Labial gland marking secretions of male Bombus lucorum bumblebees from Europe and China reveal two separate species: B. lucorum (Linnaeus 1761) and Bombus minshanicola (Bischoff 1936)

Differences in male bumblebee labial gland secretions can be used to separate species pairs. Cephalic labial gland secretions from male Bombus lucorum Linnaeus bumblebees from Europe, and male ‘B. lucorum’ bumblebees from China, were analyzed by gas chromatography/mass spectrometry (GC/MS). In the European B. lucorum L., ethyl tetradec-9-enoate was identified as the major compound (58% peak area), along with a complex mixture of straight-chain alkenols, acetates, hydrocarbons, and wax type esters. In contrast, the main component of the labial gland secretions of the Chinese ‘B. lucorum’, ethyl dodecanoate (31%), did not dominate the secretion, which additionally contained large amounts of 3,7,11-trimethyldodeca-6,10-dien-1-ol (=2,3-dihydrofarnesol, 24% peak area), ethyl octadec-9-enoate (15% peak area) and a mixture of acyclic diterpenes (alcohols, aldehydes, and acetates). Furthermore, in B. lucorum, 3,7,11-trimethyldodeca-6,10-dien-1-ol was only detected in trace amounts (0.05%) and ethyl octadec-9-enoate corresponded to only 0.8% of the mixture. These results indicate that ‘B. lucorum’ from China is a separate taxon from B. lucorum L. Further analysis revealed that ‘B. lucorum’ from China has been previously described as Bombus minshanicola Bischoff 1936 from Gansu/China. Differences in the chemical composition of male bumblebee labial gland secretions are discussed in comparison with other known bumblebee species pairs.     

Identification of Nosema bombi Fantham and Porter 1914 (Microsporidia) in Bombus impatiens and Bombus sandersoni from Great Smoky Mountains National Park (USA)

Ninety three bumble bees belonging to the genus Bombus, subgenus Pyrobombus (three Bombus vagans, seven Bombus bimaculatus, 17 B. sandersoni and 68 B. impatiens) from Great Smoky Mountains National Park were examined for microsporidia. Light microscopy of calcoflour and trichrome-stained smears, and PCR revealed infection with N. bombi in one specimen each of B. sandersoni and B. impatiens. Sizes and shapes of spores in both N. bombi isolates were similar to those described for European isolates of the microsporidium. A region of the rRNA gene from the B. impatiens isolate (1689 bp, accession GQ254295) aligned with homologous sequences from eight European isolates, with only three variable sites. Sequence variability of this region between novel isolates and the European ones was the same as among European isolates.     

The effect of overwintering temperature on the body energy reserves and phenoloxidase activity of bumblebee Bombus lucorum queens

Warming winters and changes in species composition related to the estimated global warming may cause a threat to bumblebees adapted to cold winters. During the overwintering period, their intermediary and respiratory metabolism decreases but metabolism remains responsive to temperature. The effect of temperature on diapause survival, phenoloxidase (PO) activity, and energy expenditure of the white-tailed bumblebee (Bombus lucorum) after a 4-month diapause were studied by manipulating the diapause temperature. Two overwintering temperatures were used, cold (1.8 °C) and warm (9 °C). Body fat content was used as an estimate of the remaining energy resources and PO activity as an immune function parameter of overwintering bumblebee queens. The baseline levels of PO activity were used to measure the differences in B. lucorum queen responses after overwintering in either temperature. We found a 0.4 g pre-diapause threshold weight of survival in B. lucorum. Large queens had more fat left and a higher PO activity compared to small ones after overwintering in warm conditions, but in the cold there was no effect of size on the remaining fat in the fat body of queens or their PO activity. The observed difference in energy usage appears to relate to normal size-dependent metabolism and variation in energy allocation between basic metabolism and immune functions.     

