The honey bee soluble acetylcholinesterase 1 (AmAChE1) is overexpressed under the overwintering and brood rearing-suppressed conditions. To investigate the role of AmAChE1 in regulating acetylcholine (ACh) titer, ACh concentrations both in the head (neuronal) and abdomen (non-neuronal) were analyzed. ACh titer was significantly lower in both tissues of worker bees under the overwintering and brood rearing-suppressed conditions compared to control bees. The expression levels of another two factors that regulate ACh titer, choline acetyltransferase (AmAChT) and acetylcholinesterase 2 (AmAChE2), were not altered as judged by qPCR and native PAGE, suggesting that the lower ACh titer was mainly regulated by AmAChE1. For precise verification of AmAChE1 as an ACh titer regulator, honey bees were put under brood rearing-suppressed condition to induce AmAChE1 and injected AmAChE1 dsRNA to knock down the gene. The ACh titer of AmAChE1-knocked down honey bees was 1.9 and 2.6 folds higher than that of control bees in head and abdomen, respectively. Taken together, in spite of its extremely low catalytic activity, the overexpression of AmAChE1 is likely to be related with the low level of ACh homeostasis, perhaps via ACh sequestration, under brood rearingsuppressed condition, and likely induce metabolic changes through ACh receptors-related pathways.

Small hive beetle (Aethina tumida) (SHB) is an invasive species to most northern hemisphere countries, including Korea. In an attempt to obtain basic information for efficient management of SHB, genes encoding conventional insecticide targets [voltage-sensitive sodium channel α-subunit (VSSC) and acetylcholinesterase (AChE)] were annotated and characterized following the analysis of whole transcriptomes of adults and larvae. A single VSSC gene was identified but no apparent mutations associated with pyrethroid resistance were detected. Genes encoding two AChEs (AtAChE1 and AtAChE2) were identified from the SHB transcriptome. AtAChE1 was determined to be the main catalytic enzyme, thereby being a toxicologically more relevant target. No apparent mutations associated with resistance to organophosphorus and carbamate insecticides was identified in the AtAChE1 gene, whereas the S238G mutation, originally identified from the Colorado potato beetle, was detected in the AtAChE2 gene.

The common bed bug, Cimex lectularius, possesses a cholinesterase expressed exclusively in the salivary gland (ClSChE). In this paper, we investigated the molecular structure, tissue distribution patterns, and biochemical properties of ClSChE and showed that ClSChE exists as a soluble monomeric form or a soluble dimeric form connected by a disulfide bridge. Immunohistochemical analysis confirmed that ClSChE was expressed in the epithelial cells of both the salivary gland and the duct. In addition, the secretion of monomeric ClSChE through the proboscis during feeding was detected by western blotting using a ClSChE-specific antibody. To predict the role of ClSChE injected into the tissue of an animal host, we analyzed the extent of sequestration and hydrolysis of acetylcholine (ACh)/choline (Ch) by ClSChE by ultra-performance liquid chromatography-tandem mass spectrometry. Kinetic analysis revealed that ClSChE possesses extremely low Km (high affinity to ACh) and Vmax values. These findings suggest that ClSChE functions as a sequestering enzyme specific to ACh (not to Ch) by having a very strong affinity to ACh but an extremely long turnover time.

The honey bee soluble acetylcholinesterase 1 (AmAChE1) is overexpressed under the overwintering and brood rearing-suppressed conditions. To investigate the role of AmAChE1 in regulating acetylcholine (ACh) titer, ACh concentrations in both the head (central nervous system) and abdomen (peripheral nervous system) were analyzed. ACh titer was significantly lower in both tissues of worker bees under the overwintering and brood rearing-suppressed conditions compared to control bees. Interestingly, the expression levels of choline acetyltransferase (AmChAT) and molecular marker genes of immune systems were significantly reduced in honey bee head under the same conditions. Taken together, ACh titer appears to be reduced via a cooperative interaction of the AmAChE1 overexpression and AmChAT underexpression and to be linked to reduced inmmune responses under the overwintering and brood rearing-suppressed conditions. The roles of AmAChE1 (with little catalytic activity) and AmChAT in the ACh homeostasis and signaling was discussed in the contexts of immune response and longevity regulation in honey bees.

