Hemolymph proteome changes during worker brood development match the biological divergences between western honey bees (Apis mellifera) and eastern honey bees (Apis cerana)

Background

Hemolymph plays key roles in honey bee molecule transport, immune defense, and in monitoring the physiological condition. There is a lack of knowledge regarding how the proteome achieves these biological missions for both the western and eastern honey bees (Apis mellifera and Apis cerana). A time-resolved proteome was compared using two-dimensional electrophoresis-based proteomics to reveal the mechanistic differences by analysis of hemolymph proteome changes between the worker bees of two bee species during the larval to pupal stages.

Results

The brood body weight of Apis mellifera was significantly heavier than that of Apis cerana at each developmental stage. Significantly, different protein expression patterns and metabolic pathways were observed in 74 proteins (166 spots) that were differentially abundant between the two bee species. The function of hemolymph in energy storage, odor communication, and antioxidation is of equal importance for the western and eastern bees, indicated by the enhanced expression of different protein species. However, stronger expression of protein folding, cytoskeletal and developmental proteins, and more highly activated energy producing pathways in western bees suggests that the different bee species have developed unique strategies to match their specific physiology using hemolymph to deliver nutrients and in immune defense.

Conclusions

Our disparate findings constitute a proof-of-concept of molecular details that the ecologically shaped different physiological conditions of different bee species match with the hemolymph proteome during the brood stage. This also provides a starting point for future research on the specific hemolymph proteins or pathways related to the differential phenotypes or physiology.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-563) contains supplementary material, which is available to authorized users.     

Reproduction of Varroa destructor in worker brood of Africanized honey bees (Apis mellifera)

Reproduction and population growth of Varroa destructor was studied in ten naturally infested, Africanized honeybee (AHB) (Apis mellifera) colonies in Yucatan, Mexico. Between February 1997 and January 1998 monthly records of the amount of pollen, honey, sealed worker and drone brood were recorded. In addition, mite infestation levels of adult bees and worker brood and the fecundity of the mites reproducing in worker cells were determined. The mean number of sealed worker brood cells (10,070 ± 1,790) remained fairly constant over the experimental period in each colony. However, the presence and amount of sealed drone brood was very variable. One colony had drone brood for 10 months and another for only 1 month. Both the mean infestation level of worker brood (18.1 ± 8.4%) and adult bees (3.5 ± 1.3%) remained fairly constant over the study period and did not increase rapidly as is normally observed in European honey bees. In fact, the estimated mean number of mites fell from 3,500 in February 1997 to 2,380 in January 1998. In May 2000 the mean mite population in the study colonies was still only 1,821 mites. The fertility level of mites in this study was much higher (83–96%) than in AHB in Brazil(25–57%), and similar to that found in EHB (76–94%). Mite fertility remained high throughout the entire study and was not influenced by the amount of pollen, honey or worker brood in the colonies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nosema ceranae in drone honey bees (Apis mellifera)

Nosema ceranae is a microsporidian intracellular parasite of honey bees, Apis mellifera. Previously Nosema apis was thought to be the only cause of nosemosis, but it has recently been proposed that N. ceranae is displacing N. apis. The rapid spread of N. ceranae could be due to additional transmission mechanisms, as well as higher infectivity. We analyzed drones for N. ceranae infections using duplex qPCR with species specific primers and probes. We found that both immature and mature drones are infected with N. ceranae at low levels. This is the first report detecting N. ceranae in immature bees. Our data suggest that because drones are known to drift from their parent hives to other hives, they could provide a means for disease spread within and between apiaries.     

Selection on a haploid genotype for discrimination learning performance: Correlation between drone honey bees (Apis mellifera) and their worker progeny (Hymenoptera: Apidae)

Successful bidirectional selection for discriminative olfactory learning is reported for drone honey bees (Apis mellifera). Learning performance was evaluated using a discrimination conditioning procedure that required drones to discriminate between an appetitively reinforced odorant and one that was followed by punishment. Selective breeding produced high- and low-learning-performance lines of worker progeny that diverged from performance of workers whose fathers were selected at random. Furthermore, we show that levels of sucrose-induced sensitization are not correlated to learning performance. These results corroborate earlier findings and further demonstrate the power of selection on a haploid (drone) genotype. In addition, this study now shows that the demonstrated differences in learning performance cannot be completely accounted for by alteration of sucrose-induced sensitization. Thus, using this technique, it may be possible to select for associative conditioning without a pleiotropic increase in sensitization. The honey bee will be ideally suited to these types of correlation analyses in future studies.     

