Queen Loss Research Articles

Gene expression, sperm viability, and queen (Apis mellifera) loss following pesticide exposure under laboratory and field conditions

Honey bee (Apis mellifera) queens are failing, and this may be due in part to widespread miticide use by beekeepers. The ectoparasitic mite, Varroa destructor, is a major pest of honeybees and synthetic insecticides are often used to control Varroa. Amitraz is widely used by beekeepers worldwide. Amitraz is thought to be of low toxicity to bees, but applying high doses can cause negative effects. Here, we demonstrate a sublethal dose of amitraz can affect the detoxification, cyclic adenosine monophosphate (cAMP)-dependent protein kinase, immunity, antioxidant capacity, and development of honeybee queens when queens were exposed to amitraz (2.5 ppm) under laboratory conditions. Downregulation of genes related to detoxification enzymes, cAMP-dependent protein kinase, immunity, antioxidant enzymes, and development was observed. Sublethal doses of amitraz had no effects on the viability of spermatozoa stored in spermathecae of queen when treated topically in the laboratory. Lastly, we treated colonies with three formulations of amitraz and followed queens in these colonies for 3 months to explore queen loss and/or other adverse effects of miticide treatment. Queen loss averaged about 30% over all treatment groups including untreated control colonies and no reduction in sperm viability was seen with amitraz treatments when queens remaining after 3 months were dissected. This research demonstrates that chemical exposure can affect some aspects of the queen immune system but sperm viability and queen longevity over 3 months were unaffected by colony level amitraz treatments under real-world conditions in honey bee colonies.

Israeli Acute Paralysis Virus: Honey Bee Queen⁻Worker Interaction and Potential Virus Transmission Pathways.

Queen loss or failure is an important cause of honey bee colony loss. A functional queen is essential to a colony, and the queen is predicted to be well protected by worker bees and other mechanisms of social immunity. Nevertheless, several honey bee pathogens (including viruses) can infect queens. Here, we report a series of experiments to test how virus infection influences queen–worker interactions and the consequences for virus transmission. We used Israeli acute paralysis virus (IAPV) as an experimental pathogen because it is relevant to bee health but is not omnipresent. Queens were observed spending 50% of their time with healthy workers, 32% with infected workers, and 18% without interaction. However, the overall bias toward healthy workers was not statistically significant, and there was considerable individual to individual variability. We found that physical contact between infected workers and queens leads to high queen infection in some cases, suggesting that IAPV infections also spread through close bodily contact. Across experiments, queens exhibited lower IAPV titers than surrounding workers. Thus, our results indicate that honey bee queens are better protected by individual and social immunity, but this protection is insufficient to prevent IAPV infections completely.

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.

Turning workers into false queens: the role of exogenous pheromones in regulating reproduction in worker honey bees.

One of the responses that honey bee workers can make in the event of queen loss is to develop into false queens. False queens are workers that exhibit both behavioural and physiological traits similar to those of a true queen. However, the presence of more than one false queen in a colony distorts the established hierarchies. As transformation into a false queen occurs after emergence as an adult, we tested the effect of worker mobile pheromone carriers (PCs) treated with exogenously supplied pheromones on their nestmates. The PCs carried either synthetic mandibular gland pheromones or pheromones extracted from Apis melliferacapensis parasitic workers. Only the PCs attracted retinues of workers, increased pheromone production and activated their ovaries, becoming false queens. Pheromones from A. m.capensis workers were more effective than extracts of commercially available synthetic queen pheromones in eliciting these effects. Using this simple mobile pheromone delivery system, we have shown that carrying amounts of exogenous pheromone can induce pheromone production in the carrier, resulting in the production of false queens within experimental groups. Possible implications of using this technique to modify and regulate worker reproduction in colonies are discussed.

Queen presence mediates the relationship between collective behaviour and disease susceptibility in ant colonies.

