Mellifera Drones Research Articles

Ontogeny of immunity and natural viral infection in Apis mellifera drones and workers

The most common viral diseases affecting honey bees (Apis mellifera) in Israel include deformed wing viruses (DWV-A and DWV-B) and acute paralysis viruses (ABPV and IAPV). These viruses are transmitted within and between colonies, both horizontally and vertically. All members of the colony contribute to this transmission, on the other hand individual and social immunity, particularly hygienic behaviour, may affect the outcome of the process. In this study, we evaluated the ontogeny of natural infections of DWV-A, DWV-B, ABPV and IAPV, their prevalence and loads, in workers and drones from high (H) and low (L) hygienic colonies. In parallel, we evaluated the expression of two immune genes: peptidoglycan recognition protein S2(PGRP-S2) and hymenoptaecin. The prevalence of DWV-B and IAPV increased with age and was higher in workers than in drones. ABPV was not detected in drones. The expression of both immune genes was significantly affected by age and sex. Drones from H colonies had higher expression of these genes. The increased expression of immune genes with drones’ age, particularly in hygienic colonies, suggest additional value of honey bee breeding for hygienic behaviour for sustainable beekeeping.

The Effect of Comb Cell Size on the Development of Apis mellifera Drones.

The growth and development of honeybees are influenced by many factors, one of which is the cell size of the brood comb. Larger worker bees can be obtained by being raised in bigger cells. However, whether cell size has the same effect on drone development is still unknown. Here, using 3D-printed foundations, we observed the development of drones kept in comb cells of different sizes from the late larval stage through eclosion. The results showed that drones in larger cell-size combs had heavier body weights, longer body lengths, and larger head widths, thorax widths, and abdomen widths compared to those in smaller cell-size combs. Furthermore, regardless of developmental stages, the drones' body weights increased linearly with the comb's cell size. However, the other morphological changes of drones in different developmental stages were out of proportion to the cell-size changes, resulting in smaller cells with a higher fill factor (thorax width/cell width). Our findings confirm that comb cell size affects the development of honeybees; drones become bigger when raised in large cells.

Effectiveness of glycerol, DMSO and trehalose in the process of cryopreservation of drone semen from the species apis mellifera

Cryopreservation of economically important or endangered animal species is becoming an increasingly popular practice in the field of reproductive biotechnologies. The mass extinction of honey bee (Apis Mellifera) colonies in the last decade requires the use of new chemical components in cryopreservation media for the storage of male gametes. A number of studies demonstrate conflicting data regarding the success rate and cytotoxicity of glycerol and dimethyl sulfoxide and to a lesser extent Trehalose. Dimethyl sulfoxide is an organosulfur compound with low cryotoxicity which transforms into solids below room temperature and this property makes it suitable for cryoprotectant. The kosmotropic properties of glycerol allow it to create hydrogen bonds with water molecules, making it difficult to form intracellular ice crystals in the cryoprotective media. Due to its high-water retaining properties Trehalose can be used as cryoprotectant, but its effectiveness has not yet been thoroughly researched in relation to the cryopreservation of bee drone spermatozoa. The research aims to compare the effectiveness of each of the specified cryoprotectants regarding the preservation of spermatozoa from Apiss Mellifera drones.

Prevalence of Varroa destructor and first report of Nosema sp. in Apis mellifera drone congregation areas

Varroosis and nosemosis are diseases that affect honey bees (Apis mellifera) and therefore have a great impact on beekeeping worldwide. These parasites can be dispersed by drones and, in the case of Varroa destructor (etiological agent of varroasis), its presence has been reported in areas of natural fertilization of A. mellifera, known as Drone Congregation Areas (DCA). Previously, several DCAs were identified in Argentina and progress was made in their topographic and genetic characterization, and sanitary studies were also initiated. In this study, three DCA and one apiary located in the province of Tucumán were monitored for four years, and the presence of V. destructor and Nosema sp. was evaluated. The results indicated the presence of V. destructor in the drone samples of the DCA throughout the study. Although a trend of decreasing parasitization over time was observed, no statistically significant differences were found between years or among the considered DCAs. The presence of Nosema sp. was detected for the first time in the DCA, with samples exhibiting high spore load. The results support the role of drones in the dynamics and dispersal of diseases and allow us to conclude that the analysis of DCAs represents a key tool for monitoring the health of bee populations, complementary to the analyses carried out in apiaries.