Genome-wide characterization of long intergenic non-coding RNAs (lincRNAs) provides new insight into viral diseases in honey bees Apis cerana and Apis mellifera

Background

Long non-coding RNAs (lncRNAs) are a class of RNAs that do not encode proteins. Recently, lncRNAs have gained special attention for their roles in various biological process and diseases.

Results

In an attempt to identify long intergenic non-coding RNAs (lincRNAs) and their possible involvement in honey bee development and diseases, we analyzed RNA-seq datasets generated from Asian honey bee (Apis cerana) and western honey bee (Apis mellifera). We identified 2470 lincRNAs with an average length of 1011 bp from A. cerana and 1514 lincRNAs with an average length of 790 bp in A. mellifera. Comparative analysis revealed that 5 % of the total lincRNAs derived from both species are unique in each species. Our comparative digital gene expression analysis revealed a high degree of tissue-specific expression among the seven major tissues of honey bee, different from mRNA expression patterns. A total of 863 (57 %) and 464 (18 %) lincRNAs showed tissue-dependent expression in A. mellifera and A. cerana, respectively, most preferentially in ovary and fat body tissues. Importantly, we identified 11 lincRNAs that are specifically regulated upon viral infection in honey bees, and 10 of them appear to play roles during infection with various viruses.

Conclusions

This study provides the first comprehensive set of lincRNAs for honey bees and opens the door to discover lincRNAs associated with biological and hormone signaling pathways as well as various diseases of honey bee.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1868-7) contains supplementary material, which is available to authorized users.     

Calorimetric investigations of the different castes of honey bees,Apis mellifera carnica

Summary Honey bees of different age and castes were investigated calorimetrically at 20, 25 and 30 °C. Experiments were completed by endoscopic observation of the insects in the visible and the near infrared range and by acoustical monitoring and subsequent frequency analysis of various locomotor activities. Direct calorimetric results of this paper are compared with data of indirect calorimetry from the literature using a respiratory quotient of 1.00 and 21.13 J consumed. Agreements between both methods are generally good. The results show that weight-specific heat production rates increase with age of worker bees by a factor of 5.6 at 30 °C, 3.7 at 25 °C and 40.0 at 20 °C. In groups of foragers the heat production decreases with growing group size to around 6% of the value for an isolated bee. The presence of a fertile queen or of brood reduces the heat output of a small worker group significantly. Adult drones exhibit a much higher metabolic rate (up to 19.7-fold at 20 °C) than juveniles with strong fluctuations in the power-time curves. Fertile queens show a less pronounced heat production rate than virgin queens (54% at 30 °C, 87% at 25 °C and 77% at 20 °C). Calorimetric unrest is much higher for young than for adult queens. Heat production is very low in both uncapped and capped brood and less than 30% of that of a newly emerged worker. In most cases temperature showed a significant influence on the metabolic level, although its sign was not homogeneous between the castes or even within them. Locomotor activities are easily recorded by the acoustic frequency spectrum (0–7.5 kHz) and in good agreement with endoscopic observations and calorimetric traces.Abbreviations RQ respiratory quotient - ww wet weight This paper is part of the PhD thesis of L.F.     

Virus infections in Brazilian honey bees

This work describes the first molecular-genetic evidence for viruses in Brazilian honey bee samples. Three different bee viruses, Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), and Deformed wing virus (DWV) were identified during a screening of RNAs from 1920 individual adult bees collected in a region of southeastern Brazil that has recently shown unusual bee declines. ABPV was detected in 27.1% of colony samples, while BQCV and DWV were found in 37% and 20.3%, respectively. These levels are substantially lower than the frequencies found for these viruses in surveys from other parts of the world. We also developed and validated a multiplex RT-PCR assay for the simultaneous detection of ABPV, BQCV, and DWV in Brazil.     

