNAi potential to kill this parasite. Transfection with KMP-11 dsRNA, but also adding it to the growth medium resulted in small growth reduction of the trypanosome C. mellificae, thereby showing the limited potential to apply dsRNA therapeutics to control trypanosome infection in managed honey bee species. Within bumble bees, there seems to be no application potentials against C. bombi, as we could only retrieve non-functional DCL- and AGO-related genes within the genome of this bumble bee parasite.

The Phosphofructokinase and Pyruvate Kinase Genes in Apis andreniformis and Apis cerana indica: Exon Intron Organisation and Evolution.

Genes related to carbohydrate metabolism have evolved rapidly in eusocial bees, including honey bees. However, the characterisation of carbohydrate metabolism genes has not been reported in Apis andreniformis or Apis cerana indica. This study aimed to characterise phosphofructokinase (PFK) and pyruvate kinase (PK) genes in these honey bee species and to analyse the evolution of the genus Apis using these genes. This study found the first data regarding A. andreniformis PFK and PK-like nucleotide sequences. A BLAST-n algorithm-based search showed that A. andreniformis and A. c. indica PFK and PK genes were homologous with those of Apis florea and Apis cerana cerana from Korea, respectively. Multiple alignments of PFKs from five Apis species showed many exon gains and losses, but only one among the PKs. Thus, the exon–intron organisation of the PK genes may be more conserved compare with that of the PFKs. Another evolutionary pattern indicated that more nucleotide substitutions occurred in Apis’ PK than PFK genes. Deduced PFK amino acid sequences revealed a PFK consensus pattern of 19 amino acids, while the deduced PK amino acid sequences were predicted to have barrel and alpha/beta domains. Based on these two metabolism-related genes, The Neighbour-joining and Maximum likelihood phylogenetic trees are congruent and revealed that the A. andreniformis and A. florea group were in the basal position. Apis mellifera, A. cerana, and Apis dorsata formed a monophyletic clade, although the positions of A. mellifera and A. dorsata were different in the nucleotide- and amino acid-based phylogenetic trees.

Floral diversity and foraging behaviour of little/dwarf honey bee Apis florea f. in horticultural ecosystem, Madurai District, Tamil Nadu

Among the honey bee species, little/dwarf bee Apis florea F. plays a major role in pollination of various annual and perennial crops and the successful beekeeping depends on the availability of bee foraging plants in any particular area. This study deals with the flora availability of A. florea in the study area and the results revealed that 122 plants belonging to 47 families were identified as bee foraging plants and the major bee floral families are Fabaceae (15 species/12.29%) followed by Asteraceae (13 species/10.66%), Cucurbitaceae and Euphorbiaceae (7 species/5.74% each) and Amaranthaceae (6 species/4.92%). These pasturage plants are further grouped into nectar yielders (11), pollen yielders (15) and both nectar and pollen yielders (96).

Floral visitors of canola (Brassica napus L.) hybrids in Cerrado Mineiro region, Brazil

ABSTRACT: The objective of this study was to verify floral visitors of canola in the Triângulo Mineiro region, Minas Gerais, Brazil. A completely randomized plot design with two hybrids and 10 replicates was used, totaling 20 plots. The survey of floral visitors was made during 10 days. Faunal analyses of the collected species were performed based on frequency, constancy, dominance, and diversity. A total of 2,676 individuals belonging to 13 species of bees of two families (Apidae and Halictidae) were collected. The diversity index found among hybrids was similar (1.28 in hybrid 61 and 1.50 in hybrid 433). The species Apis mellifera Linnaeus, 1758 (Hymenoptera: Apidae), Paratrigona lineata (Lepeletier, 1836) (Hymenoptera: Apidae), and Trigona spinipes (Fabricius, 1793) (Hymenoptera: Apidae) were the most abundant and constant in the crops during all the evaluated days for both hybrids. So, it is important that efforts be made to conserve natural areas close to crops in order to provide resources for the permanence of floral visitors in the area.

