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High-fat diet (HFD) often causes obesity and it has detrimental effects on the sensory system. In particular, sensory-mediated responses are crucial for maintaining energy balance, as they are involved in a metabolic regulation, however, there is still no clear explanation about the relationship between HFD-induced stress and sensory system. To gain insight on how HFD-induced stress affects olfactory sensitivity and behavioral responses, we have used a Drosophila melanogaster model for olfactory and nutrient-related signaling and accessed physiological, behavioral, and transcriptional changes. We demonstrated that lifespan and climbing ability in HFD-treated flies decreased and that olfactory sensitivity and behavioral responses to odorants were changed. Olfactory sensitivity to eight of ten odorants after 14 days on HFD treatment were reduced, while behavioral attraction was increased to benzaldehyde in flies that were treated with HFD. This behavioral and physiological modification in HFD-treated flies for 14 days was accompanied by a significant decrease in DmOrco gene expression in a peripheral olfactory organ, suggesting that is could be involved in the action of metabolic and sensory signal. Gene expression profiles of antennae showed significant differences on the olfactory receptors, odorant-binding proteins, and insulin signaling. Our results suggested that olfactory sensitivity and behavioral responses to HFD-induced stress are mediated through olfactory and nutrient-related signaling pathways.

International audience; The geographic relationships and biogeography of Apis cerana have been studied extensively, but Korean populations have not been investigated thoroughly. We sequenced the non-coding region between the tRNALeu and COII mitochondrial (mt) genes (herein named NC2) of Korean samples, along with the samples from seven Asian localities (China, Vietnam, and Thailand). Four undiscovered haplotypes were found in Korea and China, respectively. A phylogenetic analysis confirmed that Korean A. cerana belonged to the Mainland Asian group. Dominance of Japan1 haplotype in Mainland Asia including Korea suggests extensive gene flow and a common genetic origin. A newly developed non-coding region between the tRNAMet and tRNAGln mt genes (named NC1) provided nine haplotypes with twice the number of variable positions compared to those in NC2. A NC1-based phylogenetic analysis revealed the presence of two phylogenetic groups in Apis cerana from Korea suggesting two different sources.

The sense of taste is responsible for the choice of profitable food sources and nest mate recognition in honeybees. Taste detection for food sources occurs within cuticular hairs located on the antennae, on the mouthparts, and on the tarsi of the forelegs. The gustatory sensilla, which are composed of cuticular hair, respond to sugars, salts, and amino acids. In the honey bee, although sugar detection is a crucial factor in determining the acceptability of nectar and pollen for collection, little is known about the molecular and neural correlates underlying sugar perception. Here we identified the function of sugar receptors in Apis mellifera that is most closely related with sugar receptors in Drosophila melanogaster such as DmGr64a-f and DmGr5a. We determined that gustatory receptor 1 of A. mellifera (AmGr1) responded to sucrose, glucose, trehalose, and maltose in a dose-dependent manner, and that the expression patterns of AmGr1 and AmGr2 are distinct from those in Drosophila. AmGr1 alone showed full functionality, but it showed different sensitivity from a heterodimer of AmGr1 and AmGr2. That is, co-expression of AmGr1 and AmGr2 demonstrated higher sensitivity to glucose and lower sensitivity to sucrose, trehalose, and maltose compared with AmGr1 expression alone. AmGr1 and AmGr2 were co-localized or not in the antennal neurons, and especially AmGr1 was highly expressed at the distal segment of the antennae. Our study suggests that sugar receptors of the honey bee function as heterodimers (or monomer or mono-dimers), indicating that AmGr2 is required for providing honeybees with variability of sugar perceptions.

Widespread concern for the occurrence of resistant strains, along with the avoidance of the use of highly toxic insecticides and their wide environmental dispersal, highlights the need for the development of new and safer pest control agents. Natural products provide inspiration for new chemical entities with biological activities, and their analogues are good lead compounds for the development of new pest control agents. For this purpose, we evaluated the larvicidal and nematicidal activities of 48 3-acylbarbituric acid analogues against the Asian tiger mosquito, Aedes albopictus and the pine wood nematode, Bursaphelenchus xylophilus, organisms of increasing global concern. Among the 48 3-acylbarbituric acid analogues, four compounds—10, 14d, 14g and 19b—showed >90% larvicidal activity against Ae. albopictus at 10 μg/mL concentration, and one (compound 10) showed the strongest larvicidal activity against Ae. albopictus, with a LC50 value of 0.22 μg/mL. Only compound 18 showed strong nematicidal activity against pine wood nematode. Most active compounds possessed similar physicochemical properties; thus, actives typically had ClogP values of around 1.40–1.50 and rel-PSA values of 16–17% and these similar cheminformatic characteristics reflect their similar structure. This study indicates that active 3-acylbarbituric acids analogues have potential as lead compounds for developing novel mosquito control agents.

Olfactory cues are important sensory modalities on individual discrimination, perception, and efficient orientation to food sources in most insects. In honey bees, which are well known as eusocial insects, olfactory cues are mainly used to maintain a colony. Although much research has been reported on olfactory systems in honey bee olfaction, little is known about the differences between two major honey bee species, the European honey bee Apis mellifera and the Asian honey bee Apis cerana. In order to understand the differences of olfactory characteristics in the two species, we compared the distribution of sensory hairs on the antennae and antennal olfactory responses, using electron microscopy, electrophysiological recording and molecular expression level of odorant receptors. Our present study demonstrated that the antennae of A. cerana have more olfactory sensilla than A. mellifera, responding more strongly to various floral volatile compounds. At the molecular level, olfactory co-receptor (Orco), which makes heterodimers with other conventional olfactory receptors, is more abundantly expressed in the antenna of A. cerana than of A. mellifera. These findings extend our understanding of the olfactory systems and behavioral responses to various ecological and biological signals in two closely related honey bee species.