Host-seeking behaviour of tracheal mites (Acari: Tarsonemidae) on honey bees (Hymenoptera: Apidae)

The behaviour of the endoparasitic tracheal mite, Acarapis woodi (Rennie) on honey bees (Apis mellifera L.) is a challenge to observe because of its small size. Through a microscope, we videotaped this mite's movement on young bees, dead bees and bees exposed to vegetable oil. Previous studies have shown that solid vegetable oil decreases mite infestations in a bee colony. We hypothesized that the oil alters mite behaviour to the detriment of the parasite, thus helping to safeguard the host. Habitat-seeking behaviour, identified as necessary for mites to locate a new host environment, was disrupted on both dead and oil-treated bees. Questing behaviour, which is associated with transfer between hosts, increased significantly on the dead and oily bees. The behaviours of mites were significantly different between all three treatments (x ²=494.96, p<0.001 on dead bees and x ²=851.11, p<0.001 on oily bees). Both questing and seeking behaviours were significantly different on each of the thoracic treatments (F _2,66=7.88, p<0.001 and F _2,66=21.28, p<0.001) and mite questing behaviour was not altered between males and females on live or oily bees (F _1,22=0.25, p<0.62), but habitat seeking was (F _1,22=7.42, p<0.012). The male questing and habitat-seeking behaviours were observed. We conclude that oil-treated bees gained protection from habitat-seeking mites because the normal behaviour of the mites seeking an oviposition site is interrupted.     

Choice of flowers by foraging honey bees (Apis mellifera): possible morphological cues

Abstract.

• 1 Honey bees foraging for nectar on lavender (Lavandula stoechas) chose inflorescences with more of their flowers open. The number of open flowers predicted whether an inflorescence was visited by bees, inspected but rejected, or ignored. Inflorescences chosen arbitrarily by observers had numbers of open flowers intermediate between those of visited and ignored inflorescences.
• 2 Differences in morphological characters between types of inflorescence correlated with nectar volume and sugar weight per flower so that visited inflorescences had a disproportionately greater volume of nectar and weight of sugar per flower and greater variance in nectar volume.
• 3 Although there were significant associations between nectar content and the morphological characters of inflorescences, discriminant function analysis revealed discrimination on the basis of morphology rather than nectar content.
• 4 Visited inflorescences tended to have smaller than average flowers but bees tended to probe the largest flowers on visited inflorescences.
• 5 Choice of flowers within inflorescences is explicable in terms of the relationship between flower size and nectar content.

     

Absence of reproductive conflict during queen rearing in Apis cerana

Polyandry in honeybee queens (Apis) causes many patrilines (subfamilies) within a colony, which may lead to a potential conflict of interest among workers. This may be most apparent during queen rearing when nepotistic worker behavior could influence the genetics of future generations. Several studies have searched for such conflict in European honeybees (A. mellifera), but studies on other Apis species remain lacking. We investigated the presence of reproductive conflict in A. cerana japonica by comparing the patriline proportion of queen larvae to that of adult workers. We determined the patrilines of 272 workers and 57 queen larvae using four polymorphic microsatellite markers that were sampled from queenless colonies originally derived from four naturally mated queen-right colonies. The number of patrilines in each colony was 9, 12, 8, and 7, respectively, which is lower than that observed in continental Asia. We found no difference in patriline proportion between adult workers and queen larvae. Our data support neither genetic variance for royalty or existence of worker nepotism in A. cerana japonica.     

Worker policing persists in a hopelessly queenless honey bee colony (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Summary In queenright colonies of Apis mellifera, worker policing normally eliminates worker-laid eggs thereby preventing worker reproduction. However, in queenless colonies that have failed to rear a replacement queen, worker reproduction is normal. Worker policing is switched off, many workers have active ovaries and lay eggs, and the colony rears a last batch of male brood before dying out. Here we report a colony which, when hopelessly queenless, did not stop policing although a high proportion of workers had active ovaries (12.6%) and many eggs were laid. However, all these eggs and also worker-laid eggs transferred from another colony were policed. This unusual pattern was repeated eight weeks later by a second queenless colony made using worker bees from the same mother colony, which strongly suggests genetic determination.Received 19 May 2003; revised 11 September 2003; accepted 23 September 2003.     

