Natural Queen Research Articles

In-vitro queen rearing protocol for stingless bee, Tetragonula iridipennis Smith

South-East Asia falls behind in comparison to South America and Australia in beekeeping with stingless bees. This is partly contributed by the lack of proper management techniques and slow rate of natural colony multiplication in different bee species found in this part of the world. Production of natural queen bees in stingless bee colonies depends upon several factors, such as adequate nutrition, temperature regulation, seasonality, colony size, natural enemies among many others. The capacity to produce a large number of queen bees without depending upon the production of natural queen inside colonies makes in vitro rearing of stingless bee as a good help for the problem of slow colony multiplication. The present study describes in vitro queen rearing protocol for the Asian stingless bee, Tetragonula iridipennis Smith. The larval provision required for producing a queen bee was three times more than a natural worker bee. The in vitro queen bees were morphologically similar to the natural queens. The reproductive success of these in vitro queen bees also supported the proposed protocol. The technique would be useful in colony multiplication of this species at a faster rate and in developing management tactics for large-scale adoption in Indo-Malayan and Australasia regions.

Queen bee gut microbiota extends honeybee lifespan by inhibiting insulin signaling.

Queen and worker bees share the same genetic background but have vastly different lifespans. The gut microbiome regulates host health, suggesting that differences in lifespan between queen and worker bees could be related to gut bacteria. Herein, we used an innovative method to transplant gut microbiota from adult queen or worker bees to microbiota-free bees. The transplantation of queen gut microbiota to microbiota-free bees extended their lifespan. Insulin/insulin-like growth factor signaling, a highly conserved metabolic pathway related to lifespan, displayed identical expression profiles in natural queen bees and microbiota-free bees transplanted with queen microbiota. This finding significantly expands our understanding of the relationships between intestinal bacteria, host health, and the biology of aging.

A Minimally Invasive Approach Towards "Ecosystem Hacking" With Honeybees.

Honey bees live in colonies of thousands of individuals, that not only need to collaborate with each other but also to interact intensively with their ecosystem. A small group of robots operating in a honey bee colony and interacting with the queen bee, a central colony element, has the potential to change the collective behavior of the entire colony and thus also improve its interaction with the surrounding ecosystem. Such a system can be used to study and understand many elements of bee behavior within hives that have not been adequately researched. We discuss here the applicability of this technology for ecosystem protection: A novel paradigm of a minimally invasive form of conservation through “Ecosystem Hacking”. We discuss the necessary requirements for such technology and show experimental data on the dynamics of the natural queen’s court, initial designs of biomimetic robotic surrogates of court bees, and a multi-agent model of the queen bee court system. Our model is intended to serve as an AI-enhanceable coordination software for future robotic court bee surrogates and as a hardware controller for generating nature-like behavior patterns for such a robotic ensemble. It is the first step towards a team of robots working in a bio-compatible way to study honey bees and to increase their pollination performance, thus achieving a stabilizing effect at the ecosystem level.

Aptness of diverse queen cup materials for larval graft acceptance and queen bee emergence in managed honey bee (Apis mellifera) colonies

Artificial queen rearing has changed the beekeeping business in contrast to natural queen replacement, as it provides a newly mated queen into a bee colony, reduces the time between eggs not being laid, and increases the production of young bees in a colony. This study was conducted to investigate the larval acceptance rate grafted in queen cups made from different materials and to find out whether the various materials used for queen cups were suitable for the acceptance of grafted larvae in cell builder colonies. The evaluated materials included fresh bee wax (T1), old bee wax (T2), uncapping bee wax (T3), pure paraffin (T4), 50% paraffin + 50% old bee wax (T5), and brown plastic queen cups (T6) as a control. Results indicated that T3 was the material that most increased the larval acceptance (5.2 ± 0.37), followed by T4 (2.4 ± 0.5), T2 (2.2 ± 0.58), T5 (1.8 ± 0.37), T1 (1.6 ± 0.4), and minimum larval acceptance was in the T6 (1.0 ± 0.4) respectively during the spring season of the year 2020–2021. Similar findings were reported of larval acceptance rate during the spring season of 2021–2022. In addition, the wax material that least affected larval acceptance was the fresh comb bee wax. However, all materials used were coupled with a more larval acceptance rate than the control treatment during both spring seasons of 2020–2021 and 2021–2022. The larval acceptance rate was statistically significant in T3 as a compared to other queen cell cup materials during the both spring seasons (p = 0.001). These findings imply that using different types of bee wax for preparing queen cell cups during larvae grafting, particularly uncapping bee wax, may stimulate and promote grafted larvae acceptance during the queen rearing process.

