Hypertrehalosaemic peptides in the honeybee ( Apis mellifera): purification, identification and function

SUMMARYMethanolic extracts of the corpora cardiaca (CC) of foraging workers of the yellow Italian race of honey bees (Apis mellifera ligustica) contained on average 3.0 pmol/pCC (paired corpora cardiaca) of the hypertrehalosaemic hormone, Mas-AKH. The CC of A. m. ligustica nurse bees (8–10 days old) contained only about 1/20th (0.14 pmol/pCC) of the Mas-AKH as that of foragers, and very young bees (2–4 days old) contained no detectable hormone. CC extracts of A. m. ligustica virgin drones and older drones (> 8 days) also contained Mas-AKH, but much less (0.23 pmol/pCC) than foraging worker bees. CC extracts from both virgin and mated (egg laying) ligustica/carnica queens, as well as from foraging workers or drones of the brown European race of honey bees (Apis mellifera carnica) all lacked detectable Mas-AKH or any other related peptides. There was no elevation of the total blood sugar titre in any caste of either ligustica or carnica when injected with either synthetic Mas-AKH or with extracts of their respective corpora cardiaca. The amount of fat body and the amount of stored glycogen is small in all castes of both races. The crop in all castes is large and contained up to 30% of the total body weight. In conclusion, none of the castes of either race seemed to use a hormone to mobilize sugar from the fat body or other glycogen stores (such as the muscle), but rather they depend for all activities entirely on sugars in the crop.

Carniolan honey bees (Apis mellifera carnica) are considered as an indigenous subspecies in Hungary adapted to most of the ecological and climatic conditions in this area. However, during the last decades Hungarian beekeepers have recognized morphological signs of the Italian honey bee (Apis mellifera ligustica). As the natural distribution of the honey bee subspecies can be affected by the importation of honey bee queens or by natural gene flow, we aimed at determining the genetic structure and characteristics of the local honey bee population using molecular markers. All together, 48 Hungarian and 84 foreign (Italian, Polish, Spanish, Liberian) pupae and/or workers were used for mitochondrial DNA analysis. Additionally, 53 sequences corresponding to 10 subspecies and the Buckfast hybrid were downloaded from GenBank. For the nuclear analysis, 236 Hungarian and 106 foreign honey bees were genotyped using nine microsatellites. Heterozygosity values, population‐specific alleles, FST values, principal coordinate analysis, assignment tests, structure analysis, and dendrograms were calculated. Haplotype and nucleotide diversity values showed moderate values. We found that one haplotype (H9) was dominant in Hungary. The presence of the black honey bee (Apis mellifera mellifera) was negligible, but a few individuals resembling other subspecies were identified. We proved that the Hungarian honey bee population is nearly homogeneous but also demonstrated introgression from the foreign subspecies. Both mitochondrial DNA and microsatellite analyses corroborated the observations of the beekeepers. Molecular analyses suggested that Carniolan honey bee in Hungary is slightly affected by Italian and black honey bee introgression. Genetic differences were detected between Polish and Hungarian Carniolan honey bee populations, suggesting the existence of at least two different gene pools within A. m. carnica.

Simple SummaryWing venation traits are used to identify honey bee subspecies. While several wing-based tools are available, they suffer from weaknesses that were addressed by the recently developed software DeepWings©. This software allows fully automated identification of wing images in a friendly, free, and rapid manner. Here, we sought to test DeepWings© on 14,816 wing images representing 2601 colonies sampled in the native areas of three widespread subspecies in Europe: the Iberian honey bee (Apis mellifera iberiensis), the dark honey bee (Apis mellifera mellifera), both belonging to the M lineage, and the Carniolan honey bee (Apis mellifera carnica), belonging to the C lineage. DeepWings© classification of these colonies largely matched the endemic M and C lineages, with proportions of 71.4% and 97.6%, respectively. At the subspecies-level the matching proportions were 89.7% for the Iberian honey bee, 41.1% for the dark honey bee and 88.3% for the Carniolan honey bee, which can be explained by DeepWings© sometimes confounding closely related subspecies and, more importantly, by genetic pollution. A comparison between DeepWings© data and molecular data revealed that the agreement between the two is weaker when there is genetic pollution. Our results suggest that DeepWings© is a valuable tool for honey bee identification, which can be used not only for breeding and conservation but also for research purposes.DeepWings© is a software that uses machine learning to automatically classify honey bee subspecies by wing geometric morphometrics. Here, we tested the five subspecies classifier (A. m. carnica, Apis mellifera caucasia, A. m. iberiensis, Apis mellifera ligustica, and A. m. mellifera) of DeepWings© on 14,816 wing images with variable quality and acquired by different beekeepers and researchers. These images represented 2601 colonies from the native ranges of the M-lineage A. m. iberiensis and A. m. mellifera, and the C-lineage A. m. carnica. In the A. m. iberiensis range, 92.6% of the colonies matched this subspecies, with a high median probability (0.919). In the Azores, where the Iberian subspecies was historically introduced, a lower proportion (85.7%) and probability (0.842) were observed. In the A. m mellifera range, only 41.1 % of the colonies matched this subspecies, which is compatible with a history of C-derived introgression. Yet, these colonies were classified with the highest probability (0.994) of the three subspecies. In the A. m. carnica range, 88.3% of the colonies matched this subspecies, with a probability of 0.984. The association between wing and molecular markers, assessed for 1214 colonies from the M-lineage range, was highly significant but not strong (r = 0.31, p < 0.0001). The agreement between the markers was influenced by C-derived introgression, with the best results obtained for colonies with high genetic integrity. This study indicates the good performance of DeepWings© on a realistic wing image dataset.

