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PubMed2005

Characterization of a D2-like dopamine receptor (AmDOP3) in honey bee, Apis mellifera.

Dopamine is an important neurotransmitter in vertebrate and invertebrate nervous systems and is widely distributed in the brain of the honey bee, Apis mellifera. We report here the functional characterization and cellular localization of the putative dopamine receptor gene, Amdop3, a cDNA clone isolated and identified in previous studies as AmBAR3 (Apis mellifera Biogenic Amine Receptor 3). The Amdop3 cDNA encodes a 694 amino acid protein, AmDOP3. Comparison of AmDOP3 to Drosophila melanogaster sequences indicates that it is orthologous to the D2-like dopamine receptor, DD2R. Using AmDOP3 receptors expressed in HEK293 cells we show that of the endogenous biogenic amines, dopamine is the most potent AmDOP3 agonist, and that activation of AmDOP3 receptors results in down regulation of intracellular levels of cAMP, a property characteristic of D2-like dopamine receptors. In situ hybridization reveals that Amdop3 is widely expressed in the brain but shows a pattern of expression that differs from that of either Amdop1 or Amdop2, both of which encode D1-like dopamine receptors. Nonetheless, overlaps in the distribution of cells expressing Amdop1, Amdop2 and Amdop3 mRNAs suggest the likelihood of D1:D2 receptor interactions in some cells, including subpopulations of mushroom body neurons.

Amino Acid Sequence↗
PubMed2005

Pteropsin: a vertebrate-like non-visual opsin expressed in the honey bee brain.

Insects have excellent color vision based on the expression of different opsins in specific sets of photoreceptive cells. Opsins are members of the rhodopsin superfamily of G-protein coupled receptors, and are transmembrane proteins found coupled to light-sensitive chromophores in animal photoreceptors. Diversification of opsins during animal evolution provided the basis for the development of wavelength-specific behavior and color vision, but with the exception of the recently discovered non-visual melanopsins, vertebrate and invertebrate opsins have generally been viewed as representing distinct lineages. We report a novel lineage of insect opsins, designated pteropsins. On the basis of sequence analysis and intron location, pteropsins are more closely related to vertebrate visual opsins than to invertebrate opsins. Of note is that the pteropsins are missing entirely from the genome of drosophilid flies. In situ hybridization studies of the honey bee, Apis mellifera, revealed that pteropsin is expressed in the brain of this species and not in either the simple or compound eyes. It was also possible, on the basis of in situ hybridization studies, to assign different long wavelength opsins to the compound eyes (AmLop1) and ocelli (AmLop2). Insect pteropsin might be orthologous to a ciliary opsin recently described from the annelid Platynereis, and therefore represents the presence of this vertebrate-like light-detecting system in insects.

Animals↗
PubMed2005

Bee venom modulates murine Th1/Th2 lineage development.

Administration of bee venom (BV) elicits anti-inflammatory, anti-nociceptive and anti-allergic effects in various animal models. This study was designed to evaluate the direct effects of BV on helper T cell activities and on Th1/Th2 lineage development using both in vitro and in vivo conditions. In the Th1 skewed condition, BV increased the expression of IFN-gamma mRNA and enhanced the expression of T-bet on purified CD4(+) T cells from splenocytes of BALB/c mice. On the other hand, BV treatment did not alter the expression of IL-4 or GATA-3 in a Th2 driven environment. To elucidate the effects of BV on Th1/Th2 lineage development under in vivo conditions, BV was given by intraperitonial injection to BALB/c mice. It significantly increased the CD4(+) T cell population and enhanced IFN-gamma expression, while IL-4 transcripts were not altered upon in vivo activation using an anti-CD3 antibody injection. Taken together, these results imply that BV induces Th1 lineage development from CD4(+) T cells by increasing the expression of a Th1-specific cytokine, IFN-gamma. In addition, this result may be mediated by inducing a Th1-specific transcription factor, T-bet.

Animals↗
PubMed2006

Key regulators in bee venom-induced apoptosis are Bcl-2 and caspase-3 in human leukemic U937 cells through downregulation of ERK and Akt.