BACKGROUND: Insect olfactory organs possess many olfactory receptor neurons, which detect many different sets of odorants in nature. In order to feed on blood meals, stableflies locate host animals and humans using chemical cues such as 1-octen-3-ol andbutyricacid.Inthepresentstudy,behaviouralandelectroantennogram(EAG)responsepatternstorepellentvolatilesfrom essential oils (EOs) of Zanthoxylumpiperitum and Z.armatum in combination with the attractants were investigated. RESULTS: Components of the EOs such as cuminaldehyde, citronellal, neral, linalool, linalool oxide, terpinen-4-ol, 1,8-cineole, and piperitone induced remarkable repellent behaviours in the stable fly. EAG responses in the fly antenna to these chemicals showed a dose-dependent manner. The patterns of behavioural and EAG responses were significantly altered depending on the ratios of 1-octen-3-ol or butyric acid to the EOs or compounds in the air mixtures. CONCLUSION:ThepresentstudydemonstratedthattheZanthoxylumEOsdecreasedthelevelsofresponseofflightbehaviours of the stable fly towards host volatile compounds. The combinations of odorant mixtures of the attractants with the EOs and their components affect the representation of behavioural and EAG responses of the flies. The summation and integration patterns of olfactory responses measured by the EAG indicated that the peripheral olfactory networks in antennae could process the odorant complexity of different odorant mixtures between attractants and repellents. c � 2013 Society of Chemical Industry

Background Propolis (or bee glue), collected from botanical sources by honey bee, has been used as a popular natural remedies in folk medicine throughout the world. This study was conducted to assess growth inhibitory effects of ethanol extracts of propolis (EEPs) from 20 different regions in South Korea on human intestinal bacteria as well as their human β-amyloid precursor cleavage enzyme (BACE-1), acetylcholinesterase (AChE) inhibitory, antioxidant, antiproliferative, and anti-human rhinovirus activities. Methods The Bonferroni multiple-comparison method was used to test for significant differences in total polyphenol and flavonoid contents among EEP samples using SAS 9.13 program. Correlation coefficient (r) analysis of the biological activities of EEP samples was determined using their 50 % inhibition concentration or minimal inhibitory concentration values and their polyphenol or flavonoid contents in 20 native Korean EEP samples. Results The amounts of total polyphenol and flavonoids in the Korean EEP samples ranged from 49 to 239 mg gallic acid equivalent (GAE)/g EEP (Brazilian, Chinese, and Australian samples, 127–142 mg GAE/g EEP) and from 21 to 50 mg quercetin equivalent (QE)/g EEP (Brazilian, Chinese, and Australian samples, 33–53 mg QE/g EEP), respectively. Correlation coefficient analysis showed that total polyphenol contents may be negatively correlated with 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity (r = −0.872) and total flavonoid content has no correlation with the activity (r = 0.071). No direct correlation between BACE-1 inhibition, AChE inhibition, or antiproliferative activity and total polyphenol or total flavonoid content in Korean EEP samples was found. Gram-positive and Gram-negative bacteria were observed to have different degrees of antimicrobial susceptibility to the EEP samples examined, although ciprofloxacin susceptibility among the bacterial groups did not differ greatly. Conclusions Further studies will warrant possible applications of propolis as potential therapeutic BACE-1 blocker, antioxidant, antiproliferative agent, and antimicrobial agent.

Environmental toxicants like paraquat (PQ) induce the increase of oxidative stress, which is likely to lead to various neuropathological symptoms. Although the Drosophila system is applicable for anti-oxidative and neuroprotective drug discovery, this system remains to be more exploited in various aspects. In this study, using Drosophila as a model animal, we have examined the anti-oxidative and neuroprotective properties of plant-derived compounds against oxidative damage and neurotoxicity induced by paraquat, which is a widely used herbicide. In this study, paraquat showed LC 50 at 24.7 mM to adult males of Drosophila melanogaster within 24 h. Dietary feeding of curcumin, quercetin, Sanguisorba officinalis , and Zedoariae rhizoma extracts prior to paraquat exposure extended lifespan and enhanced motor activities of flies. These compounds modulated the expression level of several genes associated with anti-oxidative and anti-aging effects such as sod1 , sod2 , cat , gstD1 , and mth genes. Also, same treatments of phytochemicals to flies ameliorated other oxidative stress index factors such as ROS levels and superoxide dismutase. In contrast, no significant effects on catalase activities were observed. Additionally, the dietary feeding of phytochemical substances also reduced acetylcholine esterase activities, which were dramatically increased by paraquat treatment, implying that these phytochemicals also affected neuronal systems. Present study demonstrates that the dietary feeding of phytochemicals prior to paraquat exposure has anti-oxidative and neural protective effects, which leads to the recovery of behaviors and lifespan in fruit flies.

In all triplicate tests of six plant essential oils and of vanillin mixtures, we corroborated strong insecticidal and repellent activities against adult Aedes aegypti (L.). Essential oils with potent toxic fumigant activities also exhibited repellency. Compared with N, N-diethyl-3-methylbenzamide, 5% of the essential oil concentrations of cassia, lemongrass, lemon eucalyptus, and xanthoxylum oils did not show repellent effects. However, a composition oflemongrass oil, xanthoxylum oil, and vanillin (1:3:1, vol:vol:wt) provided 270 min of complete protection time (CPT) compared with 247.5 min CPT with 15% N, N-diethyl-3-methylbenzamide. The CPT depended on concentration, presence ofvanillin, or on both factors. When we applied a mixture of lemongrass oil: xanthoxylum oil: vanillin (1:1:1, vol:vol:wt) to the Viscopearl formulation, or porous cellulose beads, it provided gradual release of volatile compounds, thus showing >90% of repellency for 2 h. The behavioral and electrophysiological approaches we drew upon in our current study demonstrated that plant essential oil mixtures combined with vanillin showed strong and durable repellency to the mosquito. We claim that such combinations of plant essential oils and vanillin found in current study propose a viable commercial product suitable for future application in protecting a person from mosquito bites.