The success of social living can be explained, in part, by a group's ability to execute collective behaviours unachievable by solitary individuals. However, groups vary in their ability to execute these complex behaviours, often because they vary in their phenotypic composition. Group membership changes over time due to mortality or emigration, potentially leaving groups vulnerable to ecological challenges in times of flux. In some societies, the loss of important individuals (e.g. leaders, elites and queens) may have an especially detrimental effect on groups' ability to deal with these challenges. Here, we test whether the removal of queens in colonies of the acorn ant Temnothorax curvispinosus alters their ability to execute important collective behaviours and survive outbreaks of a generalist entomopathogen. We employed a split-colony design where one half of a colony was maintained with its queen, while the other half was separated from the queen. We then tested these subcolonies' performance in a series of collective behaviour assays and finally exposed colonies to the entomopathogenic fungus Metarhizium robertsii by exposing two individuals from the colony and then sealing them back into the nest. We found that queenright subcolonies outperformed their queenless counterparts in nearly all collective behaviours. Queenless subcolonies were also more vulnerable to mortality from disease. However, queenless groups that displayed more interactions with brood experienced greater survivorship, a trend not present in queenright subcolonies. Queenless subcolonies that engage in more brood interactions may have had more resources available to cope with two physiological challenges (ovarian development after queen loss and immune activation after pathogen exposure). Our results indicate that queen presence can play an integral role in colony behaviour, survivorship and their relationship. They also suggest that interactions between workers and brood are integral to colonies survival. Overall, a social group's history of social reorganization may have strong consequences on their collective behaviours and their vulnerability to disease outbreaks.

Effect of the Multiple Queens Within Colony on Some Honeybee Activities, Apis mellifera carnica and Sustainability of their Colonies

Multiple honey bee queen colonies contained two to four mated queens which able to move freely were produced by removing the mandibles of queens which trigger fighting and queen elimination within the colony. This method showed the high success, Twenty five out of twenty seven queens were accepted after their introduction in Carniolan colonies, and the success ratio reaches 92.6%. Twenty seven multiple queens introduced in nine colonies were sustainable enough for long period without losing any queen from the spring to fall in the first year. Twenty seven were over wintered successfully and no queen loss was recorded until the next fall in the second year. The multiple queen colonies which contained four queens were more efficient than the other tested multiple queen colonies in the workers brood production activity, it was recorded an average 14460 inch2/colony/ year for amount of worker brood, followed by colonies which contained three queens then colonies which contained two queens while the single queen colonies as control indicated the lowest mean amount of workers brood 5776 inch2/colony / year. Also, multiple queen colonies contained four queens were scored highest value of mean amounts 348, 9692 and 4120 inch2/colony / year for drone brood rearing, honey and pollen production, respectively. The single queen colonies indicated the lowest amount of 124, 4968 and 1864 inch2/colony / year for drones brood rearing, honey and pollen production, respectively. Also, multiple queen colonies contained four queens were scored highest value of mean amounts citrus, clover and cotton flow seasons, while single queen colonies were recorded lowest amount in same flow seasons. From the obtained results it was shown that the multiple queen colonies are more efficient for helping the beekeepers to increase the bee population and thus increase the honeybee products. In addition the multiple queens technique can be used in the commercial domain.

Context dependent bias in honeybee queen selection: swarm versus emergency queens