Molecular cloning and antibacterial activity of bombolitin isolated from the venom of a bumblebee, Bombus terrestris

Three major components of bumblebee venom are bombolitin, phospholipase A₂, and a serine protease, with bombolitin being the most abundant. Here, we describe the molecular cloning of bombolitin isolated from the venom of a bumblebee, Bombus terrestris, and demonstrate its antibacterial activity. The B. terrestris bombolitin gene consists of 2 exons encoding 56 amino acid residues. Comparative analysis shows that mature B. terrestris bombolitin consists of 18 amino acid residues, which are identical to those of B. ignitus bombolitin. B. terrestris bombolitin displayed antibacterial activity against both the Gram-negative bacterium Klebsiella pneumoniae and the Gram-positive bacterium Staphylococcus aureus, indicating that B. terrestris bombolitin may be a potential antimicrobial agent.     

Male labial gland secretions and mitochondrial DNA markers support species status of Bombus cryptarum and B. magnus (Hymenoptera, Apidae)

Summary. Spring queens of Bombus cryptarum and B. magnus from 2 localities in Brandenburg/Germany and Scotland/United Kingdom respectively were determined by morphological characteristics. The lateral border of the collare at the border of the pronotallobus or at the episternum proved to be an especially useful character. Artificial colonies were reared from safely determined spring queens and the cephalic part of the labial glands of males from these colonies were investigated by GC/MS. The investigation identified approximately 50 compounds, as a mixture of straight chain fatty acid derivatives (alcohols, esters and hydrocarbons). The labial secretions of B. cryptarum and B. magnus are significantly different. Mitochondrial cytochrome oxidase 1 (CO1) of two queens from each locality and species were sequenced. Each species from the different localities formed a cluster. Sequence divergence between B. cryptarum and B. magnus was about 30 base substitutions and approximately 0.04 in Tamura-Nei genetic distance. Bombus cryptarum and B. magnus were closer to each other than to B. lucorum and made the sister group in the topology of the tree. Both the CO1 sequences and the labial gland secretions of males of B. cryptarum from Brandenburg and of males from artificial colonies reared from safely determined spring queens from Scotland are identical. B. cryptarum has thus, for the first time, been identified as part of the British bumble bee fauna. The differences of both the labial gland secretions, used as species recognition signals, and the genetic differences established by sequencing CO1 confirm the morphological findings that B. cryptarum and B. magnus are distinct taxa which should be treated as distinct species.Received 25 March 2004; revised 13 June 2004; accepted 15 June 2004.     

Invasive Bombus terrestris (Hymenoptera: Apidae) parasitized by a flagellate (Euglenozoa: Kinetoplastea) and a neogregarine (Apicomplexa: Neogregarinorida)

The flagellate Crithidia bombi and the neogregarine Apicystis bombi have been found in individuals of Bombus terrestris, a Palaearctic species of bumble bee commercially reared and shipped worldwide for pollination services. B. terrestris has recently entered into the northwestern Patagonia region of Argentina from Chile, where it was introduced in 1998. Prevalence was 21.6% for C. bombi and 3.6% for A. bombi (n = 111). The pathogens were not detected in 441 bumble bees belonging to five of the eight known Argentine native species (Bombus atratus, Bombus morio, Bombus bellicosus, Bombus opifex, Bombus tucumanus) collected elsewhere in the country. Although the absence of natural occurrence of C. bombi and A. bombi in Argentine native bumble bees cannot be ascertained at present due to the limited surveys performed, it is important to report their detection in invasive B. terrestris. The invasion event is relatively recent and the accompanying pathogens are not species specific within the genus Bombus.     

Screening of valid reference genes for real-time RT-PCR data normalization in Hevea brasiliensis and expression validation of a sucrose transporter gene HbSUT3

Real-time RT-PCR (RT-qPCR) is a sensitive and precise method of quantifying gene expression, however, suitable reference genes are required. Here, a systematic reference gene screening was performed by RT-qPCR on 22 candidate genes in Hevea brasiliensis. Two ubiquitin-protein ligases (UBC2a and UBC4) were the most stable when all samples were analyzed together. A mitosis protein (YLS8) and a eukaryotic translation initiation factor (eIF1Aa) were the most stable in response to tapping. UBC2b and UBC1 were the most stable among different genotypes. UBC2b and a DEAD box RNA helicase (RH2b) were the most stable across individual trees. YLS8 and RH8 were most stably expressed in hormone-treated samples. Expression of the candidate reference genes varied significantly across different tissues, and at least four genes (RH2b, RH8, UBC2a and eIF2) were needed for expression normalization. In addition, examination of relative expression of a sucrose transporter HbSUT3 in different RNA samples demonstrated the importance of additional reference genes to ensure accurate quantitative expression analysis. Overall, our work serves as a guide for selection of reference genes in RT-qPCR gene expression studies in H. brasiliensis.     