Among two different acetylcholinesterase (AmAChE1 and AmAChE2) of the western honey bee, the soluble AmAChE1might be related with a stress response as judged from its over-expression in honey bee workers when brood rearingwas suppressed. In this study, to ensure the nature of AmAChE1 responding to stress factors, the expression patternsof AmAChE1 were investigated following various treatments, including varroa mite infestation, bacterial challenge, broodrearing suppression, thermal stresses, chemical treatments, ultraviolet B irradiation, starvation, water restriction and crowdingstress. In addition, transcription profiles of four heat shock protein genes known as general stress markers and vitellogeningene, which is induced in several stress conditions, were tested as positive references. In every tested condition, onlybrood rearing suppression and heat shock were related with the expression of AmAChE1.

Honey bee swarming is a natural phenomenon that occurs by changes of colony (i.e. population size and queen condition) and environment conditions. As cuticular hydrocarbons (CHCs) are known to be involved in the communication between honey bee nest-mates, we investigated and compared the CHC profiles of worker bees from individual colonies of 9-days before swarming (PPSC), a day before swarming (PSC), swarming (SG) and remaining (non-swarming) (RG). A total of 53 CHCs were identified by GC-MS, among which 11 compounds showed significantly differential expression patterns between swarming states. Before swarming (between PPSC and PSC), detection levels of 4 CHCs were significantly different, suggesting that production of some CHCs changed prior to swarming for swarming preparation. Six CHCs were deferentially produced between PSC and RG. The differential profiles of CHCs with respect to different swarming states are currently under investigation.

Perturbation of normal behaviors (e.g., nursing and foraging) in honey bee colonies by any external factors would immediately reduce the colony’s capacity for brood rearing, which can eventually lead to collapse of entire colony. To investigate the effects of brood rearing suppression in the biology of honey bee workers (nurse and forager), the gene-set enrichment analysis (GSEA) was performed for the transcriptomes of worker bees with or without their brood rearing being suppressed, from which functional profiles of pathways under influences by each condition were identified. Blocking of normal labor (i.e., nursing or foraging) induced the over-representation of pathways related with reshaping of worker bee physiology, suggesting that transition of labor is physiologically reversible. In addition, brood rearing suppression appeared to result in the reduction of neuronal excitability and aggressiveness in both forager and nurse, which would be necessary to manage the in-hive stress under unfavorable conditions

There are two different types of acetylcholinesterase (AChE1 and AChE2) in the western honeybee as in most of insects. It is suggested that soluble AmAChE1 might be related with a stress response as judged from its elevated expression level in honey bee workers when brood rearing was suppressed. In this study, to ensure the nature of AmAChE1 responding to stress factors, the expression patterns of AmAChE1 following heat shock, brood rearing suppression and chemical treatments (Imidacloprid and fluvalinate) were investigated. Also, several heat shock protein (hsp) genes (hsp10, hsp60, hsp70 and hsp90) known as general stress markers were tested as positive references. Heat shock induced expression of every tested hsp along with AmAChE1. In brood rearing-suppressed worker bees, 7 days old bees showed much higher expression level of AmAChE1 and hsp90 compared to control honey bees. However, treatment of imidacloprid and fluvalinate did not induce any apparent overexpression of these genes. These results confirm that both HSP and AmAChE1 genes generally respond to temperature and brood rearing suppression and further suggest that AmAChE1 can serve as a potential biomarker along with hsps for the detection of stress in honey bee colonies.

Two different types of acetylcholinesterae (AChE1 and AChE2) are present in majority of insects, including the Western honey bee. Out of the two honey bee AChEs (AmAChEs), the soluble AmAChE1 with little catalytic activity is widely distributed in both neuronal and non-neuronal tissues, including fat body. In this study, to identify stresss factors that can induce AmAChE expression, we tested various conditions that honey bees can encounter in natural setting, including heat shock, cold shock, bacterial challenge (Escherichia coli and Staphylococcus aureus) and Varroa mite infestations, and evaluated their effects on AmAChE expression. Among the stress factors tested, only heat shock condition induced AmAChE expression in a dose dependet manner. This finding suggests that one function of AmAChE1 is related with thermoregulations, especially against heat shock stress in honey bees.