Colonies of social Hymenoptera with multiple mated queens comprise of numerous super-sister subfamilies with high within but low between subfamily relatedness. Because fitness gain in workers is indirect and relatedness dependent, inclusive fitness theory predicts that nepotism might be adaptive, particularly with respect to queen rearing and selection of new queens. However, nepotistic queen rearing could not been detected in various eusocial insect species and instead workers preferentially rear queens from rare subfamilies after queen loss. Here, we test, whether this bias can also be observed in queen rearing in preparation for reproductive swarming in the honeybee, Apis mellifera. Colonies reared swarm and subsequently emergency queens. Genotyping of all produced queens as well as worker brood revealed a significant deviation from the expected worker subfamily distribution for emergency queens but not for swarm queens. The preference of rare subfamilies may result from worker control over queen rearing in emergency queen rearing. Based on our results it appears that workers choose a few out of a vast number of worker-destined larvae to select emergency queens at extreme selection intensity. This sets the stage for competition among larvae to compete for queen caste fate, presumably by differentially attracting nurse workers to raise them as queens. During swarm queen production, workers cannot select larvae, because the queen lays eggs into predestined cells, resulting in subfamily distributions among the queens more similar to a random subfamily sample as determined in the worker brood. SIGNIFICANCE STATEMENT: Honeybee queens mate with multiple males resulting in several coexisting subfamilies within a colony favoring nepotism by workers rearing a super-sister as a new queen. Workers feeding queen-destined larvae a special diet and thus, larvae seem to have no influence over their caste fate. Queen rearing happens under different circumstances, conditions for nursing workers to bias queen broods and for larvae to bias their caste fate might differ. We tested for subfamily bias in queen broods reared in preparation for reproductive swarming, when queens provision specific cells with eggs to be reared as queens. Within the same colonies, after dequeening, nursing workers choose from available young larvae individuals to be reared as emergency queens. We detected a subfamily bias under emergency queens, but instead of nepotism, we found evidence for larvae to selfishly bias their own caste fate.

Productivity and longevity of honey bee queens inseminated with freshly collected and diluted semen

The aim of this study was to examine whether the addition of a diluent for honey bee sperm, directly before insemination, affects the quality and vitality of queens and their brood, in comparison to queens inseminated traditionally, with fresh semen. Honey bee queens were inseminated with diluted and homogenized semen collected from a few dozen drones, in order to increase the diversity of their offspring. Queens and drones (Apis mellifera carnica) were mated within subspecies, however, only three selected lines were used. The average number of spermatozoa filling the spermatheca of queens, inseminated with semen without dilution, freshly collected from drones, from a single colony (SCS) was not significantly higher, compared to queens inseminated with semen diluted and mixed (MCS); amounting to 5.1 and 4.4 million sperm, respectively. Queens from both groups started laying eggs at a similar time after insemination, and their average life expectancy in colonies, was 20.5 and 22.6 months, respectively. The most common reason for queen losses was supersedure, and the reasons for these losses were not significantly different between groups. Brood survival rates, in both groups, did not differ significantly.

Effects of an insect growth regulator and a solvent on honeybee (Apis mellifera L.) brood development and queen viability.

Honeybee toxicology is complex because effects on individual bees are modulated by social interactions between colony members. In the present study, we applied high doses of the insect growth regulator fenoxycarb to honeybee colonies to elucidate a possible interplay of individually- and colony-mediated effects regarding honey bee toxicology. Additionally, possible effects of the solvent dimethyl sulfoxide (DMSO) were assessed. We conducted studies on egg hatching and brood development to assess brood care by nurse bees as well as queen viability. Egg hatching was determined by the eclosion rate of larvae from eggs originating from colonies (i) treated with sugar syrup only, (ii) treated with sugar syrup containing DMSO and (iii) treated with sugar syrup containing fenoxycarb (dissolved in DMSO). To evaluate brood development, combs with freshly laid eggs were reciprocally transferred between colonies, and development of brood was examined in the recipient hive. Brood reared inside DMSO- and fenoxycarb-treated colonies as well as brood from DMSO- and from fenoxycarb-exposed queens showed higher mortality than brood not exposed to the chemicals. No differences were found in egg hatching among the treatments, but there was a higher variability of eclosion rates after queens were exposed to fenoxycarb. We also observed queen loss and absconding of whole colonies. Based on our results we infer that fenoxycarb has queen- as well as nurse bee-mediated effects on brood quality and development which can lead to the queen's death. There also is an effect of DMSO on the nurse bees' performance that could disturb the colony's equilibrium, at least for a delimited timespan.

Effects of Oral Exposure to Fungicides on Honey Bee Nutrition and Virus Levels.