Bee venom (BV) has been known to inhibit proliferation and induce apoptosis in cancer cells. However, the molecular mechanisms involved in BV-induced apoptosis are still uncharacterized in human leukemic cells. In the present study, we report that BV induces apoptosis in leukemic U937 cells through downregulation of ERK and Akt signal pathway. Furthermore, BV-induced apoptosis was accompanied by downregulation of Bcl-2, activation of caspase-3 and a subsequent poly(ADP-ribose)polymerase (PARP) cleavages. The induction of apoptosis also was accompanied by the downregulation of the inhibitor of apoptosis protein (IAP) family proteins. Caspase-3 inhibitor, z-DEVD-fmk, was significantly capable of restoring cell viability and BV-induced apoptosis through caspase-3 activation was significantly attenuated in Bcl-2-overexpressing cells. These results indicate that downregulation of Bcl-2 plays a major role in the initiation as an activator of a caspase-3 involved with BV-induced apoptosis. BV also triggered the activation of p38 MAPK and JNK, and downregulation of ERK and Akt. PD98059 (an inhibitor of ERK) or LY294002 (an inhibitor of Akt), but not an inhibitor of p38 MAPK and JNK, significantly decreased cell viability and increased lactate dehydrogenase (LDH) release. The results indicated that key regulators in BV-induced apoptosis are Bcl-2 and caspase-3 in human leukemic U937 cells through downregulation of the ERK and Akt signal pathway.

Animals↗
PubMed2005

Effects of bee venom on the pro-inflammatory responses in RAW264.7 macrophage cell line.

The purpose of this study is to elucidate the molecular mechanism of anti-inflammatory effect of bee venom (BV), which has been used for the treatment of various inflammatory diseases in oriental medicine. With this aim, we examined the effects of BV on the nitric oxide (NO) production by lipopolysaccharide (LPS) or sodium nitroprusside in RAW264.7 macrophages. We further investigated the effects of BV on the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) with RT-PCR in LPS-stimulated RAW264.7 cells. BV suppressed the NO production and decreased the levels of iNOS, COX-2, NF-kappaB and MAPK mRNA in a dose-dependent manner. These results suggest that BV has an anti-inflammatory effect by inhibiting iNOS and COX-2 expression, possibly through suppression of NF-kappaB and MAPK expression.

Animals↗
PubMed2005

Temporal variation in overwintering gene expression and respiration in the solitary bee Megachile rotundata.

Partial clones of Megachile rotundata HSP90, HSP70, HSC70 and actin were developed by RT-PCR. These clones were used to generate probes to screen for the expression of their respective transcripts in heat-shocked pupae and in diapausing prepupae through post-diapausing pupae. Northern blot analysis revealed transcript sizes for MrHSP90, MrHSP70, MrHSC70, and Mractin of 3.6, 2.3, 2.5, and 1.4kb, respectively. MrHSP90 and MrHSP70 were highly upregulated in post-diapausing pupae exposed to 40 degrees C for 1h, while MrHSC70 was only slightly induced by heat shock. Levels of MrHSC70 and MrHSP90 showed little change between field collected diapausing prepupae and post-diapausing pupae. In contrast, MrHSP70 was highly upregulated in diapausing prepupae and Mractin was at or below the level of detection in diapausing prepupae. Transferring field reared overwintering prepupae in February to 25 degrees C for 3 days induced an expression pattern of MrHSP70 and Mractin more typical of post-diapausing bees, indicating the likelihood that the transition to post-diapause development had occurred prior to February. However, measurements revealed possible cyclic respiration patterns, including low respiratory quotient (RQ) values during February and March and a transition during April to more continuous respiration with elevated RQ values.

Animals↗
PubMed2005

Reproductive division of labor, dominance, and ecdysteroid levels in hemolymph and ovary of the bumble bee Bombus terrestris.