Review on Sustainable Cryopreservation and Nonfreezing Storage Solutions of European Honeybee Apis Mellifera Drone Semen

The European honeybee Apis mellifera is the main pollinator for most crops used for human consumption. However, a number of diseases, parasites, pesticides and other factors that generally result in the widely described colony collapse disorder weakens honeybee colonies. In order to maintain the existing honeybee germ lines and facilitate the creation of new disease-resistant lines, it is necessary to ensure consistent breeding work, which would also allow the long-term preservation of the unique germplasm lines. One of the most promising solutions for the preservation of honeybee germplasm is the storage of honeybee drone semen. In recent decades, there has been a renewed interest in the preservation of honeybee drone semen using both cryopreservation and non-freezing storage methods. This review looks at the latest developments in novel sperm storage technologies and compares them to well-known preservation solutions. Additionally, the most accessible and widely used solutions will be reviewed, taking into account the cost of the necessary equipment, the complexity of the methods, time consumption and resulting sperm quality.

Review of Sustainable Cryopreservation and Above-Freezing Storage Solutions of European Honey Bee Apis mellifera Drone Semen

Abstract The European honey bee Apis mellifera is the main pollinator for most crops used for human consumption. However, a number of diseases, parasites, pesticides and other factors that generally result in the widely described colony collapse disorder weakens honey bee colonies. In order to maintain the existing honey bee germ lines and facilitate the creation of new disease-resistant lines, it is necessary to ensure consistent breeding work, which would also allow the long-term preservation of the unique germplasm lines. One of the most promising solutions for the preservation of honey bee germplasm is the storage of honey bee drone semen. In recent decades, there has been a renewed interest in the preservation of honey bee drone semen using both cryopreservation and above-freezing storage methods. This review summarizes the latest developments in novel sperm storage technologies and their assessment by sperm quality and queen fertility indicators. Additionally, this article analyses the advantages and disadvantages of the reviewed solutions and outlines directions in which additional research would be needed to develop effective, available and affordable drone semen storage solutions. To the best of the authors' knowledge this is the first review of cryopreservation and above-freezing storage solutions of European honey bee Apis mellifera drone semen in the scientific literature.

Suitability of drone olfactory sensitivity as a selection trait for Varroa-resistance in honeybees

The most effective strategy against brood diseases, such as those stemming from infestation by the mite Varroa destructor, is the early detection and removal of sick brood. Recent findings suggest that genes associated with worker bee olfactory perception play a central role in Varroa-sensitive hygiene (VSH). In this study, the odour sensitivity of Apis mellifera drones was examined through proboscis extension response (PER) conditioning. Individuals sensitive/insensitive to the two Varroa-parasitised-brood odours (extract-low and extract-high) were used for breeding. Twenty-one queens from a VSH-selected line (SelQ) and nineteen queens from a nonselected line (ConQ) were single-drone-inseminated with sperm from drones that showed either sensitivity (SenD+) or insensitivity (SenD−) to the two extracts. Individual VSH behaviour in a total of 5072 offspring of these combinations (SelQ × SenD+, SelQ× SenD−, ConQ × SenD+, ConQ × SenD−) was subsequently observed in a specially designed observation unit with infrared light. The results from the video observation were also separately examined, considering the genetic origin (VSH-selected or nonselected line) of the participating queens and drones. While the drone PER conditioning results were not significantly reflected in the VSH results of the respective offspring, the genetic origin of the participating queens/drones was crucial for VSH manifestation.

Analysis of Honeybee Drone Activity during the Mating Season in Northwestern Argentina.