The toxicity of pellitorine alone or in combination with (-)-asarinin, alpha-asarone, methyleugenol, or pentadecane (1:1, 1:2, 1:3, 2:1, and 3:1 ratios) to third instars from an insecticide-susceptible KS-CP strain and -resistant DJ-CP colony of Culex pipiens pallens Coquillett was evaluated using a direct-contact mortality bioassay. The binary mixture of pellitorine and (-)-asarinin (3:1 ratio) was significantly more toxic against KS-CP larvae (0.95 mg/liter) and DJ-CP larvae (1.07 mg/liter) than either pellitorine (2.08 mg/liter for KS-CP and 2.33 mg/liter for DJ-CP) or (-)-asarinin (11.45 and 12.61 mg/liter) alone. The toxicity of the other binary mixtures (1:1, 1:2, 1:3, and 2:1 ratios) and pellitorine did not differ significantly from each other. Based on the co-toxicity coefficient (CC) and synergistic factor (SF), the three binary mixtures (1:3, 2:1, and 3:1) operated synergistically (CC, 250-390 and SF, 1.4-2.2 for KS-CP; CC, 257-279 and SF, 1.1-2.1 for DJ-CP). The binary mixtures of pellitorine and (-)-asarinin merit further study as potential larvicides for the control of insecticide-resistant mosquito populations.

The fruit fly, Drosophila melanogaster, has been intensively used as a genetic model system for basic and applied research on human neurological diseases because of advantages over mammalian model systems such as ease of laboratory maintenance and genetic manipulations. Disease-associated gene mutations, whether endogenous or transgenically-inserted, often cause phenotypes in vivo that are similar to the clinical features of the human disorder. The Drosophila genome is simpler than that of mammals, in terms of gene and chromosome number, but nonetheless demonstrates extraordinary phylogenetic conservation of gene structure and function, especially notable among the genes whose mutations cause neurodevelopmental, neuropsychiatric, or neurodegenerative disorders. In addition, its well-established neuroanatomical, developmental, and molecular genetic research techniques allow many laboratories worldwide to study complex biological and genetic processes. Based on these merits of the Drosophila model system, it has been used for screening lifespan expansion and neuroprotective activities of plant extracts or their secondary metabolites to counteract pathological events such as mitochondrial damage by oxidative stress, which may cause sporadic neurodegenerative diseases. In this review, we have summarized that the fruit fly can be used for early-stage drug discovery and development to identify novel plant-derived compounds to protect against neurodegeneration in Alzheimer's disease and Parkinson's disease, and other neurological disorders caused by oxidative stress. Thus, the Drosophila system can directly or indirectly contribute to translational research for new therapeutic strategies to prevent or ameliorate neurodegenerative diseases.

The generation of various subtypes of neurons and glial cells at the right time and place is crucial for the proper development of the vertebrate CNS. Although the mechanisms and factors for the regulation of neuronal diversity in the CNS have been well studied, the mechanisms regulating the sequential production of neuronal and glial cells from neural precursors remain poorly understood. This study shows that Tcf3, a member of the Lef/Tcf family of proteins, is required to inhibit the premature oligodendroglial fate specification of spinal cord precursors using the transgenic zebrafish, which expresses a dominant repressor form of Tcf3 under the control of a heat-shock inducible promoter. In addition, the data revealed that Tcf3 function in oligodendroglial fate specification is mediated independently of canonical Wnt signaling. Altogether, these results show a novel function for Tcf3 in regulating the timing of oligodendroglial fate specification in the spinal cord.

The repellency of Zanthoxylum armatum seed oil (ZA-SO), alone or in combination with vanillin (VA), its six major constituents, and another four major previously known Zanthoxylum piperitum fruit oil constituents, as well as aerosol products containing 5 or 10% ZA-SO and 5% VA, was evaluated against female Aedes aegypti in laboratory and field studies. Results were then compared with those of N,N-diethyl-3-methylbenzamide (DEET) as a standard. Hand in cage laboratory tests showed that 0.2, 0.1, and 0.05 mg/cm2 ZA-SO resulted in > 92% protection through 30-min postexposure and was not significantly different than 0.05 mg/cm2 DEET. Skin treated with linalool and limonene (from Z. armatum) provided > 80% repellency to female Ae. aegypti at 10-min exposure, whereas cuminaldehyde, citronellal, geranyl acetate, and cuminyl alcohol (from Zanthoxylum piperitum) provided > 90% protection during this same time period. Only cuminaldehyde and citronellal provided complete protection comparable to DEET at 10-min postexposure. After that time, repellency of all plant constituents to mosquitoes was considerably decreased (< approximately 65%). An increase in repellency and duration of effectiveness was produced by a binary 1:4 mixture of ZA-SO and VA (0.05:0.2 mg/cm2) that was significantly more effective than 0.05 mg/cm2 DEET through 90 min. In field tests, an aerosol formulation containing 5 or 10% ZA-SO plus 5% VA gave 100% repellency at 60-min postexposure. Although these formulations were equal to the level of protection afforded by 10% DEET, repellency to the binary ZA-SO aerosol formulations at 90 min was significantly less effective than DEET. However, mixtures formulated from ZA-SO and VA merit further study as potential repellents for protection of humans and domestic animals from biting and nuisance caused by mosquitoes.