Phylogenetic Uniqueness of Honeybee Apis Cerana from the Korean Peninsula Inferred from The Mitochondrial, Nuclear, and Morphological Data

Abstract Apis cerana is an Eastern honeybee species distributed throughout Asia and closely related to the Western honeybee species Apis mellifera distributed across all of Africa, Europe and Western Asia, and subdivided into thirty confirmed subspecies. Currently, A. cerana is an endangered bee species in contrast to A. mellifera. We sequenced and annotated the complete mitochondrial genome of A. cerana from the Jeollanam-do province of South Korea and uploaded to the DDBJ/Genbank database(AP018431). MtDNA sequence is 15.925 bp long, has 84% AT-content and 16% GC-content and contains 22 tRNA genes, 13 protein-coding genes, two ribosomal RNA genes, one AT-rich region and four non-coding intergenic regions (NC1-4). All protein-coding genes are started by ATT and ATG codons, except the genes ATP8 and ND4, which started by ATC and ATA, respectively, and are stopped by the common codons TAA and TAG. A comparative analysis of the whole mtDNA sequences of A. cerana from Korea and Taiwan, A. c. cerana from China and A. c. japonica from Japan showed that the genetic divergence of the Korean A. cerana sample from subspecies A. c. cerana (2.57%) and A. c. japonica (2.58%) matched to the level of genetic divergence of mtDNA between animal subspecies (0.8-8%). Based on the comparative analysis of complete mtDNA (~16,000 bp), two nuclear gene VG and EF1- α sequences (~8,000 bp) and morphological measurements (six parameters), we assumed that Korean A. cerana, Chinese A. c. cerana and Japanese A. c. japonica are different subspecies at an early stage of sub-speciation and could be called further as subspecies of Apis cerana koreana.

Apis cerana Beekeeping and Sacbrood Disease Management in Vietnam: Review

Beekeeping status of Apis cerana with emphasis of experiences overcoming sacbrood virus disease are presented. Social bee fauna are rich in Vietnam with 6 honeybee species (Apis laboriosa, Apis dorsata, Apis mellifera, Apis cerana, Apis andrenifomis, Apis florea); 8 stingless bee species (Trigona laeviceps, Trigona ventralis, Trigona pagdeni, Trigona gressitti, Trigona fuscobalteata, Trigona capenteri, Trigona scintillans Trigona iridipenis) and 2 bumble bee species (Bumbus haemorrhoidalis, B. breviceps). All of them are native except A. mellifera which was introduced in1887. These bees are slated for conservation by the Ministry of Agriculture & Rural Development. Honey and other bee products are mainly harvested from 3 species including A. cerana, A. mellifera and A. dorsata. The manageable species (A. cerana and A. mellifera) are increasing in number, reaching about 1,500,000 beehives. Vietnam is the second largest honey exporter in Asia, with a total of about 48,000 tons of honey exported to the international market in 2014. A. cerana plays an important role in poverty alleviation in mountainous and remote areas of Vietnam. Honeybee suffers from various diseases of Sacbrood virus disease (SBV), European foulbrood (EFB), Nosema, and parasitic mites of Tropilaelaps mercedes and Varroa destructor. Most of these diseases can be resolved with biocontrol methods. For the parasitic mites, Vietnamese beekeepers usually apply formic acid.

Marked interspecific differences in the neuroanatomy of the male olfactory system of honey bees (genus Apis).

All honey bee species (genus Apis) display a striking mating behavior with the formation of male (drone) congregations, in which virgin queens mate with many drones. Bees' mating behavior relies on olfactory communication involving queen-but also drone pheromones. To explore the evolution of olfactory communication in Apis, we analyzed the neuroanatomical organization of the antennal lobe (primary olfactory center) in the drones of five species from the three main lineages (open-air nesting species: dwarf honey bees Apis florea and giant honey bees Apis dorsata; cavity-nesting species: Apis mellifera, Apis kochevnikovi, and Apis cerana) and from three populations of A. cerana (Borneo, Thailand, and Japan). In addition to differences in the overall number of morphological units, the glomeruli, our data reveal marked differences in the number and position of macroglomeruli, enlarged units putatively dedicated to sex pheromone processing. Dwarf and giant honey bee species possess two macroglomeruli while cavity-nesting bees present three or four macroglomeruli, suggesting an increase in the complexity of sex communication during evolution in the genus Apis. The three A. cerana populations showed differing absolute numbers of glomeruli but the same three macroglomeruli. Overall, we identified six different macroglomeruli in the genus Apis. One of these (called MGb), which is dedicated to the detection of the major queen compound 9-ODA in A. mellifera, was conserved in all species. We discuss the implications of these results for our understanding of sex communication in honey bees and propose a putative scenario of antennal lobe evolution in the Apis genus.