Simple SummaryWhen we think of social insects, we mostly neglect males and their biology. Honeybee males or drones are an example. Like queens, drones are founders of the colony, but unlike queens and workers, their activity is not well-known. Drones exit the hive to mate as their only goal, in spite of the high chances of getting lost, being preyed upon and starving; and only if they are lucky, they will die when mating. These nuptial flights take place in Drone Congregation Areas during spring and summer, and we know that their exit time is in the afternoons. Although, drone activity at other hours of the day has not been studied. Using three methods (direct observation, video records and microchip tagging) we evaluated drone activity during the whole day in Northwest Argentina. We detected 24 h of activity for the first time. Surprisingly, several drones were active at dawn, morning, and late morning. Most of them were active in the afternoon, as previously reported. The activity at dawn and during the morning could be a normal pattern in drones around the world, or it could be a result of abnormal factors, such as environmental variables altering their biological clocks.Males in Hymenopteran societies are understudied in many aspects and it is assumed that they only have a reproductive function. We studied the time budget of male honey bees, drones, using multiple methods. Changes in the activities of animals provide important information on biological clocks and their health. Yet, in nature, these changes are subtle and often unobservable without the development and use of modern technology. During the spring and summer mating season, drones emerge from the hive, perform orientation flights, and search for drone congregation areas for mating. This search may lead drones to return to their colony, drift to other colonies (vectoring diseases and parasites), or simply get lost to predation. In a low percentage of cases, the search is successful, and drones mate and die. Our objective was to describe the activity of Apis mellifera drones during the mating season in Northwestern Argentina using three methods: direct observation, video recording, and radio frequency identification (RFID). The use of RFID tagging allows the tracking of a bee for 24 h but does not reveal the detailed activity of drones. We quantified the average number of drones’ departure and arrival flights and the time outside the hive. All three methods confirmed that drones were mostly active in the afternoon. We found no differences in results between those obtained by direct observation and by video recording. RFID technology enabled us to discover previously unknown drone behavior such as activity at dawn and during the morning. We also discovered that drones may stay inside the hive for many days, even after initiation of search flights (up to four days). Likewise, we observed drones to leave the hive for several days to return later (up to three days). The three methods were complementary and should be considered for the study of bee drone activity, which may be associated with the diverse factors influencing hive health.

Differential Viral Distribution Patterns in Reproductive Tissues of Apis mellifera and Apis cerana Drones.

Honeybee drones are male bees that mate with virgin queens during the mating flight, consequently transferring their genes to offspring. Therefore, the health of drones affects the overall fitness of the offspring and ultimately the survivability of the colony. Honeybee viruses are considered to be a major threat to the health of honeybees. In the present study, we demonstrated the pattern of common honeybee viruses in various tissues of drones in the western honeybee, Apis mellifera, and the eastern honeybee, Apis cerana. Drones were collected during the mating flight and analyzed using quantitative real-time (qRT-PCR) to detect the presence of seven honeybee viruses. The qRT-PCR result revealed that three honeybee viruses, namely Black Queen Cell Virus (BQCV), Deformed Wing Virus (DWV), and Chinese Sacbrood Virus (CSBV), were detected in the reproductive tissues of A. mellifera and A. cerana drones. The results from qRT-PCR showed that the Israeli Acute Paralysis Virus (IAPV) was only detected in A. mellifera drone body tissues. Moreover, the prevalence of DWV and BQCV in the drones collected from A. mellifera colonies was significantly higher than that of A. cerana. In addition, virus multiple infections were higher in A. mellifera drones compared to those in A. cerana. CSBV was found predominantly in the reproductive tissues of A. cerana drones. This study is the first report describing the presence of the CSBV in reproductive tissues of A. mellifera drones. Our results may reflect the preference of honeybee viruses in honeybee species and may provide a piece of interesting evidence for understanding the virus transmission in A. cerana.

Juvenile hormone pathway in honey bee larvae: A source of possible signal molecules for the reproductive behavior of Varroa destructor.

The parasitic mite Varroa destructor devastates honey bee (Apis mellifera) colonies around the world. Entering a brood cell shortly before capping, the Varroa mother feeds on the honey bee larvae. The hormones 20‐hydroxyecdysone (20E) and juvenile hormone (JH), acquired from the host, have been considered to play a key role in initiating Varroa's reproductive cycle. This study focuses on differential expression of the genes involved in the biosynthesis of JH and ecdysone at six time points during the first 30 hr after cell capping in both drone and worker larvae of A. mellifera. This time frame, covering the conclusion of the honey bee brood cell invasion and the start of Varroa's ovogenesis, is critical to the successful initiation of a reproductive cycle. Our findings support a later activation of the ecdysteroid cascade in honey bee drones compared to worker larvae, which could account for the increased egg production of Varroa in A. mellifera drone cells. The JH pathway was generally downregulated confirming its activity is antagonistic to the ecdysteroid pathway during the larva development. Nevertheless, the genes involved in JH synthesis revealed an increased expression in drones. The upregulation of jhamt gene involved in methyl farnesoate (MF) synthesis came into attention since the MF is not only a precursor of JH but it is also an insect pheromone in its own right as well as JH‐like hormone in Acari. This could indicate a possible kairomone effect of MF for attracting the mites into the drone brood cells, along with its potential involvement in ovogenesis after the cell capping, stimulating Varroa's initiation of egg laying.