Honey bee swarming is a natural phenomenon that occurs when the colony encounters changes in the in-hive (i.e. population size and queen condition) and environmental conditions. To better understand the molecular basis of swarming, we conducted the transcriptomic profiles of worker bees between before swarming [pre-swarming colony (PSC)] and after swarming [swarming group (SG) and remaining group (RG)]. Based on the gene set enrichment analysis (GSEA), we predicted the biological processes associated with swarming. In addition, we analyzed the composition of cuticular hydrocarbons (CHCs) by gas chromatography-mass spectrometry and compared their profiles between different bee groups. GSEA results showed that there were a little differences between PSC and RG while many of the pathways related with metabolism and protein processing were down regulated in SG relative to PSC and RG. CHCs profiling revealed a similar CHCs composition between PSC and RG but some differences in CHCs composition (i.e. heneicosane, octacosane, octacosanol) were detected between SG and RG. These differences in gene pathway and CHC composition were discussed with respect to physiological changes and social communication.

Acetylcholinesterase 1 (AmAChE1) has low catalytic activity and is abundantly expressed in both neuronal and non-neuronal tissues. In previous experiments, we observed that AmAChE1 is rarely expressed in summer while highly expressed in winter. Through additional experiments, the expression of AmAChE1 was suggested to be associated with brood rearing status. Under the assumption that abnormal suppression of brood rearing activity may result in stressful condition in honey bee social community, it was further suggested that AmAChE1 is likely involved in stress management particularly during winter. We hypothesized that the increased docility usually observed in overwintering bees is likely an outcome of stress management in colony, which is mediated by AmAChE1 expression. To verify this, worker bees expressing abundant AmAChE1 were collected in early winter and injected with Amace1 dsRNA to knockdown Amace1. Then, the behavioral activity of the bees was investigated using the EthoVison video tracking system. Honey bees injected with Amace1 dsRNA showed significantly increased motility, which was strongly correlated with the suppressed expression level of AmAChE1 in the abdomen. No apparent reduced expression of AmAChE1 in the head was observed perhaps due to the limited efficacy of RNA interference in the blood-brain-barrier. Our finding suggests that behavioral activity can be regulated, at least, by AmAChE1 expression level in non-neuronal tissue (i.e., fatbody) perhaps via metabolic alteration.

Human body and head lice are obligatory human ectoparasites. Although both body and head lice belong to a single species, Pediculus humanus, only body lice are known to be a vector of several bacterial diseases. The higher vector competence of body lice is assumed to be due to their weaker immune response than that of head lice. To test this hypothesis, immune reactions were compared between body and head lice following infections by two model bacteria, Staphylococcus aureus and Escherichia coli, and a human pathogen, Bartonella quintana. Following dermal or oral challenge, the number of these bacteria increased both in hemocoel and alimentary tract of body lice but not in head lice and the viability of the B. quintana was significantly higher in body louse feces, the major route of infection to human. In addition, body lice showed the lower basal/induced transcription level of major immune genes, cytotoxic reactive oxygen species and phagocytosis activity compared with head lice. These findings suggest that a reduced immune response may be responsible, in part, for the increased proliferation and excretion of viable bacteria which are associated with the high level of human infectivity seen in body versus head lice.

Tropilaelaps mercedesae is an ectoparasite of immature honey bees belonging to the genus Tropilaelaps (Acari: Laelapidae). T. mercedesae has become a major threat to the Western honey bee Apis mellifera in Asia, including Korea, and is expanding its geographical range to northern regions due to global warming. To establish gene resources of T. mercedesae, the whole transcriptome was analyzed by RNA sequencing. An mRNA-focused library was generated from total RNA extracted from the mixed stages using the TruSeq RNA Library Preparation kit and sequenced using the HiSeq 2000 platform. A total of 6.0 Gb reads were obtained with 85% Q30 value. Trimmed sequence data were de novo assembled using the CLC Assembly Cell v 4.2. A total of 64,868 non-duplicate contigs were finally obtained and annotated by the Blast2GO using the NCBI nr database. The most abundant species in the resulting 14,336 Blast hits (22.1%) was Metaseiulus occidentalis, a predatory mite, followed by Ixodes scapularis and Tribolium castaneum, suggesting that the T. mercedesae transcriptome matches well with closely related other arthropod species, including mites and ticks. In order to provide basic information for efficient control and monitoring of potential resistance in T. mercedesae, acaricide target genes were annotated and characterized. One voltage-sensitive sodium channel gene encoding the molecular target of fluvalinate, a pyrethroid acaricide most widely used for the control of T. mercedesae, was identified and its molecular properties were investigated. In addition, other acaricide target genes, including acetylcholinesterase and glutamate (or GABA)-gated chloride channel, were identified and characterized.