The components of Origanum vulgare L. essential oil showing insecticidal activity and repellency against red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae), adults were analysed by GC-MS. All constituents were identified, and the main components were carvacrol (67.2%), p-cymene (16.2%), γ-terpinene (5.5%), thymol (4.9%), and linalool (2.1%). In a vapor phase fumigant assay, the origanum oil was more effective in closed conditions (LD50 = 0.055 mg/cm3) than in open conditions (LD50 > 0.353 mg/cm3). This suggests that toxicity is exerted largely in the vapor phase. Based on 24-h LD50 values, the toxicity of caryophyllene oxide (0.00018 mg/cm3) was comparable with that of dichlorvos (0.00007 mg/cm3). In addition, thymol, camphene, α-pinene, p-cymene, and γ-terpinene showed good insecticidal activity (LD50 = 0.012–0.195 mg/cm3). In repellency tests using 9 constituents of origanum oil, caryophyllene oxide showed complete repellency at 0.03 mg/cm2. Hydrogenated monoterpenoids, such as thymol, α-pinene, carvacrol, and myrcene, elicited strong repellency at 0.03 and 0.006 mg/cm2. Repellency depended on both time and concentration. These results indicate that origanum oil and its components could be potential candidates as a fumigant and repellent for managing T. castaneum adults.

Heat sensitivity is a sensory modality that plays a critical role in close-range host-seeking behaviors of adult female Anopheles gambiae, the principal Afrotropical vector for human malaria. An essential step in this activity is the ability to discriminate and respond to increases in environmental temperature gradients through the process of peripheral thermoreception. Here, we report on the characterization of the anopheline homolog of the transient receptor potential (TRP) A1/ANKTM1 channel that is consistent with its role as a heat-sensor in host-seeking adult female mosquitoes. We identify a set of distal antennal sensory structures that specifically respond to temperature gradients and express AgTRPA1. Functional characterization of AgTRPA1 in Xenopus oocytes supports its role in the molecular transduction of temperature gradients in An. gambiae, providing a basis for targeting mosquito heat responses as a means toward reducing malaria transmission.

Summary Background Many species of mosquitoes, including the major malaria vector Anopheles gambiae , utilize carbon dioxide (CO 2 ) and 1-octen-3-ol as olfactory cues in host-seeking behaviors that underlie their vectorial capacity. However, the molecular and cellular basis of such olfactory responses remains largely unknown. Results Here, we use molecular and physiological approaches coupled with systematic functional analyses to define the complete olfactory sensory map of the An. gambiae maxillary palp, an olfactory appendage that mediates the detection of these compounds. In doing so, we identify three olfactory receptor neurons (ORNs) that are organized in stereotyped triads within the maxillary-palp capitate-peg-sensillum population. One ORN is CO 2 -responsive and characterized by the coexpression of three receptors that confer CO 2 responses, whereas the other ORNs express characteristic odorant receptors (AgORs) that are responsible for their in vivo olfactory responses. Conclusions Our results describe a complete and highly concordant map of both the molecular and cellular olfactory components on the maxillary palp of the adult female An. gambiae mosquito. These results also facilitate the understanding of how An. gambiae mosquitoes sense olfactory cues that might be exploited to compromise their ability to transmit malaria.

We report a floating electrode-based bioelectronic tongue mimicking insect taste systems for the detection and discrimination of umami substances. Here, carbon nanotube field-effect transistors with floating electrodes were hybridized with nanovesicles containing honeybee umami taste receptor, gustatory receptor 10 of Apis mellifera (AmGr10). This strategy enables us to discriminate between l-monosodium glutamate (MSG), best-known umami tastant, and non-umami substances with a high sensitivity and selectivity. It could also be utilized for the detection of MSG in liquid food such as chicken stock. Moreover, we demonstrated the synergism between MSG and disodium 5'-inosinate (IMP) for the umami taste using this platform. This floating electrode-based bioelectronic tongue mimicking insect taste systems can be a powerful platform for various applications such as food screening, and it also can provide valuable insights on insect taste systems.

The human β-amyloid (Aβ) cleaving enzyme (BACE-1) is a target for Alzheimer’s disease (AD) treatments. This study was conducted to determine if acacetin extracted from the whole Agastache rugosa plant had anti-BACE-1 and behavioral activities in Drosophila melanogaster AD models and to determine acacetin’s mechanism of action. Acacetin (100, 300 and 500 μM) rescued amyloid precursor protein (APP)/BACE1-expressing flies and kept them from developing both eye morphology (dark deposits, ommatidial collapse and fusion and the absence of ommatidial bristles) and behavioral (motor abnormalities) defects. The reverse transcription polymerase chain reaction analysis revealed that acacetin reduced both the human APP and BACE-1 mRNA levels in the transgenic flies, suggesting that it plays an important role in the transcriptional regulation of human BACE-1 and APP. Western blot analysis revealed that acacetin reduced Aβ production by interfering with BACE-1 activity and APP synthesis, resulting in a decrease in the levels of the APP carboxy-terminal fragments and the APP intracellular domain. Therefore, the protective effect of acacetin on Aβ production is mediated by transcriptional regulation of BACE-1 and APP, resulting in decreased APP protein expression and BACE-1 activity. Acacetin also inhibited APP synthesis, resulting in a decrease in the number of amyloid plaques.