Morphological characterization of the Apis mellifera drones in the Southern Urals

The results of the morphological characterization of Apis mellifera drones are presented in order to identify the biopotential of the “clean” native population in bee yards in 12 regions of the Republic of Bashkortostan. The presence of Is, I, O morphotypes – dark and gray in drones, is established. The O morphotype (dark) corresponds to the Central Russian subspecies, and the drones characterized by the O color (gray) can be related to the subspecies with a gray color of abdominal terga. The analysis of morphometric features in drones is identified as Apis mellifera mellifera allowed to distinguish the following characteristics: O (dark) morphotype with brown (coffee hue) or black hairs color on the abdomen; proboscis length (average) – 3.81 ± 0.08 … 3.97 ± 0.03 mm, cubital index – 1.36 ± 0.07 … 1.45 ± 0.09, wing length (front right) – 11.98 ± 0.49…12.08 ± 0.45 mm, wing width (front right) – 3.72 ± 0.21 … 3.92 ± 0.23 mm, tergite length (third) – 2.90 ± 0.12 … 3.51 ± 0.01 mm, tergite width (third) – 6.40 ± 0.02 … 6.44 ± 0.04 mm, sternite length (third) – 2.62 ± 0.03 … 2.66 ± 0.05 mm, sternite width (third) – 4.52 ± 0.10 … 4.62 ± 0.07 mm, tarsal index – 49.59 ± 0.89 … 52.15 ± 1.93%.

Influence of RNA interference-mediated reduction of Or11 on the expression of transcription factor Kr-h1 in Apis mellifera drones

9-Oxo-2-decenoic acid (9-ODA, the predominant component of honeybee queen mandibular pheromones) acts as a sex pheromone attracting drones during mating flights in midair. Odorant receptor 11 (Or11), which is located on the membrane of antennal olfaction receptor neurons in bees, can specifically recognize 9-ODA. At present, the molecular pathway of honeybee drones responding to 9-ODA is still unclear. Studies have demonstrated that 9-ODA could downregulate the expression of Kruppel-homolog1 (Kr-h1, a transcription factor related to the regulation of reproduction and division of labor mediated by juvenile hormone) gene in the mushroom of honeybee brain. We speculate that Kr-h1 may be the downstream gene of Or11, which is involved in the pathway of drones responding to 9-ODA. Therefore, we analyzed the influence of 9-ODA on the expression of Or11 and Kr-h1 in the antennae of sexually immature (4 days) and mature (14 days) male honeybees (Apis mellifera) by quantitative polymerase chain reaction (qPCR). The results demonstrated that 9-ODA significantly downregulated the expression of Or11 and Kr-h1 in the antennae of sexually immature and mature drones. Additionally, siRNA-Or11 was injected into the antennae and brain tissues of 8-day-old drone pupae, and the expression patterns of Or11, Kr-h1 and Broad-Complex (Br–c, downstream gene of Kr-h1) were determined by qPCR at 72 h. The RNAi-induced knockdown of Or11 significantly decreased the expression of Or11, Kr-h1 and Br–c in the antennae and brains of drones. This study suggests that the transcription factor Kr-h1 is downstream of Or11, Kr-h1, which may play an important role in the signal transduction process of drones responding to 9-ODA.

Drone production, semen viability and spermatozoa longevity of Africanized Apis mellifera

Characteristics correlated with beekeeping production, less influenced by the environment and that can be controlled by management techniques, can help in the selection of colonies with higher production capacity, aiming to improve breeding programs. This research was carried out to evaluate the production of Africanized Apis mellifera drones and the longevity of spermatozoa from different genetic groups when supplemented with protein or not. Two genetic groups were used: one selected for royal jelly production from the Africanized honeybee breeding program and another without genetic selection. In both groups, the number of drone brood and the quality of semen were evaluated every 30 days, for five months. Statistical analysis was performed using Bayesian Inference. Statistical difference was found for the drone production among the treatments, and colonies without genetic selection produced more males (187.80 ± 11.15) than the selected colonies (93.07 ± 8.88). The selected colonies for royal jelly production presented greater efficiency in the reproductive cycle of males, because they produced fewer drones than colonies without genetic selection, however, with 31% greater semen viability.