Assessment of synthetic acaricide residues in Royal Jelly

In this study, Royal Jelly was produced from colonies under chemical treatment of coumaphos (Perizin and Checkmite+) and tau-fluvalinate (Apistan), using artificial plastic queen cells. The contamination level was assessed 42 days after the application. The application of CheckMite + strips during the production led to higher levels of acaricide residues in the final product, in contrast to the residues from Perizin and Apistan. For this reason, the CheckMite + strips were further applied in order to assess the contamination of the product after a long period (292 days after the application). For the specific sampling, natural (center and adjacent) and plastic cells were used. The results showed that the contamination of Royal Jelly produced in plastic queen cells decreased with time; one month after removing the strips, residues were not detectable. On the contrary, the contamination of samples collected from natural queen cells was high, particularly from those adjacent to the strips, where even 292 days after the strips’ removal, coumaphos concentration was 2.25 ± 0.50 mg kg−1. The maximum concentration (12.52 ± 2.24 mg kg−1) was detected in natural queen cells. Τo our knowledge, this is the highest contamination that has ever been recorded in a food product. Our results suggest that coumaphos may be transferred from wax to Royal Jelly even in low concentrations. Also, the findings of this research provide data for setting safe intervals between acaricide treatment and Royal Jelly production, raising at the same time awareness about the impact of wax contamination on honey bees’ health.

Contribution Concerning the Behaviour Knowledge before Natural Swarming in Honeybees (Apis mellifera carpathica, Linnaeus)

In this study we followed the way in which evolves the behaviour of some honeybee families (Apis mellifera carpathica, Linnaeus), maintained in vertical beehives (modified Dadant type), in conditions of stationary bee keeping, having in view the natural queen cell’s build rhythm after the second acacia tree harvest (Robinia pseudoacacia L.) in Jucu area of Cluj County. There were taken into observation 20 biological units with queen bees of different ages. It was monitored the development rhythm of natural queen cells, which are in attendance by the working bees, and to put into evidence the swarming instinct strength, so in bee keeping practice to be known and to be avoided the natural swarming. The determination of occupied surfaces with covered brood and larval was effected with the Netz framework. The aim of our study was to understand how the swarming in honeybees occurs. By detailed observation, we want to describe and to explain the behaviour of honeybees before the swarming process. The obtained results are quantified in absolute values and expressed graphically in relative values, but in the next three apicultural years (2020, 2021 and 2022), the experiment will be repeated for a variance analysis, which will provide a statistical assurance. We hope that these data will give contribution for the apicultural practice, keeping attention on queen bees’ importance during active season and on careful supervision on development rhythm of the honeybee units during the natural swarming.

A Route to Direct Fitness: Natural and Experimentally Induced Queen Succession in the Tropical Primitively Eusocial Wasp Ropalidia marginata

Insect societies are hallmarks of cooperation because one or a few queens monopolize reproduction and several non-reproductive workers cooperatively raise brood. However, the loss of the queen exposes a colony to potential reproductive conflict, which is resolved only after a new queen takes over. We studied queen succession in natural and experimental colonies of the primitively eusocial wasp Ropalidia marginata to understand the proximate behavioral strategies involved in the resolution of this conflict. Previous work has shown that in this species, experimental queen removal always results in only one worker becoming hyper-aggressive and taking over the colony as its next queen, without ever being challenged. Here we show that even during natural queen turnover, one and only one worker becomes hyper-aggressive and takes over as the next queen, without being challenged. During natural queen turn-over, aggression of the successor may sometimes begin before the loss of the old queen and may sometimes decline more rapidly, unlike in the case of experimental queen removal. The successor begins to lay eggs sooner after a natural queen turn-over as compared to experimental queen removal. This is expected because workers might detect the gradual decline of the queen preceding her disappearance. Because queen succession is expected to be more prevalent in tropical perennial species, we expect natural selection to have favored such an orderly queen succession so that a route to direct fitness is available without significant reduction in cooperation.