In mosquitoes, precise and efficient finding of a host animal is crucial for survival. One of the poorly understood aspects of mosquito blood-feeding behavior is how these insects target an optimal site in order to penetrate the skin and blood vessels without alerting the host animal. Here we provide new findings that a piercing structure of the mouthpart of the mosquitoes, the stylet, is an essential apparatus for the stage in blood feeding. Indeed, the stylet possesses a number of sensory hairs located at the tip of the stylet. These hairs house olfactory receptor neurons that express two conventional olfactory receptors of Aedes aegypti (AaOrs), AaOr8 and AaOr49, together with the odorant co-receptor (AaOrco). In vivo calcium imaging using transfected cell lines demonstrated that AaOr8 and AaOr49 were activated by volatile compounds present in blood. Inhibition of gene expression of these AaOrs delayed blood feeding behaviors of the mosquito. Taken together, we identified olfactory receptor neurons in the stylet involved in mosquito blood feeding behaviors, which in turn indicates that olfactory perception in the stylet is necessary and sufficient for mosquitoes to find host blood in order to rapidly acquire blood meals from a host animal.

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.

Background The honey bee is an important model system for increasing understanding of molecular and neural mechanisms underlying social behaviors relevant to the agricultural industry and basic science. The western honey bee, Apis mellifera, has served as a model species, and its genome sequence has been published. In contrast, the genome of the Asian honey bee, Apis cerana, has not yet been sequenced. A. cerana has been raised in Asian countries for thousands of years and has brought considerable economic benefits to the apicultural industry. A cerana has divergent biological traits compared to A. mellifera and it has played a key role in maintaining biodiversity in eastern and southern Asia. Here we report the first whole genome sequence of A. cerana. Results Using de novo assembly methods, we produced a 238 Mbp draft of the A. cerana genome and generated 10,651 genes. A.cerana-specific genes were analyzed to better understand the novel characteristics of this honey bee species. Seventy-two percent of the A. cerana-specific genes had more than one GO term, and 1,696 enzymes were categorized into 125 pathways. Genes involved in chemoreception and immunity were carefully identified and compared to those from other sequenced insect models. These included 10 gustatory receptors, 119 odorant receptors, 10 ionotropic receptors, and 160 immune-related genes. Conclusions This first report of the whole genome sequence of A. cerana provides resources for comparative sociogenomics, especially in the field of social insect communication. These important tools will contribute to a better understanding of the complex behaviors and natural biology of the Asian honey bee and to anticipate its future evolutionary trajectory. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-16-1) contains supplementary material, which is available to authorized users.

SUMMARYUsing antennal movements as an indicator of learning and retention, an associative learning paradigm has been developed to investigate associative memory between visual and olfactory stimuli. Experiments were performed on the restrained cockroach Periplaneta americana, which normally moves its antennae towards a localized odor source. Such `antennal projection responses'(APRs) are exploited to demonstrate long-term memory, where an APR is elicited by a conditioned stimulus (CS; green light point source) paired with a spatially coincident odor [the unconditioned stimulus (US)]. Association of the CS with the US is established after five trials. Before training, a visual cue alone does not elicit an APR. This behavior is elicited by a visual cue only after pairing it with an odor stimulus. The acquired APR to the green light cue persists for up to 72 h, indicative of long-term memory. This paradigm is thus suitable for future studies of neural correlates of learning and memory on restrained animals.

SUMMARY Spatial learning abilities were tested in restrained cockroaches by observing antennal projection responses towards the positions of a learned visual cue perceived monocularly by one eye in the context of a second stimulus provided to the contralateral eye. Memory of the position of the conditioning stimulus relative to the contralateral reference stimulus was tested by altering the relative positions of the two stimuli. Memory of the conditioning stimulus is retained if the angle between the conditioning stimulus and the contralateral reference stimulus is maintained. The results suggest that during learning the insect recognizes spatial relationships between the conditioning stimulus and the contralateral reference stimulus. Possible mechanisms, such as retinotopic matching versus angular matching, are discussed.

Inhibitor cysteine knot (ICK) peptides exhibit ion channel blocking, insecticidal, and antimicrobial activities, but currently, no functional roles for bee-derived ICK peptides have been identified. In this study, a bee (Apis cerana) ICK peptide (AcICK) that acts as an antifungal peptide and as an insecticidal venom toxin was identified. AcICK contains an ICK fold that is expressed in the epidermis, fat body, or venom gland and is present as a 6.6-kDa peptide in bee venom. Recombinant AcICK peptide (expressed in baculovirus-infected insect cells) bound directly to Beauveria bassiana and Fusarium graminearum, but not to Escherichia coli or Bacillus thuringiensis. Consistent with these findings, AcICK showed antifungal activity, indicating that AcICK acts as an antifungal peptide. Furthermore, AcICK expression is induced in the fat body and epidermis after injection with B. bassiana. These results provide insight into the role of AcICK during the innate immune response following fungal infection. Additionally, we show that AcICK has insecticidal activity. Our results demonstrate a functional role for AcICK in bees: AcICK acts as an antifungal peptide in innate immune reactions in the body and as an insecticidal toxin in venom. The finding that the AcICK peptide functions with different mechanisms of action in the body and in venom highlights the two-pronged strategy that is possible with the bee ICK peptide.