Sublethal exposure to fungicides can affect honey bees (Apis mellifera L.) in ways that resemble malnutrition. These include reduced brood rearing, queen loss, and increased pathogen levels. We examined the effects of oral exposure to the fungicides boscalid and pyraclostrobin on factors affecting colony nutrition and immune function including pollen consumption, protein digestion, hemolymph protein titers, and changes in virus levels. Because the fungicides are respiratory inhibitors, we also measured ATP concentrations in flight muscle. The effects were evaluated in 3- and 7-d-old worker bees at high fungicide concentrations in cage studies, and at field-relevant concentrations in colony studies. Though fungicide levels differed greatly between the cage and colony studies, similar effects were observed. Hemolymph protein concentrations were comparable between bees feeding on pollen with and without added fungicides. However, in both cage and colony studies, bees consumed less pollen containing fungicides and digested less of the protein. Bees fed fungicide-treated pollen also had lower ATP concentrations and higher virus titers. The combination of effects we detected could produce symptoms that are similar to those from poor nutrition and weaken colonies making them more vulnerable to loss from additional stressors such as parasites and pathogens.

Honey bee colonies provided with natural forage have lower pathogen loads and higher overwinter survival than those fed protein supplements

Malnutrition is a major cause of colony losses. In managed hives, bees are fed protein supplements (PS) during pollen shortages. If bees were provided with natural forage instead of PS, would they have lower pathogen levels and higher queen and colony survival? We addressed this question by either providing colonies with forage (Brassica rapa—rapini) or feeding them PS from November to February. Soluble protein concentrations in the PS were lower than the rapini pollen as were levels of most amino acids. Nurse bees digested less of the protein in PS than the pollen. Hemolymph protein titers in nurse bees and colony growth did not differ between those fed PS or foraging on rapini. However, colonies fed PS had higher levels of black queen cell virus and Nosema and greater queen losses, indicating that natural forage might improve overwintering survival.

Honeybee colony disorder in crop areas: the role of pesticides and viruses.

As in many other locations in the world, honeybee colony losses and disorders have increased in Belgium. Some of the symptoms observed rest unspecific and their causes remain unknown. The present study aims to determine the role of both pesticide exposure and virus load on the appraisal of unexplained honeybee colony disorders in field conditions. From July 2011 to May 2012, 330 colonies were monitored. Honeybees, wax, beebread and honey samples were collected. Morbidity and mortality information provided by beekeepers, colony clinical visits and availability of analytical matrix were used to form 2 groups: healthy colonies and colonies with disorders (n = 29, n = 25, respectively). Disorders included: (1) dead colonies or colonies in which part of the colony appeared dead, or had disappeared; (2) weak colonies; (3) queen loss; (4) problems linked to brood and not related to any known disease. Five common viruses and 99 pesticides (41 fungicides, 39 insecticides and synergist, 14 herbicides, 5 acaricides and metabolites) were quantified in the samples.The main symptoms observed in the group with disorders are linked to brood and queens. The viruses most frequently found are Black Queen Cell Virus, Sac Brood Virus, Deformed Wing Virus. No significant difference in virus load was observed between the two groups. Three acaricides, 5 insecticides and 13 fungicides were detected in the analysed samples. A significant correlation was found between the presence of fungicide residues and honeybee colony disorders. A significant positive link could also be established between the observation of disorder and the abundance of crop surface around the beehive. According to our results, the role of fungicides as a potential stressor for honeybee colonies should be further studied, either by their direct and/or indirect impacts on bees and bee colonies.

Orphaning does not affect the colony productivity of the primitive eusocial wasp Polistes snelleni

In colonies of primitively eusocial wasps, some dominant workers become successive queens and inherit queenship after the death of the foundress queens. Although workers in many species do not mate, workers of Polistes snelleni are capable of mating and female production. In this study, we removed foundress queens from colonies of P. snelleni to evaluate the effects of queen loss on the dominant–subordinate relationships among the remaining workers and the productivity of colonies in the species. The foundress queens were the sole egg layers in almost all of the queenright colonies. The frequency of dominance behaviour among the wasps in the queenright colonies was significantly less than in the orphan colonies. The frequency of dominance behaviour in the successive queens after queen removal was significantly more than in the foundress queens. Multiple workers had developed ovaries, including the successive queens in 66.7 % (10/15) of the orphan colonies after queen removal. The orphan colonies produced significantly more cells and eggs than the queenright colonies. Our results suggest that the reproductive potential of the successive queens in the orphan colonies is not lower than that of the foundress queens, and that the productivity of the orphan colonies is maintained rather than causing potential conflict over direct reproduction among workers.