A case of natural queen succession in a captive colony of naked mole-rats, Heterocephalus glaber

Naked mole-rats occur in large colonies where usually a single queen monopolizes reproduction. Queen succession occurs from within usually as a result of aggressive encounters with subordinate females that queue for reproductive succession following colony instability, which inevitably results in death of either the queen or the challenging conspecific. We monitored a queen succession following the death of the breeding male in a colony of the naked mole-rat, Heterocephalus glaber, prior to, during as well as after replacement of the original breeding female. The response of the pituitary luteotrophs was investigated in the non-reproductive females during this period of instability by the administration of endogenous gonadotrophin releasing hormone (GnRH) and evaluating the subsequent luteinising hormone (LH) response in the blood. Larger and older non-breeding females engaged in aggressive encounters that culminated in death. The new breeding successor which arose from within the colony was a large female who continued to procreate. The six non-breeding females that were killed during reproductive takeover were larger and older females which exhibited elevated basal circulating LH concentrations as well as increased pituitary sensitivity as measured by the amount of releasable LH to an exogenous GnRH challenge. By contrast, non-breeding females that survived the succession were smaller and younger animals with reduced basal and GnRH challenged LH concentrations. Likewise, five non-breeding males which were heavier and older than those non-breeding males which survived were killed. These animals did not, however, show elevated basal or exogenous GnRH challenged LH concentrations when compared to the surviving males. The non-breeding animals of both sexes which survived the reproductive takeover event represented individuals which posed a minimal threat to the new successor and hence promoted the continuation of the marked reproductive skew that is prevalent in this highly inbred colonial subterranean hystricomorph.

An advance in the in vitro rearing of stingless bee queens

Stingless bees are a diverse group of highly eusocial tropical bees and potential pollinators of natural and crop environments. Nevertheless, large-scale breeding for agricultural purposes still needs development. A bottleneck is the small number of queens generally found in the colonies of most species. A proposed solution is to develop in vitro rearing of stingless bee queens, which includes a technical support due to the massive liquid feeding of larvae. In this study, we tested the efficacy of in vitro queen-rearing techniques in terms of queen size, survival, and fecundity. We found that humidity must be kept high during the first 6 days of larval development and reduced thereafter to 75 %. In the most effective treatments, we obtained up to 97.9 % queen survival. We also found that queens not different in size from natural queens could be produced if sufficient larval food is provided. In vitro queens were able to mate and lay viable eggs. This methodology is also useful for several research appliances, like pesticide effect on bees, pollen quality effect in caste determination, and the use of genetic sources for selection programs, among others.

Horizontal transmission of Nosema ceranae (Microsporidia) from worker honeybees to queens (Apis mellifera)

Horizontal transmission from worker honeybees to queens is confirmed in a laboratory essay as a possible route of Nosema ceranae infection in field colonies and pathological repercussions on honeybee queens are described. Lesions are only detected in the epithelial ventricular layer of the infected queens and death occurs within 3 weeks when the nurse workers are experimentally and collectively infected with approximately 5000 viable spores per bee. These data suggest that the higher number of infected house bees, the higher risk of transmission to queens. The presented data may explain the role of house honeybees in natural queen infection, although it is probably that a high proportion of infected house bees must be required to infect the queen.