Nutritional Composition of Apis mellifera Drones from Korea and Denmark as a Potential Sustainable Alternative Food Source: Comparison Between Developmental Stages.

We compared nutrient compositions of honey bee (Apis mellifera) drones of different developmental stages from two different populations—the Italian honey bee reared in Korea and Buckfast bees from Denmark. Analyses included amino acid, fatty acid, and mineral content as well as evaluations of antioxidant properties and haemolysis activities. The compositions of total amino acids, and thus protein content of the insects, increased with development. A similar trend was observed for minerals presumably due to the consumption of food in the adult stage. In contrast, total fatty acid amounts decreased with development. Altogether, seventeen amino acids, including all the essential ones, except tryptophan, were determined. Saturated fatty acids dominated over monounsaturated fatty acids in the pupae, but the reverse held true for the adults. Drones were found to be rich in minerals and the particularly high iron as well as K/Na ratio was indicative of the nutritional value of these insects. Among the three developmental stages, adult Buckfast drones exhibited the highest antioxidant activity. Bearing in mind the overall high nutritional value, i.e., high amino acids, minerals and less fatty acids, late pupae and adult drones can be useful for human consumption while the larvae or early pupal stage can be recommended as feed. However, owing to their relatively high haemolysis activity, we advocate processing prior to the consumption of these insects.

Body mass of honey bee drones developing in constant and in changing temperatures

The body mass of honey bee (Apis mellifera) drones was investigated during a post-capping development under four temperature regimes: constant low temperature (32 °C), constant high temperature (35 °C), low temperature in the early stage followed by high temperature in the later stage, and high temperature in the early stage followed by low temperature in the later stage. The temperature regimes had significant influence on the drones’ body mass. The heaviest drones were obtained when the temperature was high at the early stage and low at the later stage of the post-capping period. The most lightweight drones occurred at constant high temperature. The body mass of drones was changing significantly over their adult life and was highest at the age of five days. After this age, the body mass tended to decrease until the end of life. These results suggest that the highest body mass of drones can be obtained by keeping them during the post-capping development in changing and not constant temperatures.

Influence of physicochemical factors on environmental availability and distribution of semiochemicals that affect Varroa destructor and phylogenetically close organisms: classification by VHWOC PCA-clustering

Varroa destructor parasites Apis mellifera larvae following the interception of the semiochemicals involved in bee communication; thus, the semiochemical availability and distribution pathways take place within different physicochemical environments. The structure of 172 molecules with semiochemical activity on Varroa destructor was used to compute the representative physicochemical descriptors of the thermodynamic partition among different physicochemical environments: vapor pressure (V), Henry's coefficient (H), water solubility constant (W), octanol-water partition coefficient (O) and organic carbon partition coefficient (C); VHWOC. The principal component analysis (PCA) and hierarchical clustering of VHWOC descriptors allowed us to establish the trend in availability and distribution of the semiochemicals resulting in a 4 classes model of physicochemical environments: Class 1, Soluble/Volatile; Class 2, Soluble; Class 3, Contact; Class 4, Adsorbed/Volatile. Our results suggest that semiochemicals can transit between different thermodynamic equilibrium phases depending on environment conditions. The classification prediction of the model was tested on 6 new molecules obtained from ketonic extracts of L5 Apis mellifera drone larvae; locating them in class 4, which was consistent with their molecular structure. This study can be the starting point for the design of synthetic semiochemicals or for the control of Varroa destructor. In addition, the method can be used in the analysis of other semiochemical groups.