Background Alzheimer’s disease (AD) is the most common type of presenile and senile dementia. The human β-amyloid precursor cleavage enzyme (BACE-1) is a key enzyme responsible for amyloid plaque production, which implicates the progress and symptoms of AD. Here we assessed the anti-BACE-1 and behavioral activities of curcuminoids from rhizomes of Curcuma longa (Zingiberaceae), diarylalkyls curcumin (CCN), demethoxycurcumin (DMCCN), and bisdemethoxycurcumin (BDMCCN) against AD Drosophila melanogaster models. Methods Neuro-protective ability of the curcuminoids was assessed using Drosophila melanogaster model system overexpressing BACE-1 and its substrate APP in compound eyes and entire neurons. Feeding and climbing activity, lifespan, and morphostructural changes in fly eyes also were evaluated. Results BDMCCN has the strongest inhibitory activity toward BACE-1 with 17 μM IC50, which was 20 and 13 times lower than those of CCN and DMCCN respectively. Overexpression of APP/BACE-1 resulted in the progressive and measurable defects in morphology of eyes and locomotion. Remarkably, supplementing diet with either 1 mM BDMCCN or 1 mM CCN rescued APP/BACE1-expressing flies and kept them from developing both morphological and behavioral defects. Our results suggest that structural characteristics, such as degrees of saturation, types of carbon skeleton and functional group, and hydrophobicity appear to play a role in determining inhibitory potency of curcuminoids on BACE-1. Conclusion Further studies will warrant possible applications of curcuminoids as therapeutic BACE-1 blockers.

Insect-derived antimicrobial peptides (AMPs) have diverse effects on antimicrobial properties and pharmacological activities such as anti-inflammation and anticancer properties. Naturally occurring genetic polymorphism have a direct and/or indirect influence on pharmacological effect of AMPs, therefore information on single nucleotide polymorphism (SNP) occurring in natural AMPs provides an important clue to therapeutic applications. Here we identified nucleotide polymorphisms in melittin gene of honey bee populations, which is one of the potent AMP in bee venoms. We found that the novel SNP of melittin gene exists in these two honey bee species, Apis mellifera and Apis cerana. Nine polymorphisms were identified within the coding region of the melittin gene, of which one polymorphism that resulted in serine (Ser) to asparagine (Asp) substitution that can potentially effect on biological activities of melittin peptide. Serine-substituted melittin (Mel-S) showed more cytotoxic effect than asparagine-substituted melittin (Mel-N) against E. coli. Also, Mel-N and Mel-S had different inhibitory effects on the production of inflammatory factors such as IL-6 and TNF-α in BV-2 cells. Moreover, Mel-S showed stronger cytotoxic activities than Mel-N peptide against two human ovarian cancer cell lines. Using carbon nanotube-based transistor, we here characterized that Mel-S interacted with small unilamellar liposomes more strongly than Mel-N. Taken together, our present study demonstrates that there exist different characteristics of the gene frequency and the biological activities of the melittin peptide in two honey bee species, Apis mellifera and A. cerana.

Insect-derived Kazal-type serine protease inhibitors exhibit thrombin, elastase, plasmin, proteinase K, or subtilisin A inhibition activity, but so far, no functional roles for bee-derived Kazal-type serine protease inhibitors have been identified. In this study, a bee (Apis cerana) venom Kazal-type serine protease inhibitor (AcKTSPI) that acts as a microbial serine protease inhibitor was identified. AcKTSPI contained a single Kazal domain that displayed six conserved cysteine residues and a P1 threonine residue. AcKTSPI was expressed in the venom gland and was present as a 10-kDa peptide in bee venom. Recombinant AcKTSPI Kazal domain (AcKTSPI-Kd) expressed in baculovirus-infected insect cells demonstrated inhibitory activity against subtilisin A (Ki 67.03 nM) and proteinase K (Ki 91.53 nM), but not against α-chymotrypsin or trypsin, which implies a role for AcKTSPI as a microbial serine protease inhibitor. However, AcKTSPI-Kd exhibited no detectable inhibitory effects on factor Xa, thrombin, tissue plasminogen activator, or elastase. Additionally, AcKTSPI-Kd bound directly to Bacillus subtilis, Bacillus thuringiensis, Beauveria bassiana, and Fusarium graminearum but not to Escherichia coli. Consistent with these findings, AcKTSPI-Kd showed antibacterial activity against Gram-positive bacteria and antifungal activity against both plant-pathogenic and entomopathogenic fungi. These findings constitute molecular evidence that AcKTSPI acts as an inhibitor of microbial serine proteases. This paper provides a novel view of the antimicrobial functions of a bee venom Kazal-type serine protease inhibitor.

Olfactory sensitivity exhibits daily fluctuations. Several studies have suggested that the olfactory system in insects is modulated by both biogenic amines and neuropeptides. However, molecular and neural mechanisms underlying olfactory modulation in the periphery remain unclear since neuronal circuits regulating olfactory sensitivity have not been identified. Here, we investigated the structure and function of these signaling pathways in the peripheral olfactory system of the American cockroach, Periplaneta americana, utilizing in situ hybridization, qRT-PCR, and electrophysiological approaches. We showed that tachykinin was co-localized with the octopamine receptor in antennal neurons located near the antennal nerves. In addition, the tachykinin receptor was found to be expressed in most of the olfactory receptor neurons in antennae. Functionally, the effects of direct injection of tachykinin peptides, dsRNAs of tachykinin, tachykinin receptors, and octopamine receptors provided further support for the view that both octopamine and tachykinin modulate olfactory sensitivity. Taken together, these findings demonstrated that octopamine and tachykinin in antennal neurons are olfactory regulators in the periphery. We propose here the hypothesis that octopamine released from neurons in the brain regulates the release of tachykinin from the octopamine receptor neurons in antennae, which in turn modulates the olfactory sensitivity of olfactory receptor neurons, which house tachykinin receptors.