To determine whether ecdysteroids are associated with reproductive division of labor in Bombus terrestris, we measured their levels in hemolymph and ovaries of queens and workers. Queens heading colonies had large active ovaries with high ecdysteroid content, whereas virgin gynes and mated queens before and after diapause had undeveloped ovaries with low ecdysteroid content. The hemolymph ecdysteroid titer was rather variable, but in a pooled analysis of mated queens before and after diapause versus colony-heading queens, ecdysteroid titers were higher in the latter group. In workers, agonistic behavior, ovarian activity, ovarian ecdysteroid content, and hemolymph ecdysteroid titers were positively correlated, and were lowest when a queen was present. In queenless workers, ecdysteroid levels were elevated in dominant workers, and were also influenced by the presence of brood and by group demography; hormone levels were higher in bees kept in larger groups. These findings are consistent with the premise that in B. terrestris the ovary is the primary site of ecdysteroid synthesis, and they show that ecdysteroids levels vary with the social environment.

Animals↗
PubMed2004

In vivo dynamics of an immune response in the bumble bee Bombus terrestris.

Concepts from evolutionary ecology have recently been applied to questions of immune defences. However, an important but often neglected aspect is the temporal dynamics of the simple immune measures used in ecological studies. Here, we present observations for workers of the bumble bee Bombus terrestris on the dynamics of the phenoloxidase (PO) system, antibacterial activity, and the total number of haemocytes following a challenge with immune elicitors (LPS, Laminarin), over a time-span ranging from 1min to 14 days. The dynamics of the PO measurement showed a complex pattern and was correlated with haemocyte counts. Antibacterial activity, on the other hand, increased sharply between 2 and 24h post-challenge followed by a slow decrease. Surprisingly, the effects of a challenge lasted up to 14 days.

Adjuvants, Immunologic↗
PubMed2007

Development of a multiplex RT-PCR for the simultaneous detection of three viruses of the honeybee (Apis mellifera L.): acute bee paralysis virus, Black queen cell virus and Sacbrood virus.

A single-step multiple-target (multiplex) reverse transcription-PCR (RT-PCR) was developed for the simultaneous detection and differentiation of three economically important viruses of the honeybee Apis mellifera L.: Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV) and Sacbrood virus (SBV). Three compatible sets of primers, specific for each virus, were designed in conserved regions of the viral genomes for use in a one-step (single tube) RT-PCR assay. The individual RT-PCR assays and the combined multiplex assay were optimized for highest sensitivity and specificity. The multiplex RT-PCR assay was tested on field samples collected from Austrian honeybee colonies. All three viruses were detected, and their identity was confirmed by sequencing of the PCR products. The described multiplex RT-PCR proved to be an accurate tool for rapid simultaneous detection of ABPV, BQCV and SBV directly in honeybee specimens.

Animals↗
PubMed2004

Acupoint stimulation with diluted bee venom (apipuncture) alleviates thermal hyperalgesia in a rodent neuropathic pain model: involvement of spinal alpha 2-adrenoceptors.

UNLABELLED: Chemical acupuncture with diluted bee venom (DBV), termed apipuncture, has been traditionally used in oriental medicine to treat several inflammatory diseases and chronic pain conditions. In the present study we investigated the potential antihyperalgesic and antiallodynic effects of apipuncture in a rat neuropathic pain model. DBV (0.25 mg/kg, subcutaneous) was injected into the Zusanli acupoint 2 weeks after chronic constrictive injury (CCI) of the sciatic nerve. Between 5 and 45 minutes after DBV injection, we observed a significant reduction in the thermal hyperalgesia induced by CCI, but apipuncture failed to reduce CCI-induced mechanical allodynia. We subsequently examined whether this antihyperalgesic effect of apipuncture was related to the activation of spinal opioid receptors and/or alpha2-adrenoceptors. Intrathecal pretreatment with naloxone (10 microg/rat), an opioid receptor antagonist, did not reverse the antihyperalgesic effect of apipuncture, whereas pretreatment with idazoxan (40 microg/rat), an alpha2-adrenoceptor antagonist, completely blocked the effect of apipuncture. These results indicate that DBV-induced apipuncture significantly reduces the thermal hyperalgesia generated by CCI and also suggest that this antihyperalgesic effect is dependent on the activation of alpha2-adrenoceptors, but not opioid receptors, in the spinal cord. PERSPECTIVE: The antinociceptive effect of apipuncture was evaluated in a rodent neuropathic pain model. The relieving effect of apipuncture on thermal hyperalgesia was found to be mediated by spinal alpha2-adrenoceptors, but not opioid receptors. These data suggest that apipuncture might be an effective alternative therapy for patients with painful peripheral neuropathy, especially for those who are poorly responsive to opioid analgesics.