小峰熊蜂蜂毒磷脂酶A2基因的克隆及表达分析

磷脂酶A2 （phospholipase A2, PLA2）是蜂毒主要成分, 也是蜂毒的主要过敏原, 在熊蜂个体和群体防御方面具有重要功能。为了探究熊蜂A2基因的生物学功能, 本研究以小峰熊蜂Bombus hypocrita为材料进行了蜂毒PLA2基因的克隆、 鉴定与表达特性分析。结果表明： 该基因全长为2 272 bp, GenBank登录号为KF214771, 由4个外显子和3个内含子组成, 编码区（CDS）长为543 bp, 共编码180个氨基酸残基。氨基酸序列相似性分析显示, 成熟的小峰熊蜂PLA2（含有136个氨基酸）与其他蜂类PLA2的氨基酸序列相似性较高, 均包含10个保守的半胱氨酸残基、 1个保守的Ca2+结合位点和1个酶活性中心。基于PLA2氨基酸序列的系统进化树分析表明, 熊蜂属Bombus与蜜蜂属Apis在不同分支上, 属单系群, 且蜜蜂属分化较早。荧光定量PCR结果表明, PLA2基因在小峰熊蜂各日龄均有表达, 且随日龄增长, 表达量呈先上升后下降的趋势, 10日龄时出现峰值, 其表达量显著高于其他日龄（P<0.05）。半定量PCR结果表明, PLA2基因在毒腺、 卵巢、 中肠中表达量较高, 在足、 触角、 食道腺中表达量较低, 在脂肪体、 肌肉、 神经、 气管、 复眼、 脑中未表达。本研究探明了小峰熊蜂PLA2的基因结构及其表达特性, 丰富了熊蜂PLA2的生物学基础, 为进一步深入研究熊蜂PLA2生物学功能和作用机制以及开发蜂毒生物制剂等鉴定了基础。     

Reduced cephalic labial glands in the male bumblebees of the subgenus Rhodobombus Dalla Torre (Hymenoptera: Apidae: Bombus Latreille)

Until now, all males of bumblebees are known to attract conspecific females by marking places with a chemical signal secreted by their cephalic labial gland. The specific combination of patrolling flight and scentmarking is of outmost importance to bumblebees as it is their main species specific recognition system. We report here the lack of that recognition system in species of the subgenus Rhodobombus by comparing the morphology and histology of the cephalic labial glands of Bombus (Rhodobombus) mesomelas with those of a well known species, B. (Bombus) terrestris (L.) The cephalic labial glands are much smaller in B. mesomelas than in other bumblebees species and most likely non-functional. This morphology is also observed in B. pomorum and B. brodmanni which also belong to the subgenus Rhodobombus. Our morphological observations are consistent with the chemical analyses of the secretions in B. mesomelas and B. pomorum which are very limited and of most unusual composition for a bumblebee. In addition, whereas other species are thought to use their barbae mandibularis to spread their secretion onto the substrate, these structures are absent in all Rhodobombus. All these observations would mean that the males of Rhodobombus do not attract females from far away by the use of their cephalic labial gland secretions.     

Selection of Reference Genes for Gene Expression Studies Related to Intramuscular Fat Deposition in Capra hircus Skeletal Muscle