BackgroundResearch into the genetic diversity of honey bee (Apis melliferaL.) populations has become increasingly significant in recent decades, primarily due to population declines attributed to human activities and climate change. As a species of great importance, breeding programs that leverage understanding of genomic diversity could offer solutions to mitigate these challenges. The objective of this study was to examine the genomic diversity and population structure of Carniolan honey bees (Apis mellifera carnica) using the Illumina SNP chip on a large honey bee sample collected from Central and South-Eastern European countries. The study also aims to offer recommendations for future breeding programs.ResultsOur analysis involved Discriminant Analysis of Principal Components (DAPC), heterozygosity, admixture analysis, fixation indices (FST), Neighbour-Joining tree, gene flow and Isolation-by-distance analysis. DAPC indicated distinct separation between the Carniolan and Italian honey bee (Apis mellifera ligustica) populations, whereas the admixture analysis revealed varying levels of gene flow and genetic admixture within the Carniolan honey bee populations, demonstrating closer relationships between specific geographic regions (confirmed by Isolation-by-distance analysis). Furthermore, the research of heterozygosity, genomic inbreeding, pairwise FST values, and Neighbour-Joining tree provided insights into the patterns of genetic differentiation and similarity among the populations of Carniolan honey bee within its natural habitat. We have observed genetic homogeneity of the Carniolan honey bee population when considered in a broader genetic/geographical context. However, the Carniolan honey bee has sufficient genetic diversity in its geographical home range that needs to be carefully monitored and maintained.ConclusionsThis study provides important insights into the genetic composition, differentiation, and relationships among Carniolan honey bee populations in Central and South-Eastern European countries. The findings are crucial for conservation efforts, breeding programs, and sustainable beekeeping practices. They emphasise the importance of considering genetic factors and population structure in the breeding and management of honey bees. By understanding these genetic relationships, we can develop strategies to preserve genetic diversity, improve breeding outcomes, and ensure the resilience of honey bee populations in the face of environmental changes and challenges. This knowledge can also inform policy makers and stakeholders on best practices to maintain healthy bee populations, which are vital for ecosystem services and agricultural productivity.

Adipokinetic hormones (AKHs) of sphingid Lepidoptera, including the identification of a second M. sexta AKH

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A comparative study on hypertrehalosaemic hormones in the Hymenoptera: sequence determination, physiological actions and biological significance

The natural diversity of the honey bee (Apis mellifera) includes five evolutionary lineages and 26 subspecies, currently described that come from Africa, Europe, and the Middle East. They were introduced by humans to almost every continent and each of them has adapted favorably to the environmental and climatic conditions of their geographical areas of origin, which has led to greater genetic diversity and hybridization processes between subspecies. The genetic diversity of honey bees in Chile was characterized by the presence of European subspecies, such as Apis mellifera mellifera and Apis mellifera ligustica, but no updated information is available. The objective of this study was to assess the current genetic characterization of honey bees in the coastal zone of the O’Higgins Region, due to its national importance in beekeeping, using geometric morphometrics. Samples were taken from five counties taking samples of five apiaries from each one, including 3 colonies per apiary with a total of 30 bees per colony. The results indicate that there is evidence of hybridization between the subspecies Apis mellifera carnica and Apis mellifera ligustica, which reveals that the genetic pattern of the region has changed. This raises the questions as to whether this hybridization with predominantly Apis mellifera carnica is the most suitable for the ecological conditions of the region and how this could affect colony productivity and local beekeeping.

Background: Adipokinetic hormones (AKHs) regulate important physiological processes in insects. AKHs are short peptides with blocked termini and Trp in position 8. Often, proline occupies position 6. Few post-translational modifications have been found, including hydroxyproline ([Hyp6]) and kynurenine. Our recent data suggest that the Hyp- and Kyn-containing AKHs occur more often than originally thought and we here investigate if they are natural or artifactual. Methods: From crude extracts of the corpora cardiaca (CC) of various insect species, AKHs were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-MS). Synthetic [Hyp6]-AKHs were tested in an in vivo metabolic assay. Freshly dissected Periplaneta americana and Blaberus atropos CCs (with precautions taken against oxidation) were analyzed. B. atropos CC were placed into a depolarizing saline and the released AKHs were measured. Results: Hyp was detected in several decapeptides from cockroaches. The modified form accompanied the AKH at concentrations below 7%. The [Hyp6]-AKHs of B. atropos were present in fresh CC preparations and were shown to be releasable from the CC ex vivo. Synthetic [Hyp6]-containing peptides tested positively in a hypertrehalosemic bioassay. Hydroxyprolination was also detected for Manto-CC from the termite Kalotermes flavicollis and for Tetsu-AKH of the grasshopper, Tetrix subulata. Oxidized Trp-containing forms of Nicve-AKH were found in species of the burying beetle genus Nicrophorus. Conclusions: Trp oxidation is known to occur easily during sample handling and is likely the reason for the present findings. For hydroxyprolination, however, the experimental evidence suggests endogenous processes.