The Asian cavity-nesting honey bee, Apis cerana, the workers ovaries is very active in queenless colonies. We investigated the duration of egg laying by laying workers, egg-laying behavior, determine egg load in ovaries, estimate oviposition rate nad number of eggs per cell in three queenless colonies of Korean A. cerana. The number of eggs laid by laying workers at different times on the manipulated comb was checked and counted. We observed that the laying workers spent, on average, 109.2 seconds per cell. During oviposition, the laying workers showed two types of behaviors, viz; a still phase, and a recovery phase. On average, 4.0 of worker eggs per cell per week was recorded. The workers laid three different shapes of eggs in a cell. The laying workers ovaries retained 1 to 4 mature eggs. This suggests that a laying worker may lay from one to four eggs per oviposition. We did not observe false-queen in Korean A. cerana in three queenless colonies. In this study, we reported for the first time that one worker cell of A. cerana retains up to 33 eggs in queenless condition. The queenless colonies build the comb and lay eggs in new comb, and also reared the new queen without successful development of the queen larva from laying worker eggs.

Insect olfactory organs possess many olfactory receptor neurons, which detect many different sets of odorants in nature. In order to feed on blood meals, stable flies locate host animals and humans using chemical cues such as 1-octen-3-ol and butyric acid. In the present study, behavioural and electroantennogram (EAG) response patterns to repellent volatiles from essential oils (EOs) of Zanthoxylum piperitum and Z. armatum in combination with the attractants were investigated.Components of the EOs such as cuminaldehyde, citronellal, neral, linalool, linalool oxide, terpinen-4-ol, 1,8-cineole, and piperitone induced remarkable repellent behaviours in the stable fly. EAG responses in the fly antenna to these chemicals showed a dose-dependent manner. The patterns of behavioural and EAG responses were significantly altered depending on the ratios of 1-octen-3-ol or butyric acid to the EOs or compounds in the air mixtures.The present study demonstrated that the Zanthoxylum EOs decreased the levels of response of flight behaviours of the stable fly towards host volatile compounds. The combinations of odorant mixtures of the attractants with the EOs and their components affect the representation of behavioural and EAG responses of the flies. The summation and integration patterns of olfactory responses measured by the EAG indicated that the peripheral olfactory networks in antennae could process the odorant complexity of different odorant mixtures between attractants and repellents.

The honeybee is an important insect species in global ecology, agriculture, and alternative medicine. While chymotrypsin and trypsin inhibitors from bees show activity against cathepsin G and plasmin, respectively, no anti-elastolytic role for these inhibitors has been elucidated. In this study, we identified an Asiatic honeybee (Apis cerana) chymotrypsin inhibitor (AcCI), which was shown to also act as an elastase inhibitor. AcCI was found to consist of a 65-amino acid mature peptide that displays ten cysteine residues. When expressed in baculovirus-infected insect cells, recombinant AcCI demonstrated inhibitory activity against chymotrypsin (K(i) 11.27 nM), but not trypsin, defining a role for AcCI as a honeybee-derived chymotrypsin inhibitor. Additionally, AcCI showed no detectable inhibitory effects on factor Xa, thrombin, plasmin, or tissue plasminogen activator; however, AcCI inhibited human neutrophil elastase (K(i) 61.05 nM), indicating that it acts as an anti-elastolytic factor. These findings constitute molecular evidence that AcCI acts as a chymotrypsin/elastase inhibitor.

We investigated the molecular and kinetic properties of two acetylcholinesterases (AmAChE1 and AmAChE2) from the Western honey bee, Apis mellifera. Western blot analysis revealed that AmAChE2 has most of catalytic activity rather than AmAChE1, further suggesting that AmAChE2 is responsible for synaptic transmission in A. mellifera, in contrast to most other insects. AmAChE2 was predominately expressed in the ganglia and head containing the central nervous system (CNS), while AmAChE1 was abundantly observed not only in the CNS but also in the peripheral nervous system/non-neuronal tissues. Both AmAChEs exist as homodimers; the monomers are covalently connected via a disulfide bond under native conditions. However, AmAChE2 was associated with the cell membrane via the glycophosphatidylinositol anchor, while AmAChE1 was present as a soluble form. The two AmAChEs were functionally expressed with a baculovirus system. Kinetic analysis revealed that AmAChE2 has approximately 2,500-fold greater catalytic efficiency toward acetylthiocholine and butyrylthiocholine than AmAChE1, supporting the synaptic function of AmAChE2. In addition, AmAChE2 likely serves as the main target of the organophosphate (OP) and carbamate (CB) insecticides as judged by the lower IC(50) values against AmAChE2 than against AmAChE1. When OP and CB insecticides were pre-incubated with a mixture of AmAChE1 and AmAChE2, a significant reduction in the inhibition of AmAChE2 was observed, suggesting a protective role of AmAChE1 against xenobiotics. Taken together, based on their tissue distribution pattern, molecular and kinetic properties, AmAChE2 plays a major role in synaptic transmission, while AmAChE1 has non-neuronal functions, including chemical defense.

Many insecticides have been used for managing stored products insect pests, especially coleopteran insects such as beetles and weevils because most of them have cosmopolitan distribution and are destructive insects damaging various stored cereals, legumes and food stuffs. Approximately one-third of the worldwide food production has been economically affected, valued annually at more than 100 billion USD, by more than 20,000 species of field and storage insect pests, which can cause serious post-harvest losses from up to 9% in developed countries to 43% of the highest losses occur in developing African and Asian countries (Jacobson, 1982; Pimentel, 1991). Among the most serious economic insect pests of grains, internal feeders such as Rhyzopertha dominica and Sitophilus oryzae are primary insect pests (Phillips & Throne, 2010). The former lays eggs outside the kernel and hatching larvae intrude into it to complete development to the adult stage, and the latter lays eggs directly inside the kernel. The other commonly found insects in shelled kennel are Sitophilus zeamais and Sitotroga cerealella. In addition, external feeders such as Tribolium castaneum, Cryptolestes ferrugineus, and Oryzaephilus surinamensis are commonly found insect pests in wheat or maize. Especially, R. dominica, S. oryzae, and S. cerealella are major internal feeding pests of rice. Most of them belong to the order of Coleoptera.