No facultative worker policing in the honey bee (Apis mellifera L.)

Kin selection theory predicts that in colonies of social Hymenoptera with multiply mated queens, workers should mutually inhibit ("police") worker reproduction, but that in colonies with singly mated queens, workers should favor rearing workers' sons instead of queens' sons. In line with these predictions, Mattila et al. (Curr Biol 22:2027-2031, 2012) documented increased ovary development among workers in colonies of honey bees with singly mated queens, suggesting that workers can detect and respond adaptively to queen mating frequency and raising the possibility that they facultative police. In a follow-up experiment, we test and reject the hypothesis that workers in single-patriline colonies prefer worker-derived males and are able to reproduce directly; we show that their eggs are policed as strongly as those of workers in colonies with multiply mated queens. Evidently, workers do not respond facultatively to a kin structure that favors relaxed policing and increased direct reproduction. These workers may instead be responding to a poor queen or preparing for possible queen loss.

Functionality of Varroa-Resistant Honey Bees (Hymenoptera: Apidae) When Used in Migratory Beekeeping for Crop Pollination

Two types of honey bees, Apis mellifera L. (Hymenoptera: Apidae), bred for resistance to Varroa destructor Anderson & Trueman were evaluated for performance when used in migratory crop pollination. Colonies of Russian honey bees (RHB) and outcrossed bees with Varroa-sensitive hygiene (VSH) were managed without miticide treatments and compared with colonies of Italian honey bees that served as controls. Control colonies were managed as groups which either were treated twice each year against V. destructor (CT) or kept untreated (CU). Totals of 240 and 247 colonies were established initially for trials in 2008 and 2009, respectively. RHB and VSH colonies generally had adult and brood populations similar to those of the standard CT group regarding pollination requirements. For pollination of almonds [Prunus dulcis (Mill.) D.A.Webb] in February, percentages of colonies meeting the required six or more frames of adult bees were 57% (VSH), 56% (CT), 39% (RHB), and 34% (CU). RHB are known to have small colonies in early spring, but this can be overcome with appropriate feeding. For later pollination requirements in May to July, 94-100% of colonies in the four groups met pollination size requirements for apples (Malus domestica Borkh.), cranberries (Vaccinium macrocarpon Aiton), and lowbush blueberries (Vaccinium angustifolium Aiton). Infestations with V. destructor usually were lowest in CT colonies and tended to be lower in VSH colonies than in RHB and CU colonies. This study demonstrates that bees with the VSH trait and pure RHB offer alternatives for beekeepers to use for commercial crop pollination while reducing reliance on miticides. The high frequency of queen loss (only approximately one fourth of original queens survived each year) suggests that frequent requeening is necessary to maintain desired genetics.

Differential Proteomics in Dequeened Honeybee Colonies Reveals Lower Viral Load in Hemolymph of Fertile Worker Bees

The eusocial societies of honeybees, where the queen is the only fertile female among tens of thousands sterile worker bees, have intrigued scientists for centuries. The proximate factors, which cause the inhibition of worker bee ovaries, remain largely unknown; as are the factors which cause the activation of worker ovaries upon the loss of queen and brood in the colony. In an attempt to reveal key players in the regulatory network, we made a proteomic comparison of hemolymph profiles of workers with completely activated ovaries vs. rudimentary ovaries. An unexpected finding of this study is the correlation between age matched worker sterility and the enrichment of Picorna-like virus proteins. Fertile workers, on the other hand, show the upregulation of potential components of the immune system. It remains to be investigated whether viral infections contribute to worker sterility directly or are the result of a weaker immune system of sterile workers.