The identification of suitable reference genes is critical for obtaining reliable results from gene expression studies using quantitative real-time PCR (qPCR) because the expression of reference genes may vary considerably under different experimental conditions. In most cases, however, commonly used reference genes are employed in data normalization without proper validation, which may lead to incorrect data interpretation. Here, we aim to select a set of optimal reference genes for the accurate normalization of gene expression associated with intramuscular fat (IMF) deposition during development. In the present study, eight reference genes (PPIB, HMBS, RPLP0, B2M, YWHAZ, 18S, GAPDH and ACTB) were evaluated by three different algorithms (geNorm, NormFinder and BestKeeper) in two types of muscle tissues (longissimus dorsi muscle and biceps femoris muscle) across different developmental stages. All three algorithms gave similar results. PPIB and HMBS were identified as the most stable reference genes, while the commonly used reference genes 18S and GAPDH were the most variably expressed, with expression varying dramatically across different developmental stages. Furthermore, to reveal the crucial role of appropriate reference genes in obtaining a reliable result, analysis of PPARG expression was performed by normalization to the most and the least stable reference genes. The relative expression levels of PPARG normalized to the most stable reference genes greatly differed from those normalized to the least stable one. Therefore, evaluation of reference genes must be performed for a given experimental condition before the reference genes are used. PPIB and HMBS are the optimal reference genes for analysis of gene expression associated with IMF deposition in skeletal muscle during development.     

Reference gene selection for quantitative real-time PCR normalization: Application in the caprine mammary gland

In dairy animals, gene expression analysis has become increasing key to understand the biological processes occurring in mammary gland development that shape future milk potential. Selecting high-stability reference genes is crucial to interpret real-time qPCR data. This study investigated the expression stability of five top-ranked candidate reference genes in the goat mammary gland through three assays comparing different experimental conditions (physiological states, sample types and experimental treatments). The expression stability of genes including β-actin, glyceraldehyde-3-phosphate dehydrogenase, 18S rRNA, cyclophilin A and ribosomal protein large P0 was analyzed. Normalization for each experimental condition expression data revealed a different reference gene. Nevertheless, in our various assays, genes encoding for ribosomal proteins, 18S rRNA and RPLP0 presented the best expression stability. This result has been confirmed using a combined analysis of stability on the three assays. All genes showed the same distribution within and among the three assays and a different distribution between Ct variability and GeNorm normalization. In addition, the application on Catenin B1 expression using an inappropriate reference gene confirmed erroneous variations in interpretation. To conclude, there is no single ideal reference gene for caprine mammary gland studies and we recommend using a panel of top-ranked reference genes, including RPLP0, at the beginning of each experiment to validate the most stable(s) gene(s).     

Cryptic species diversity in a widespread bumble bee complex revealed using mitochondrial DNA RFLPs

Cryptic species diversity is thought to be common within the class Insecta, posing problems for basic ecological and population genetic studies and conservation management. Within the temperate bumble bee (Bombus spp.) fauna, members of the subgenus Bombus sensu stricto are amongst the most abundant and widespread. However, their species diversity is controversial due to the extreme difficulty or inability morphologically to identify the majority of individuals to species. Our character-based phylogenetic analyses of partial CO1 (700 bp) from 39 individuals spread across their sympatric European ranges provided unequivocal support for five taxa (3–22 diagnostic DNA base pair sites per species). Inclusion of 20 Irish specimens to the dataset revealed ≥2.3% sequence divergence between taxa and ≤1.3% within taxa. We developed a PCR-RFLP based method for unequivocally distinguishing amongst the four cryptic European taxa of this subgenus, B. cryptarum, B. lucorum, B. magnus and B. terrestris, and used it to analyse 391 females of the former three species collected across Ireland, all of which could be unambiguously assigned to species. Bombus lucorum was the most widely distributed and abundant of the cryptarum–lucorum–magnus species complex, comprising 56% of individuals, though it was significantly less abundant at higher altitudes (>200 m) whilst B. cryptarum was relatively more abundant at higher altitudes. Bombus magnus was rarely encountered at urban sites. Both B. lucorum and B. terrestris are nowadays reared commercially for pollination and transported globally. Our RFLP approach to identify native fauna can underpin ecological studies of these important cryptic species as well as the impact of commercial bumble bees on them.     

Characterization of Neutral Lipase BT-1 Isolated from the Labial Gland of Bombus terrestris Males

Background

In addition to their general role in the hydrolysis of storage lipids, bumblebee lipases can participate in the biosynthesis of fatty acids that serve as precursors of pheromones used for sexual communication.