Acupuncture Points↗
PubMed2006

Bee venom injection significantly reduces nociceptive behavior in the mouse formalin test via capsaicin-insensitive afferents.

UNLABELLED: Peripheral bee venom (BV) administration produces 2 contrasting effects, nociception and antinociception. This study was designed to evaluate whether the initial nociceptive effect induced by BV injection into the Zusanli acupoint is involved in producing the more prolonged antinociceptive effect observed in the mouse formalin test, and whether capsaicin-sensitive primary afferents are involved in these effects. BV injection into the Zusanli point increased spinal Fos expression but not spontaneous nociceptive behavior. BV pretreatment 10 minutes before intraplantar formalin injection dose-dependently attenuated nociceptive behavior associated with the second phase of the formalin test. The destruction of capsaicin-sensitive primary afferents by resiniferatoxin (RTX) pretreatment selectively decreased BV-induced spinal Fos expression but did not affect BV-induced antinociception. Furthermore, BV injection increased Fos expression in tyrosine hydroxylase immunoreactive neurons in the locus caeruleus, and this expression was unaltered by RTX pretreatment. Finally, BV's antinociception was blocked by intrathecal injection of 10 microg idazoxan, and this effect was not modified by RTX pretreatment. These findings suggest that subcutaneous BV stimulation of the Zusanli point activates central catecholaminergic neurons via capsaicin-insensitive afferent fibers without induction of nociceptive behavior. This in turn leads to the activation of spinal alpha2-adrenoceptors, which ultimately reduces formalin-evoked nociceptive behaviors. PERSPECTIVE: This study demonstrates that BV acupuncture produces a significant antinociception without nociceptive behavior in rodents, which is mediated by capsaicin-insensitive afferents and involves activation of central adrenergic circuits. These results further suggest that BV stimulation into this acupuncture point might be a valuable alternative to traditional electrical or mechanical acupoint stimulation.

Acupuncture Analgesia↗
PubMed2004

Changes of 5-HT receptor subtype mRNAs in rat dorsal root ganglion by bee venom-induced inflammatory pain.

The reverse transcriptase polymerase chain reaction (RT-PCR) technique was used to examine the changes of the expression of 5-hydroxytryptamine (5-HT) receptors in the rat lumbar dorsal root ganglion (DRG) following subcutaneous bee venom (BV) injection into the plantar surface of the unilateral hindpaw. In the DRG ipsilateral to the BV injection, significant increase of mRNA levels of 5-HT(1A), 5-HT(1B), 5-HT(2A) and 5-HT(3) receptor subtypes were observed at 1 and 4h after the BV injection, while increase of 5-HT(2C), 5-HT(4), 5-HT(6) and 5-HT(7) receptor subtype mRNAs was detected at 4h only. No such changes were observed in the expressions of 5-HT(1D), 5-HT(1F) and 5-HT(5A) receptor subtype mRNAs. Upregulation of 5-HT(1A), 5-HT(1B) and 5-HT(2A) receptor subtype mRNAs was also observed in the contralateral DRG at 4 h. The presence of 5-HT(1E), 5-HT(2B) and 5-HT(5B) receptor subtype mRNAs was not detected in the rat DRG. The present results suggest that different sets of 5-HT receptor subtypes work at different stages of the inflammatory pain induced by subcutaneous BV injection.

Animals↗
PubMed2006

Substantial role of locus coeruleus-noradrenergic activation and capsaicin-insensitive primary afferent fibers in bee venom's anti-inflammatory effect.