Ant Queen Egg-Marking Signals: Matching Deceptive Laboratory Simplicity with Natural Complexity

BackgroundExperiments under controlled laboratory conditions can produce decisive evidence for testing biological hypotheses, provided they are representative of the more complex natural conditions. However, whether this requirement is fulfilled is seldom tested explicitly. Here we provide a lab/field comparison to investigate the identity of an egg-marking signal of ant queens. Our study was based on ant workers resolving conflict over male production by destroying each other's eggs, but leaving queen eggs unharmed. For this, the workers need a proximate cue to discriminate between the two egg types. Earlier correlative evidence indicated that, in the ant Pachycondyla inversa, the hydrocarbon 3,11-dimethylheptacosane (3,11-diMeC27) is more abundant on the surface of queen-laid eggs.MethodologyWe first tested the hypothesis that 3,11-diMeC27 functions as a queen egg-marking pheromone using laboratory-maintained colonies. We treated worker-laid eggs with synthetic 3,11-diMeC27 and found that they were significantly more accepted than sham-treated worker-laid eggs. However, we repeated the experiment with freshly collected field colonies and observed no effect of treating worker-laid eggs with 3,11-diMeC27, showing that this compound by itself is not the natural queen egg-marking pheromone. We subsequently investigated the overall differences of entire chemical profiles of eggs, and found that queen-laid eggs in field colonies are more distinct from worker-laid eggs than in lab colonies, have more variation in profiles, and have an excess of longer-chain hydrocarbons.ConclusionsOur results suggest that queen egg-marking signals are significantly affected by transfer to the laboratory, and that this change is possibly connected to reduced queen fertility as predicted by honest signaling theory. This change is reflected in the worker egg policing response under field and laboratory conditions.

66 DEVELOPMENT OF IN VITRO-PRODUCED CAT EMBRYOS AFTER VITRIFICATION AND NONSURGICAL EMBRYO TRANSFER: PRELIMINARY RESULTS

There are no refereed reports on vitrification of domestic cat embryos derived from in vitro-matured oocytes and transferred using a nonsurgical embryo transfer technique. The aim of this study was to verify the effects of vitrification on the in vitro and in vivo developmental ability of in vitro-produced (IVP) cat blastocysts. Oocytes recovered from minced ovaries were matured, fertilized, and cultured in vitro as previously reported (Merlo B et al. 2005 Theriogenology 63, 2032–2039). On Day 7 of in vitro culture (IVC), blastocysts were selected and vitrified in straws (Cristal ET 0.25 mL, 133 mm, IMV-Technologies, Paillette Crista, France). For vitrification (modified from Campos-Chillòn LF et al. 2006 Theriogenology 65, 1200–1214), the embryos were transferred in 1 mL of V1 [ethylene glycol 3.5 m in HEPES synthetic oviductal fluid (HSOF)] for 3 min, and then in 10 μL of V2 (ethylene glycol 7 m, galactose 0.5 m, Ficoll 70 18% in HSOF) for 20 s. Finally, the embryos were loaded in straws preloaded with 190 μL of dilution solution (galactose 0.5 m in HSOF). Straws were heat sealed and immediately plunged into liquid nitrogen. Vitrified embryos were warmed in air for 10 s, and then in a waterbath at 37°C for 30 s. For developmental ability and in vitro evaluation, 27 embryos were warmed and immediately examined: 25 re-expanded, 2 did not re-expand, and 1 had damaged zona pellucida. Re-expanded embryos were cultured in SOF plus amino acids, 16 mg mL–1 BSA, and 5% fetal bovine serum at 38.5°C in 5% O2, 5% CO2, 90% N2. After 24 h of IVC, only 4 blastocysts were expanded, and after 48 h, embryos were clearly degenerated or shrunk. in vivo developmental ability was tested by nonsurgical embryo transfer of 8 vitrified-warmed embryos and 6 IVP fresh embryos into 2 natural estrus queens, injected with 200 IU of hCG i.m. (Day 0) for induction of ovulation. Ovulation was confirmed by plasmatic progesterone assay on Day 5. Nonsurgical embryo transfer was made on Day 8 using the catheter proposed by Zambelli et al. 2001 for transcervical insemination in the cat. The catheter was connected to a 1-mL syringe and loaded with the embryos. Then, it was inserted in the vagina and transrectally guided into the uterus, where the embryos were deposited. To assess pregnancy status, abdominal ultrasonography was done on recipients on Day 13, 25, and 40. On Day 13, an embryonic vesicle was observed in both queens, although a smaller diameter than expected was detected in the recipient of the vitrified embryos. On Day 25, a viable embryo was detected only in the recipient of fresh IVP embryos. On Day 40, the gestational chamber was still present but no sign of a viable embryo was detected. Further studies are in progress to improve the nominal incidence of pregnancy and frequency of embryo survival after vitrification. Nevertheless, the preliminary results obtained using an AI catheter for nonsurgical embryo transfer are encouraging, and the improvement of the technique could make it reliable in the cat. Supported by Animal Stem Cells Laboratory, Regione Emilia Romagna, PRRIITT Project Number M-404AIWTSV.