Asymmetrical coexistence of Nosema ceranae and Nosema apis in honey bees

Globalization has provided opportunities for parasites/pathogens to cross geographic boundaries and expand to new hosts. Recent studies showed that Nosema ceranae, originally considered a microsporidian parasite of Eastern honey bees, Apis cerana, is a disease agent of nosemosis in European honey bees, Apis mellifera, along with the resident species, Nosema apis. Further studies indicated that disease caused by N. ceranae in European honey bees is far more prevalent than that caused by N. apis. In order to gain more insight into the epidemiology of Nosema parasitism in honey bees, we conducted studies to investigate infection of Nosema in its original host, Eastern honey bees, using conventional PCR and duplex real time quantitative PCR methods. Our results showed that A. cerana was infected not only with N. ceranae as previously reported [Fries, I., Feng, F., Silva, A.D., Slemenda, S.B., Pieniazek, N.J., 1996. Nosema ceranae n. sp. (Microspora, Nosematidae), morphological and molecular characterization of a microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera, Apidae). Eur. J. Protistol. 32, 356-365], but also with N. apis. Both microsporidia produced single and mixed infections. Overall and at each location alone, the prevalence of N. ceranae was higher than that of N. apis. In all cases of mixed infections, the number of N. ceranae gene copies (corresponding to the parasite load) significantly out numbered those of N. apis. Phylogenetic analysis based on a variable region of small subunit ribosomal RNA (SSUrRNA) showed four distinct clades of N. apis and five clades of N. ceranae and that geographical distance does not appear to influence the genetic diversity of Nosema populations. The results from this study demonstrated that duplex real-time qPCR assay developed in this study is a valuable tool for quantitative measurement of Nosema and can be used to monitor the progression of microsprodian infections of honey bees in a timely and cost efficient manner.     

Kin recognition of worker brood by worker honey bees,Apis mellifera L.

Experimental colonies of honey bees consisting of two patrilines were observed as they reared worker brood. Seven behavior patterns that relate to brood care were recorded. Worker bees biased the care they provided to eggs and larvae destined to become workers on the basis of brood patrilines. Both patrilineal and antipatrilineal preferences in various behavioral patterns were observed. There was variation among colonies that may have been the result of the frequencies of brood of each patriline and the total amount of brood available to be reared. In addition, there were some differences between workers of the two patrilines in the way that they cared for the two patrilines of brood.     

Transcriptome differences in the hypopharyngeal gland between Western Honeybees (Apis mellifera) and Eastern Honeybees (Apis cerana)

Background

Apis mellifera and Apis cerana are two sibling species of Apidae. Apis cerana is adept at collecting sporadic nectar in mountain and forest region and exhibits stiffer hardiness and acarid resistance as a result of natural selection, whereas Apis mellifera has the advantage of producing royal jelly. To identify differentially expressed genes (DEGs) that affect the development of hypopharyngeal gland (HG) and/or the secretion of royal jelly between these two honeybee species, we performed a digital gene expression (DGE) analysis of the HGs of these two species at three developmental stages (newly emerged worker, nurse and forager).

Results

Twelve DGE-tag libraries were constructed and sequenced using the total RNA extracted from the HGs of newly emerged workers, nurses, and foragers of Apis mellifera and Apis cerana. Finally, a total of 1482 genes in Apis mellifera and 1313 in Apis cerana were found to exhibit an expression difference among the three developmental stages. A total of 1417 DEGs were identified between these two species. Of these, 623, 1072, and 462 genes showed an expression difference at the newly emerged worker, nurse, and forager stages, respectively. The nurse stage exhibited the highest number of DEGs between these two species and most of these were found to be up-regulated in Apis mellifera. These results suggest that the higher yield of royal jelly in Apis mellifera may be due to the higher expression level of these DEGs.