Several lines of evidence indicate significant interactions between the immune and nervous systems. Our recent study reveals that 'bee venom (BV) induced anti-inflammatory effect' (BVAI) was produced by sympathetic preganglionic neuronal activation and subsequent adrenomedullary catecholamine release in a zymosan-induced inflammation model. However, the specific peripheral input and the supraspinal neuronal systems that are involved in this BVAI remain to be defined. Here we show that subcutaneous BV injection into left hind limb significantly reduces zymosan-induced leukocyte migration and that this effect is completely inhibited by denervation of the left sciatic nerve. This BVAI was not affected by the destruction of capsaicin-sensitive primary afferent fibers using either neonatal capsaicin or resiniferatoxin (RTX) pretreatment. BV injection into the left hind limb significantly increased Fos expression in the contralateral locus coeruleus (LC) in non-inflamed mice. In zymosan-inflamed mice, BV injection produced a further increase in LC Fos expression as compared with non-inflamed mice. This BV-induced Fos increase in the LC was not affected by RTX pretreatment. Pharmacological blockage of central noradrenergic activity by either central chemical sympathectomy (i.c.v. 6-hydroxydopamine) or alpha2 adrenoceptor antagonism (i.c.v. idazoxan) completely blocked BVAI. Taken together, these results suggest that BVAI is mediated by peripheral activation of capsaicin-insensitive primary afferent fibers and subsequent central noradrenergic activation including the LC.

Adrenergic Agents↗
PubMed2004

Evaluation of atlas selection strategies for atlas-based image segmentation with application to confocal microscopy images of bee brains.

This paper evaluates strategies for atlas selection in atlas-based segmentation of three-dimensional biomedical images. Segmentation by intensity-based nonrigid registration to atlas images is applied to confocal microscopy images acquired from the brains of 20 bees. This paper evaluates and compares four different approaches for atlas image selection: registration to an individual atlas image (IND), registration to an average-shape atlas image (AVG), registration to the most similar image from a database of individual atlas images (SIM), and registration to all images from a database of individual atlas images with subsequent multi-classifier decision fusion (MUL). The MUL strategy is a novel application of multi-classifier techniques, which are common in pattern recognition, to atlas-based segmentation. For each atlas selection strategy, the segmentation performance of the algorithm was quantified by the similarity index (SI) between the automatic segmentation result and a manually generated gold standard. The best segmentation accuracy was achieved using the MUL paradigm, which resulted in a mean similarity index value between manual and automatic segmentation of 0.86 (AVG, 0.84; SIM, 0.82; IND, 0.81). The superiority of the MUL strategy over the other three methods is statistically significant (two-sided paired t test, P < 0.001). Both the MUL and AVG strategies performed better than the best possible SIM and IND strategies with optimal a posteriori atlas selection (mean similarity index for optimal SIM, 0.83; for optimal IND, 0.81). Our findings show that atlas selection is an important issue in atlas-based segmentation and that, in particular, multi-classifier techniques can substantially increase the segmentation accuracy.

Algorithms↗
PubMed2006

Effects of caffeine on olfactory and visual learning in the honey bee (Apis mellifera).

Although caffeine is known to improve alertness and arousal in humans and other mammals, its impacts on specific behaviours, including complex cognitive processes, remain controversial. We reasoned that the availability of an easily manipulable, but behaviourally complex invertebrate organism with a simpler nervous system would be beneficial to this field of research. We used a popular behavioural model, the honeybee, to evaluate the effects of caffeine on (1) the development of olfactory learning and (2) the performance in complex learning paradigms, including a 'delayed-match-to-sample' task and visual associative learning. To evaluate the efficacy of caffeine treatment, a variety of doses (0.4-400 ng/1 mg of body mass) were applied topically to tethered individuals. Behavioural testing was performed with either tethered or free-flying adult honeybees. We show that caffeine has marked cognitive effects in this species. In young honeybees, it reduces the age at which restrained individuals are able to learn an olfactory associative task, whereas in older, free-flying bees, caffeine improves both motivation and cognitive performance in complex learning tasks. Our results suggest that the honeybee model may be useful in explaining caffeine-related behavioural changes not only in this species, but also in mammalian systems.

Animals↗
PubMed2004

Structure and ultrastructure of spermatozoa in Meliponini (stingless bees) (Hymenoptera: Apidae).