This paper investigated the genetic diversity of MDH Ⅱ in 5 different honeybee species by means of IEF-PAGE technology. The genotype, genotype frequency, allele frequency, as well as the homozygous and heterozygous degree of MDHI Ⅱ were analyzed in this study, leading us to discover the polymorphism of MDH Ⅱ in these western honeybees. Allelea was found to be the dominant loci in Apis mellifera carnica (79.7%), and the allele b frequency was predominant in Apis mellifera ligustica from Jiangshan (53.8%), while allele c showed to have a higher frequency in Apis mellifera ligustica of high royal jelly bee (64.2%) than the other. Genetic heterosis of the high royal jelly bee is the highest at 67.3%, but it was low in Apis mellifera carnica. These results showed to be significantly different among the different honeybees regarding genotype, genotype frequency, gene frequency, gene heterosis, and gene homogeneity of MDH Ⅱ.

Carniolan honeybee (

ABSTRACT. An attempt was made to separate glycogen phosphorylase activating hormone (GPAH) and adipokinetic hormone (AKH) from the corpora cardiaca (CC) of the moth Manduca sexta (Lepidoptera: Sphingidae) by separating extracts of CC on various chromotographic media, but it was not possible to conclude whether GPAH and AKH are activities of one or of two different peptides. Both activities elute together from glass beads, from Sephadex G‐25 and from Sephadex LH‐20 columns. In the separation experiments with glass beads and G‐25 the activities eluted as a single peak, but using LH‐20 we found two peaks exhibiting both activities. The major peak eluted at 1.25 × Vt, which is very similar to locust AKH, while the smaller second peak eluted at O.74 ×Vt. Cross injections of CC extracts from M. sexta into Locusta migratoria and CC extracts from L. migratoria into M. sexta suggest that GPAH and the AKH from M. sexta are not identical with the decapeptide AKH from locusts.

We conducted a statewide study, in June and July 2016, of feral colonies and domesticated hives to understand the current and potential impacts of invasive parasites and pests on honey bee (Apis mellifera) populations in Texas. We identify the subspecies for each colony sampled in addition to reporting the presence of parasites. The most common subspecies found were Apis mellifera scutellata, Apis mellifera carnica, and Apis mellifera ligustica. We confirmed the presence of Apis mellifera syriaca and Apis mellifera macedonica in Texas. Varroa destructor and Nosema ceranae were the most common parasites, found in eight and nine ecoregions, respectively. Aethina tumida was found in only 4 of the 10 ecoregions.

SummaryWorker honey bee samples were collected from six different locations spread throughout the Republic of Macedonia. Morphometric analyses were performed on the front right wing venation of workers (n=1800) and measurements were obtained for a total of 21 characteristics. The bees were examined according to the apiaries from which they originated (n=6) and in equal groups (n=300). Our aim was to determine the diversity of the sampled workers, as well as to examine the relationship with other honey bee races. The racial relationship of the total sample of analysed bees expressed in percentages were: Apis mellifera macedonica: 15.87%; Apis mellifera ligustica. 12.70%; Apis mellifera carnica: 10.32%; Apis mellifera caucasica: 4.76%; Apis mellifera mellifera 4.76%. The variability in honey bee populations found thus provides a suitable basis for the selection of large number of subpopulations with desired biological or production characteristics for introducing a National Bee Breeding Programme in the Republic of Macedonia.

Bee venom contains the allergic enzymes phospholipases A2 (PLA2) and hyaluronidase. These enzymes have been extensively studied as therapeutic modalities because of their proven effects in pharmaceutical and clinical applications. The cDNA cloning of PLA2 and hyaluronidase was amplified by RT-PCR from the total RNA of the venom gland of a honeybee (Apis mellifera carnica). The lengths of the PLA2 and hyaluronidase of Apis mellifera ligustica were 504 and 1146bp, respectively. The genes of PLA2 and hyaluronidase shared 90.94% and 96.65% homologies with A. mellifera ligustica and Apis cerana cerana, respectively. Some similar PLA2 and hyaluronidase were also found in the venom of other bee species, We analyzed their sequences and compared them with those of other sources. A notable finding was that the two genes differed from those of A. mellifera ligustica and A. cerana cerana. The positions of the disulfide bonds of PLA2 and hyaluronidase were also completely different from those previously reported. We used the available sequences to construct a phylogenetic tree and discovered that these two genes of A. mellifera carnica belonged to the western honeybee, and was more closely related to that of A. mellifera ligustica than to any other insect.