Results

We studied the temporal dynamics of lipolytic activity in crude extracts from the cephalic part of Bombus terrestris labial glands. Extracts from 3-day-old males displayed the highest lipolytic activity. The highest lipase gene expression level was observed in freshly emerged bumblebees, and both gene expression and lipase activity were lower in bumblebees older than 3 days. Lipase was purified from labial glands, further characterized and named as BT-1. The B. terrestris orthologue shares 88% sequence identity with B. impatiens lipase HA. The molecular weight of B. terrestris lipase BT-1 was approximately 30 kDa, the pH optimum was 8.3, and the temperature optimum was 50°C. Lipase BT-1 showed a notable preference for C8-C10 p-nitrophenyl esters, with the highest activity toward p-nitrophenyl caprylate (C8). The Michaelis constant (K_m) and maximum reaction rate (V_max) for p-nitrophenyl laurate hydrolysis were K_m = 0.0011 mM and V_max = 0.15 U/mg.

Conclusion

This is the first report describing neutral lipase from the labial gland of B. terrestris. Our findings help increase understanding of its possible function in the labial gland.     

Ultrastructure and Immunofluorescence of the midgut of Bombus morio (Hymenoptera: Apidae: Bombini)

Bumblebees are widely distributed across the world and have great economic and ecological importance as pollinators in the forest as well as in agriculture. The insect midgut consists of three cell types, which play various important roles in digestion, absorption, and hormone production. The present study characterized the anterior and posterior midgut regions of the bumblebee, Bombus morio. The digestive, regenerative and endocrine cells in the midgut showed regional differences in their number, nuclear size, as well as the size of the striated border. Ultrastructurally, the digestive cells contained many mitochondria and long microvilli; however, in the anterior midgut region, these cells showed dilated basal labyrinths with a few openings for the hemocoel, whereas the labyrinths of the basal posterior region remained inverse characteristics. Thus, the characterization of the midgut of B. morio supported an ecto-endoperitrophic circulation, contributing to a better understanding of the digestive process in this bee.     

Exocrine gland secretions of virgin queens of five bumblebee species (Hymenoptera: Apidae, Bombini)

Secretions of three different glands (mandibular gland, labial gland, and Dufour's gland) of virgin queens of five bumblebee species (Bombus lucorum, B. lapidarius, B. hypnorum, B. pascuorum, and B. terrestris) were analysed. Around 200 compounds were identified in the secretions. The compositions of the secretions of labial and mandibular glands were species-specific. Dufour's gland of all species produced mainly hydrocarbons, both saturated and unsaturated, the proportions of which differed quantitatively between the species studied.     

Tissue antigens from the third instar larva of Drosophila melanogaster

Antibodies were prepared against the soluble proteins from six tissues of Drosophila larvae. These were used to analyse the antigens in different tissues and at different developmental stages. The results suggest (1) the pattern of antigens determines the characteristics of a tissue, (2) salivary gland antigens are sequestered by the imaginal disks, (3) not all pupal glue antigens are synthesized in the salivary glands, and (4) most larval serum antigens are synthesized by the fat body.     

Biochemical evidence of DNA transport from the silk gland to the fat body of the silkworm, Bombyx mori

β-DNA, a component of DNA found in the pupal fat body of the silkworm, Bombyx mori, has the same GC content but a smaller molecular weight than typical silkworm DNA (α-DNA). Its origin and time of synthesis were studied by MAK column chromatography of phenol extracts after labelling with radioactive precursors.The DNA components of the fat body changed greatly during the early pupal stage, the β-DNA showing a striking increase relative to α-DNA. Thymidine-6-³H and phosphoric acid-³²P injected into the animals 1 day before analysis caused labelling of α-DNA, but not of β-DNA of the fat body, indicating that β-DNA was not synthesized during the stage of its appearance in the fat body.On the other hand, injection of thymidine-6-³H into 2-day-old fifth instar larvae, when DNA of the silk gland was being actively synthesized, gave high incorporation of the isotope into β-DNA of the pupal fat body. The sudden appearance of highly labelled β-DNA in the fat body during the early pupal stage as well as the occurrence of β-DNA in both the silk gland and fat body suggested that DNA might move from the silk gland to the fat body.It is possible that the fat body stores DNA as a nutrient from the degenerating silk gland.