Intraspecific queen parasitism in a highly eusocial bee

Insect societies are well-known for their advanced cooperation, but their colonies are also vulnerable to reproductive parasitism. Here, we present a novel example of an intraspecific social parasitism in a highly eusocial bee, the stingless bee Melipona scutellaris. In particular, we provide genetic evidence which shows that, upon loss of the mother queen, many colonies are invaded by unrelated queens that fly in from unrelated hives nearby. The reasons for the occurrence of this surprising form of social parasitism may be linked to the fact that unlike honeybees, Melipona bees produce new queens in great excess of colony needs, and that this exerts much greater selection on queens to seek alternative reproductive options, such as by taking over other nests. Overall, our results are the first to demonstrate that queens in highly eusocial bees can found colonies not only via supersedure or swarming, but also by infiltrating and taking over other unrelated nests.

The Challenge Hypothesis in an Insect: Juvenile Hormone Increases during Reproductive Conflict following Queen Loss in Polistes Wasps

The challenge hypothesis was proposed as a mechanism for vertebrates to optimize their testosterone titers by upregulating testosterone during periods of aggressive competition. Here we test key predictions of the challenge hypothesis in an independently evolved endocrine system: juvenile hormone (JH) in a social insect. We assess how social conflict influences JH titers in Polistes dominulus wasps. Aggressive conflict was produced by removing the queen from some colonies (experimental) and a low-ranked worker from other colonies (control). Queen removal produced competition among workers to fill the reproductive vacancy. Workers upregulated their JH titers in response to this social conflict; worker JH titers were higher in queenless than in queenright colonies. Furthermore, JH titers were associated with an individual's ability to dominate rivals; the worker that took over as the replacement queen had a substantially higher JH titer than did other workers. Finally, JH titers were positively associated with aggression in queenless colonies, but there was no relationship between JH and aggression in stable, queenright colonies. Overall, these results match key predictions of the challenge hypothesis and parallel much of the work on testosterone in vertebrates. Social modulation of hormone titers is not confined to a particular endocrine system but is likely to be an adaptive feature of endocrine systems across diverse taxa.

Maternity of emergency queens in the Cape honey bee, Apis mellifera capensis

During reproductive swarming, some workers of the Cape honey bee, Apis mellifera capensis, lay eggs in queen cells, many of which are reared to maturity. However, it is unknown if workers are able to lay in queen cells immediately after queen loss during an episode of emergency queen rearing. In this study we experimentally de-queened colonies and determined the maternity of larvae and pupae that were reared as queens. This allowed us to determine how soon after queen loss workers contribute to the production of new queens. We were further interested to see if workers would preferentially raise new queens from queen-laid brood if this was introduced later. We performed our manipulations in two different settings: an apiary setting where colonies were situated close together and a more natural situation in which the colonies were well separated. This allowed us to determine how the vicinity of other colonies affects the presence of parasites. We found that workers do indeed contribute to queen cell production immediately after the loss of their queen, thus demonstrating that some workers either have activated ovaries even when their colony has a queen or are able to activate their ovaries extremely rapidly. Queen-laid brood introduced days after queen loss was ignored, showing that workers do not prefer to raise new queens from queen brood when given a choice. We also detected non-natal parasitism of queen cells in both settings. We therefore conclude that some A. m. capensis genotypes specialize in parasitizing queen cells.

Ecological competition favours cooperation in termite societies

Conflict and competition lie at the heart of the theories of both ecology and sociobiology. Despite this, the interaction between societal conflicts on one hand and ecological competition on the other remains poorly understood. Here, we investigate this interaction in two ecologically similar sympatric termite species, Cryptotermes secundus Hill and Cryptotermes domesticus Haviland. We manipulated the incidence of king and queen loss (within-species conflict) and the incidence of cohabitation of the two species (between-species competition) in a series of 2 year experiments. Manipulation alone had no detectable effect and most colonies survived the 2-year period. In contrast, promoting both within- and between-species conflict caused the great majority of colonies to die. Moreover, the resulting colony loss was much more rapid in the conflict-ridden C. domesticus than in C. secundus. Our data suggest that ecological competition among species can greatly exacerbate the impact of internal conflicts, thereby promoting the evolution of within-species cooperation.