Honey Bees Species Differentiation using Geometric Morphometric on Wing Venations

Wings are the most interesting part for morphometric study in any species of insect, including honey bees. The geometric morphometric is a strong analytical method to identify the species of insect, therefore we used wing venations to explore the wing variations among five honey bee species in Indonesia, i.e. A. mellifera, A. cerana, A. dorsata, A. andreniformis, and A. koschevnikovi. We used Thin Plate Splin (TPS) software to conduct the landmark digitation on wing venation and geometric morphometric analysis based on the 19 anatomically points of venation through tpsdig2 and tpsRelw software to describe the variations of wing venations. Our result of Relative Warp ordination plot showed variations of wing venation of five honey bee species, particularly on the five points. Grid deformations show the difference patterns among the five species of honey bees as well. Moreover, based on phylogenetic tree by using Neighbour Joining approach, the morphometric geometric wings venation is also able to separate the species of honey bees; thus, a possibility method to differentiate the honey bee species in Indonesia.

Complete genome sequence of sacbrood virus isolated from Asiatic honey bee Apis cerana indica in India.

We determined the complete genome sequence of a sacbrood virus (SBV) infecting Indian honey bee (Apis cerana indica) from Tamil Nadu, India named as AcSBV-IndTN1. The genome of AcSBV-IndTN1 comprised of 8740 nucleotides, encoding a single large ORF containing 2849 amino acids flanked by 5' and 3' untranslated regions. Results of phylogenetic tree analysis based on complete genomes of SBV isolates indicated that the virus isolates from India isolated from the Asiatic honey bee A. cerana (AcSBVs) formed a separate group along with six Vietnam isolates and three Chinese isolates. The AcSBV-IndTN1 isolate showed closer genetic relationship with other isolates from India. The second major group had both AcSBVs and AmSBVs (virus isolated from European honey bee, Apis mellifera SBV) of Korea, China and Vietnam. The third and a distantly related group had AmSBVs of Australia, UK, USA and Korea. The results obtained from phylogenetic analysis were further supported with evolutionary distance analysis. AcSBV-IndTN1 isolate open reading frame had 95-99% amino acid sequence similarity with other Indian isolates and 92-96% with AcSBVs and AmSBVs of other geographical locations. In addition, sequence difference count matrix ranged from 154 to 907nt among all the SBV isolates. This suggests that the virus isolates have evolved significantly in different geographical locations but isolates on different hosts in a given location/country are closely related. The high similarity in the genome among the AcSBV and AmSBV isolates indicate possible cross-infections and recombination of SBV isolates in Asian continent where both the honey bee species are reared in close proximity. Gene flow between SBV population indicating that an infrequent gene flow occur between them. The pattern of molecular diversity in SBV population revealed that the occurrence of recent population expansion of SBV. To the best of our knowledge this is the first report of the complete nucleotide sequence of AcSBV from Tamil Nadu, India. This study provided an opportunity to establish the molecular evolution of SBV isolates and shall be useful in the development of diagnostics and effective disease control strategies.

An update of Africanization in honey bee (Apis mellifera) populations in Buenos Aires, Argentina

European and African honey bee populations have been separated and influenced by different environments. Furthermore, Apis mellifera is the only species of honey bees that evolved in Europe, the Middle East, and Africa, where several subspecies are recognized. A. m. scutellata was introduced in Brazil in 1956, resulting in the spread of African bees throughout South and Central America. The aim of the present study was to determine the prevalence and geographical distribution of Africanized bees in Buenos Aires province (Argentina) employing a mtDNA cytochrome b polymorphism. A total of 430 colonies were sampled between 2013 and 2014; eighteen out of them (4,2%) belonged to African linage. Our results confirm that European haplotypes are the most prevalent in Buenos Aires Province, and that the process of African gene introgression remains stable since 2005.

Discrimination of the entomological origin of honey according to the secretions of the bee (Apis cerana or Apis mellifera)

The eastern honeybee Apis cerana and the western honeybee Apis mellifera are the two most economically valuable honeybee species used in apiculture. In market, the price of Apis cerana honey (ACH) is usually several times higher than that of Apis mellifera honey (AMH) due to the production limit, resulting in wide adulteration and counterfeiting of ACH by AMH. In the present study, we compared honeybee secretions in these two kinds of honey, and found significant differences in protein profiles and hydrocarbon components. The SDS-PAGE pattern showed three species-specific bands with molecular weights between 15.0 and 29.4 KDa in ACH, and six species-specific bands in AMH with molecular weights between 13.8 and 33.1 KDa. The GC–MS-MS detection of the petroleum ether extracts of the two kinds of honey showed that 17-Pentatriacontene and Hentriacontane were the characteristic constituents of ACH and AMH, respectively. These two methods constitute a system to satisfy different needs for entomological authentication of honey samples.