In spite of their importance in the reproduction of phanerograms, few bees have received an adequate description of their sperm ultrastructure. In this study, we concluded that it was possible to define a characteristic pattern for Meliponini spermatozoa. However, we have also found species-specific differences that could be used, for example, in a taxonomic or phylogenetic evaluation. Meliponini spermatozoa are made up of a head and a flagellar region. The head includes an acrosome containing the perforatorium, covered by the acrosomal vesicle and a nucleus. In transverse sections, the acrosome is circular at the tip but becomes triangular as it nears the nucleus. The perforatorium base penetrates into a small cavity in the nuclear tip. The flagellum consists of an axoneme, a pair of mitochondrial derivatives, a centriolar adjunct and a pair of accessory bodies. The axoneme has a typical 9+9+2 microtubule pattern. In the final portion, the axoneme becomes gradually disorganized with the accessory microtubules terminating last. The mitochondrial derivatives are asymmetric in length and diameter. In cross-section, they are ellipsoidal and the larger one has a paracrystalline region. The centriolar adjunct begins at the nuclear base and extends parallel to the axoneme up to the anterior end of the smaller mitochondrial derivative.

Acrosome↗
PubMed2006

Effects of bee venom on protease activities and free radical damages in synovial fluid from type II collagen-induced rheumatoid arthritis rats.

The effect of bee venom acupuncture (BVA) (api-toxin) on the development of type II collagen (CII)-induced arthritis (CIA) in rats has been studied. We have compared the levels of activity of a comprehensive range of cytoplasmic, lysosomal and matrix protease types, together with the levels of free radical-induced protein damage (determined as protein carbonyl derivative) in synovial fluid from CIA-treated, BVA-treated and normal rats. Many protease types showed significantly increased activity in CIA compared with normal rats. BVA (5 and 10 microl/100g) significantly reduced these enzyme activities by some 80% each, but levels of plasma proteases activity (including those enzyme types putatively involved in the immune response, such as dipeptidyl aminopeptidase IV and proline endopeptidase) in CIA, BVA (5 microl/100g)-treated and normal plasma samples were not significantly different. The level of free radical induced damage to synovial fluid proteins was approximately three-fold higher in CIA compared with normal rats. However, BVA (5 microl/100g) significantly decreased the level of reactive oxygen free radical species (ROS) induced oxidative damage to synovial fluid proteins. It was concluded that activation of proteolytic enzymes and free radicals are likely to be of equal potential importance as protein damaging agents in the pathogenesis of rheumatoid arthritis (RA), and the development of novel therapeutic strategies for the latter disorder should include both protease inhibitory and free radical scavenging elements. In addition, the protease inhibitory element should be designed to inhibit the action of a broad range of enzymatic mechanistic types (cysteine, serine, metallo proteinases and peptidases). In conclusion, BVA is considered to be an effective RA modulator, inhibiting protease activities and removing ROS.

Acupuncture↗
PubMed2005

Microarray analysis of gene expression in chondrosarcoma cells treated with bee venom.

Bee venom (BV) has a broad array of clinical applications in Korean medicine, including treatment of inflammatory conditions such as arthritis. The final common pathway of many arthropathies is the destruction of articular cartilage and consequent loss of articular function. Chondrocyte dysfunction plays a key role in the pathogenesis of such disorders. To explore the global gene expression profiles in a human chondrocyte-like cell line treated with BV, microarray analysis was performed. The HTB-94 human chondrosarcoma cells were treated with BV, lipopolysaccharide (LPS), or both. Of the 344 genes profiled in this study, with a cut-off level of 4-fold change in the expression, (1) 35 were downregulated following BV treatment, (2) 16 were upregulated and 7 downregulated following LPS treatment, and (3) 32 were downregulated following co-stimulation of BV and LPS. The results of the present study shows that treatment of BV reversed the LPS-induced upregulation of such genes as interleukin-6 (IL-6) receptor, matrix metalloproteinase 15 (MMP-15), tumor necrosis factor (ligand) superfamily-10, caspase-6 and tissue inhibitor of metalloproteinase-1 (TIMP-1). It is thought that microarrays will play an ever-growing role in the advance of our understanding of the pharmacologic actions of BV in the treatment of arthritis.

Bee Venoms↗