BACKGROUND: The repellency to stable fly, Stomoxys calcitrans (L.), of Zanthoxylum piperitum (L.) DC pericarp steam distillate (ZP-SD), Zanthoxylum armatum DC seed oil (ZA-SO) and their constituents alone or in combination with Calophyllum inophyllum L. nut oil (CI-NO), as well as six aerosol formulations containing ZP-SD or ZP-SO and CI-NO, was compared with that of a synthetic repellent, DEET, using an exposed human hand bioassay. RESULTS: At 0.20 mg cm−2, ZP-SD treatments resulted in 91 and 68% repellency following 30 and 60 min exposure respectively, while DEET gave 100 and 84% repellency. ZA-SO was less effective than ZP-SD. At 0.2 mg cm−2, the most active constituents, cuminaldehyde, cuminyl alcohol, limonene and methyl cinnamate, gave 82, 74, 74 and 64% repellency at 30 min respectively, but DEET gave 100 and 87% repellency at 30 and 60 min. An increase in effectiveness and duration of repellency was produced by binary mixtures of ZP-SD, ZA-SO or bioactive constituents (each 0.01 mg cm−2) and CI-NO (0.99 mg cm−2). The repellency of aerosols containing 2.5% ZP-SD or 2.5% ZA-SO and 2.5% CI-NO was comparable with that of 5% DEET aerosol. CONCLUSION: Mixtures formulated from ZP-SD, ZA-SO or bioactive constituents and CI-NO could be useful as potential repellents for the control of stable fly populations in light of global efforts to reduce the level of highly toxic synthetic repellents. Copyright © 2010 Society of Chemical Industry

Scanning electron microscopic observations on the Korean haplotype of Varroa destructor collected from Apis mellifera was indentifed by mtDNA analysis to describe external morphology K-type V. destructor. SEM micrographs showed that the K-type V. destructor is more oval than Japan/Thai- Vietnam type and has three kinds of setae viz, serrated, spiny, and smooth marginal setae. The marginal setae are somewhat flexible. The K-type V. destructor is larger than V. jacobsoni and V. rindereri. The peritreme of K-type V. destructor is smaller than V. jacobsoni and V. rindereri. No sternal pores were observed in the K-type V. destructor, which is present in V. jacobsoni and V. rindereri. The K-type V. destructor has a more metapodal setae than V. jacobsoni and V. rindereri.

The proboscis is an important head appendage in insects that has primarily been thought to process gustatory information during food intake. Indeed, in Drosophila and other insects in which they have been identified, most gustatory receptors are expressed in proboscis neurons. Our previous characterization of the expression of AgOR7, a highly conserved odorant receptor (OR) of the Afrotropical malaria vector mosquito Anopheles gambiae in the labellum at the tip of the proboscis was suggestive of a potential olfactory function in this mosquito appendage. To test this hypothesis, we used electrophysiological recording and neuronal tracing, and carried out a molecular characterization of candidate OR expression in the labellum of A. gambiae . These studies have uncovered a set of labial olfactory responses to a small spectrum of human-related odorants, such as isovaleric acid, butylamine, and several ketones and oxocarboxylic acids. Molecular analyses indicated that at least 24 conventional OR genes are expressed throughout the proboscis. Furthermore, to more fully examine AgOR expression within this tissue, we characterized the AgOR profile within a single labial olfactory sensillum. This study provides compelling data to support the hypothesis that a cryptic set of olfactory neurons that respond to a small set of odorants are present in the mouth parts of hematophagous mosquitoes. This result is consistent with an important role for the labellum in the close-range discrimination of bloodmeal hosts that directly impacts the ability of A. gambiae to transmit malaria and other diseases.

The yellow fever mosquito, Aedes aegypti, is a vector for transmitting dengue fever and yellow fever. In this study, we assessed the histopathological and molecular effects of pellitorine, an isobutylamide alkaloid, on the third instar of Ae. aegypti larvae. At 5 mg/l concentration of pellitorine, the whole body of the treated larvae became dark in color, particularly damaged thorax and abdominal regions. Pellitorine was targeted mainly on midgut epithelium and anal gills, indicating variably dramatic degenerative responses of the midgut through a sequential epithelial disorganization. The anterior and posterior midgut was entirely necrosed, bearing only gut lumen residues inside the peritrophic membranes. Pellitorine caused comprehensive damage of anal gill cells and branches of tracheole and debris was found in hemolymph of the anal gills. RT-PCR analysis indicates that the compound inhibited gene expression encoding V-type H(+)-ATPase and aquaporine 4 after treatment with 2.21 mg/l pellitorine. These results verify that pellitorine merits further study as a potential larvicide with a specific target site and a lead molecule for the control of mosquito populations.

The olfactory-driven blood-feeding behaviour of female Aedes aegypti mosquitoes is the primary transmission mechanism by which the arboviruses causing dengue and yellow fevers affect over 40 million individuals worldwide. Bioinformatics analysis has been used to identify 131 putative odourant receptors from the A. aegypti genome that are likely to function in chemosensory perception in this mosquito. Comparison with the Anopheles gambiae olfactory subgenome demonstrates significant divergence of the odourant receptors that reflects a high degree of evolutionary activity potentially resulting from their critical roles during the mosquito life cycle. Expression analyses in the larval and adult olfactory chemosensory organs reveal that the ratio of odourant receptors to antennal glomeruli is not necessarily one to one in mosquitoes.