Conclusions

In this study, we investigated the DEGs between the HGs of two sibling honeybee species (Apis mellifera and Apis cerana). Our results indicated that the gene expression difference was associated with the difference in the royal jelly yield between these two species. These results provide an important clue for clarifying the mechanisms underlying hypopharyngeal gland development and the production of royal jelly.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-744) contains supplementary material, which is available to authorized users.     

Brood-cell size does not influence the susceptibility of honey bees (Apis mellifera) to infestation by tracheal mites (Acarapis woodi)

Tracheal mites have been associated with the condition in honey bees that devastated colonies in Britain and Ireland in the early 1900s. The first outbreak of this condition, that became known as the ‘Isle of Wight’ disease, coincided with the period when brood-cell size was increased from about 5.0 mm to about 5.5 mm in width. We undertook an inoculation experiment over a 7-day period to establish if the act of increasing the brood-cell size could have triggered the onset of tracheal mites in honey bees. The standard-sized cells used had a cell width of 5.44 mm and the small-sized cells a width of 5.07 mm. Using callow (newly emerged) bees, from three colonies that had mixed cell sizes, we compared the susceptibility of bees reared in standard-sized cells with that of those raised in small-sized cells. The results indicated similar levels of female mite abundance (0.49 vs. 0.52 mites per bee) and mean fecundity (4.33 vs. 4.22 offspring per female mite), and produced no evidence of any difference in the overall susceptibility between the bees raised in the standard-sized cells versus small-sized brood cells.     

Nosema ceranae is a long-present and wide-spread microsporidian infection of the European honey bee (Apis mellifera) in the United States

Honey bee samples collected between 1995 and 2007 from 12 states were examined for the presence of Nosema infections. Our results showed that Nosema ceranae is a wide-spread infection of the European honey bee, Apis mellifera in the United States. The discovery of N. ceranae in bees collected a decade ago indicates that N. ceranae was transferred from its original host, Apis cerana to A. mellifera earlier than previously recognized. The spread of N. ceranae infection in A. mellifera warrants further epidemiological studies to identify conditions that resulted in such a widespread infection.     

Phylogenetic analysis of Starmerella apis in honey bees (Apis mellifera)

Honey bees are among the most effective pollinators that promote plant reproduction. Bees are highly active in the pollen collection season, which can lead to the transmission of selected pathogens between colonies. The clade Starmerella comprises yeasts that are isolated mainly from bees and their environment. When visiting plants, bees can come into contact with Starmerella spp. The aim of this study was to determine the prevalence and phylogenetic position of S. apis in bee colonies. Bee colonies were collected from nine apiaries in three regions. Ten colonies were sampled randomly from each apiary, and pooled samples were collected from the central part of the hive in each colony. A total of 90 (100%) bee colonies from nine apiaries were examined. Starmerella apis was detected in 31 (34.44%) samples, but related species were not identified. The 18S rRNA amplicon sequences of S. apis were compatible with the GenBank sequences of Starmerella spp. from India, Japan, Syria, Thailand, and the USA. The amplicon sequences of S. apis were also 99.06% homologous with the sequences deposited in GenBank under accession numbers JX515988 and NG067631 .This is the first study to perform a phylogenetic analysis of S. apis in Polish honey bees.     

Survival of the mite Varroa jacobsoni Oud. (Mesostigmata: Varroidae) in broodless colonies of the honey bee Apis mellifera L. (Hymenoptera: Apidae)

Varroa mite free colonies of the honey bee Apis mellifera L. were artificially infested, with either parasitized bees or infested worker brood. Queens were kept in cages to provide broodless conditions during the experiment. Parasites that fell to the bottom of the hive were monitored at 3–4 days intervals for three months. An acaricide treatment was used to recover mites still alive after this time period. Survivorship at each interval was calculated and life table functions of the phoretic mite cohorts were obtained. Trends in survival of Varroa cohorts showed maximum lifespans ranging from 80 to 100 days. Life expectancy of these phoretic cohorts at the beginning of the experiment ranges between 19 to 41, with a mean of 31 days.