Flight activity of honey bee (Apis mellifera) drones

Compared to the queen or the workers, the biology of honey bee Apis mellifera L. drones is poorly known. Available information on drone activity is based mainly on direct observations during a limited period of time and for a restricted time of the day. Complete registers of the flight activity of honey bee drones are lacking. We studied the activity of A. mellifera drones during their entire life in spring and summer by using an optical bee counter at the entrance of the hive. Drones were active in the afternoon, with most flights occurring between 14:00 and 18:00. Short orientation flights were performed at 6–9 days old, and longer mating flights of 30 min were performed from the age of 21 days onward during the spring and from the age of 13 days onward during the summer. Our registers show that 50 and 80% of the drones remained faithful to their colony (did not drift) in spring and summer, respectively. The present study confirms existing information, but also reveals unknown aspects about drone biology.

Efficiency of Artificial Insemination for Breeding Apis cerana in Korea

Control mating is very important aspect in bee breeding programs. The technique of artificial insemination is the possible one that can surely control mating of the selected drones with the virgin queen. This is the first time applied this technique with A. cerana in Korea. Altogether 18 queens were artificially inseminated, and 2,000 drones of A. cerana both from Korea and Vietnam were used to evaluate amount of semen collection. Semen from Apis mellifera also was collected as a comparison. Semen of A. cerana is much difficult to separate from mucus in comparing with A. mellifera. The average amount of semen can be collected from one Korean A. cerana drone was 0.09μl and 0.1μl of Vietnamese one, whereas the A. mellifera was more than 6 times (0.58μl semen per A. mellifera drone). Obtaining 1μl of semen have to collect from 11.94 drones that successful semen ejection and have to kill 17 drones of Korean A. cerana. Queens artificially inseminated with 4μl of semen (once insemination) or 8μl of semen (twice insemination, each with 4μl of semen) started laying egg later than naturally mated queens 5.3 and 2.5 days, respectively. The onsets of oviposition of artificially inseminated queens were 12.5 to 15.3 days. Queens received twice inseminations started laying eggs 2.8 days earlier than those received only once insemination. Artificially inseminated queens produced exclusively brood and were similar as the naturally mated ones. The brood production of the queens received once insemination with 4μl of semen was insignificantly different than those received twice inseminations or naturally mated ones, suggesting that one artificial insemination with 4μl of semen is favorable.

The Ability of Honey Bee Drones to Ejaculate

Abstract The effectiveness of two methods of collecting semen from honeybee Apis mellifera drones was compared, and the reasons for problems with ejaculating semen were analysed. Among 275 drones, 100 were stimulated to release semen using a manual method, 100 with the use of chloroform, and from 75 drones the reproductive organs were dissected for analysis and evaluation. It was found that the principal causes of problems that drones had with ejaculating their semen were anatomical changes or a delay in the development of the mucus glands. It was also found that the method employing chloroform was less efficient in the first phase of eversion of the endophallus, compared with the manual method. The method with the use of chloroform allows the determination of the proportion of drones, which do not evert the endophallus because of poor or delayed development of mucus glands, as well as the proportion of drones which evert the organ, but do not ejaculate semen because of the absence of semen in the seminal vesicles.

Protein and Peptide Composition of Male Accessory Glands of Apis mellifera Drones Investigated by Mass Spectrometry.

In honeybees, reproductive females usually mate early in their life with more than 10 males in free flight, often within 10 minutes, and then store male gametes for up to five years. Because of the extreme polyandry and mating in free flight special adaptations in males are most likely. We present here the results of an investigation of the protein content of four types of male reproductive glands from the Western honeybee (Apis mellifera) drone, namely seminal vesicles (secretion in ejaculate), as well as bulbus, cornua and mucus glands (secretions for the mating plug). Using high resolution and accuracy mass spectrometry and a combination of database searching and de novo sequencing techniques it was possible to identify 50 different proteins in total, inside all mentioned glands, except in the mucus gland. Most of the proteins are unique for a specific gland type, only one of them (H9KEY1/ATP synthase subunit O) was found in three glands, and 7 proteins were found in two types of glands. The identified proteins represent a wide variety of biological functions and can be assigned to several physiological classes, such as protection, energy generation, maintaining optimal conditions, associated mainly with vesicula seminalis; signaling, cuticle proteins, icarpin and apolipoproteins located mainly in the bulbus and cornua glands; and some other classes. Most of the discovered proteins were not found earlier during investigation of semen, seminal fluid and tissue of reproductive glands of the bee drone. Moreover, we provide here the origin of each protein. Thus, the presented data might shed light on the role of each reproductive gland.