Biology ofApis dorsatain Vietnam

This paper studies the biology of A. dorsata in the Melaleuca forest of Vietnam. The width of 158 A. dorsata combs was from 43–162 cm, and the height from 23–90 cm. About 3–4 weeks after nesting, a colony could store 4.1 ± 2.6 kg of honey. The thickness of the worker brood comb was 3.3 cm and of the drone was 3.7 cm, but that of the honey comb was possibly 19.0 cm. The width of 10 continuous drone cells (58.1 ± 1.4 cm, n = 90) is significantly larger than that of worker cells (55.7 ± 1.7, n = 330). The depth of drone cells was 19.0 ± 0.4 mm (n = 90) and of worker cells was 17.4 ±1.5 mm (n = 225). The weights of the newly emerged worker, drone and queen bees were 159.9 ± 15.5 mg (n = 30), 168.5 ± 10.4 mg (n = 30), and 272.0 ± 40.1 mg (n = 9), respectively. Development time from egg to adult was 19.7 ± 0.1 days (n = 30) for workers, 23.7 days for drones, and 16.5 days for queen bees. When a colony reached 121 cm × 57 cm in size, it built 9.7 ± 2.4 (n = 32 nests) natural queen cells. The volume of a queen cell was 1.300 ± 0.159 mL (n = 28).

Improved technique for introducing four-day old virgin queens to mating hives that uses artificial and natural queen cells for introduction

Improved technique for introducing four-day old virgin queens to mating hives that uses artificial and natural queen cells for introduction

Improved technique for introducing four-day old virgin queens to mating hives that uses artificial and natural queen cells for introduction

SummaryWe compared the acceptance of 4-day old virgin queens introduced into mating nucleus hives using natural and artificial queen cells versus a wooden 3-hole mailing cage, a standard introduction method. The queen cell methods gave high acceptance (95% and 93% for natural and artificial, respectively) even though the queen was released from the queen cell approximately 10 minutes after being introduced into the mating hive. By contrast, success using mailing cages was significantly lower (47% and 73%) when the queen was released from her cage after 1 hour or 48 hours, respectively. The equal success rates of the reused and artificial queen cells suggests that high success is not due to chemicals present in natural queen cells transferring to the queens. To further investigate why queen cells give higher introduction success than cages, we introduced virgin queens into queenless observation hives. Workers attacked only 1 of 12 queens leaving a queen cell whereas 5 out of 6 queens leaving a cage were attacked.

Gene expression profiles underlying alternative caste phenotypes in a highly eusocial bee, Melipona quadrifasciata

To evaluate caste-biased gene expression in Melipona quadrifasciata, a stingless bee, we generated 1278 ESTs using Representational Difference Analysis. Most annotated sequences were similar to honey bee genes of unknown function. Only few queen-biased sequences had their putative function assigned by sequence comparison, contrasting with the worker-biased ESTs. The expression of six annotated genes connected to caste specificity was validated by real time PCR. Interestingly, queens that were developmentally induced by treatment with a juvenile hormone analogue displayed an expression profile clearly different from natural queens for this set of genes. In summary, this study represents an important first step in applying a comparative genomic approach to queen/worker polyphenism in the bee.

Comparison of Some Characteristics of Queen Honey Bees (Apis mellifera L.) Reared by Using Doolittle Method and Natural Queen Cells

Dodologlu, A., Emsen, B. and Gene, F. 2004. Comparison of some characteristics of queen honey bees (Apis mellifera L.) reared by using doolittle method and natural queen cells. J. Appl. Anim. Res., 26: 113–115. This study was conducted to examine some characteristics of queen bees raised by using Doolittle method and natural queen cells in Erzurum conditions. The average lengths of the sealed queen cells, queen weights at emergence, diameters of spermatheca and numbers of spermatozoid were higher in case of honey bees reared by Doolittle method as compared to natural queen cell method. However, the differences between the two methods regarding pre-oviposition period were not significant. It is concluded that queen bees reared by grafting method (Doolittle method) were superior to queen bees reared by using natural queen cells.

Effects of relatedness on queen competition within honey bee colonies

The influence of relatedness on the pre- and post-emergent survival of honey bee queens was investigated. Workers did not preferentially rear sisters over non-siblings under conditions of natural queen replacement. After queen emergence, however, there was a significant effect of a queen's relatedness to the workers on her survivorship during fights with rival queens. The mechanism of this bias towards related queens is unknown, and several hypotheses are discussed. The difference in post-emergent survivability suggests that kin selection may operate during competition among adult queens at this crucial stage of honey bee reproduction.

Shellfish tainting – a means of monitoring petroleum-contaminated effluents

SynopsisWater from the separation plant at Sullom Voe is discharged into the sea under agreed conditions from a diffuser situated to the north of Calback Ness, close to natural scallop (Pecten maximus) beds.As part of the arrangements to monitor potential effects of the discharge, Torry Research Station was asked to assess, for petroleum taint, shellfish which would be sampled at intervals from Sullom Voe. To avoid depletion of the natural shellfish beds, queen scallops (Chlamys opercularis) or scallops, whichever were available from other areas, were set out in cages suspended (or on the bottom) 100–1250 m eastwards of the outfall for a period, and then recovered, frozen, and assessed subsequently at the laboratory by a trained taste panel, and compared with the same species unexposed.The cages were laid on 10 occasions from March 1980 to February 1987. Initially, no petroleum taint was detected. In August 1980 and May 1981, a different flavour, but not recognisable as a petroleum taint, was detected at 800–1000 m from the outfall after three days exposure. In May 1982, 33% of the queen scallops in the cage on the bottom, 100 m south east of the diffuser, were dead after six days exposure, and taint was detected at the threshold level in shellfish in the cage at 300 m. In May 1983, taint and petroleum derived hydrocarbons were detected in the sample from the cage on the bottom, 100 m south east of the diffuser, after seven days exposure. In May 1984, taint was again detected at the bottom, 200 m south east of the outfall, after 10 days exposure.Enquiries established that the diffuser was not operating with maximum efficiency. Subsequently, monitoring revealed an improvement. Taint assessment proved to be a sensitive detector of petroleum contamination.

Queen Succession in the Social Wasp, Polistes annularis

AbstractOutcomes of conflicts among social animals can strongly affect individual fitness and therefore partly determine how sociality evolves. In the social wasp, Polistes annularis, conflicts over egg laying result in a linear dominance hierarchy discernible from which wasps attack which others. We investigated the structure and maintenance of dominance hierarchies in colonies containing both nest foundresses and workers. We also investigated the outcome of a potential conflict between foundresses and workers over the identity of the female which becomes queen after the original queen disappears. To investigate queen replacement we recorded individuals' behavior before and after removal of the queen from 13 nests. This experiment simulated natural queen disappearance which occurs frequently. All foundresses were ranked over workers; high ranking females were most aggressive and directed most of their attacks to the female directly beneath them in the hierarchy. This hierarchy determined succession to queenship; the beta female became queen within 3 h. Therefore a foundress became the new queen whenever one was present. There was no evidence of conflict between workers and foundresses when a foundress became queen. This chance of becoming queen contributes to the expected fitness of a female that accepts a subordinate role in spring.