The acetylcholinesterase 1 (AmAChE1) of the honey bee is known to be abundantly expressed both in the central and peripheral nervous systems. AmAChE1 exists mostly in the soluble form with little catalytic activity and has non-neuronal functions. Our preliminary observation showed that AmAChE1 expression fluctuated between the forages and nurses. A more systematic expression profiling of AmAChE1 over a year cycle on a monthly basis revealed that AmAChE1 was predominantly expressed during the winter months with being moderately expressed during the rainy summer time. However, no significant difference in AmAChE1 expression was noticed between the nurse and forager workers. Interestingly, AmAChE1 expression was inhibited when bees were allowed for brooding by placing overwintering bee hives in strawberry green houses with the supplement of pollen diets whereas it was resumed when the bee hives were removed from the green houses, thereby suppressed brooding. To confirm whether brooding status is a main determining factor for the suppression of AmAChE1 expression, active bee hives were placed in a screen tent, thereby hindering foraging, until brooding was completely suppressed, and then allowed to restore brooding by removing the screen. The AmAChE1 expression in the head was up-regulated when brooding was suppressed whereas its expression was down-regulated when brooding was resumed. These finding demonstrates that AmAChE1 expression in the central nervous system (i.e., head) is related with brooding status of honey bee. To understand the connection between the AmAChE1 expression and other pathways related with brooding, currently in progress are the analyses of head transcriptomes of honey bee workers with or without their brooding suppressed.

Leptotrombidium pallidum is the major vector mites for Orientia tsutsugamushi, the causative agent of scrub typhus. To understand the molecular mechanism of L. pallidum, we sequenced the whole genome using Illumina sequencing technology. Totally four genomic libraries with different insert sizes ranging from 280 bp to 8 kb were used to generate 45.1 Gb of genome in the combination of paired-end and mate-pairs sequencing reads. Quality filtering and correction of paired-end reads for very small and/or bad-quality sequences yielded 26.9 Gb of high-quality sequences, which are used to estimate the genome size as 175 Mbusing kmer methods and assembled into a 193.7 Mb genomic sequence scaffolds with N50 length of 92,945 bp. Furthermore, 94% of CEGMA completeness score were obtained from genome scaffold assembly. To facilitate gene annotation, we used a combination of de novo and homology based tools to predict gene models in the chigger mite genome. A combination of evidence-based and de novo approaches predicted 15,842 high-confidence protein-coding genes with an average transcript length of 1,511 bp and 2.4 exons per gene which corresponds to about 12.4% total gene length. Bacterial endosymbiosis are very common in mite species and can range from mutualistic to pathogenic associations. Henceforth, the endosymbionts in L. pallidum were predicted using the NCBI microbial draft genomes and mitochondrial genome. Besides, this L. pallidum draft genome can be used as a significant reference for comparative genomic studies across mite species.

Leptotrombidium pallidum is the major vector mite for Orientia tsutsugamushi, the causative agent of scrub typhus, in Asian countries, including Korea. The genome size of L. pallidum was previously estimated to be 191 ± 7 Mb (Kim et al., 2014). Genomic DNA (gDNA) was extracted from a single female from a 9-generation inbred L. pallidum colony and used for whole genome amplification (WGA). The resulting amplified gDNA was used for the construction of paired-end and mate-pair libraries and sequenced using Illumina platforms (HiSeq2000 and MiSeq). An unamplified gDNA sample extracted from 20 female mites was also used for sequencing in parallel. More than 45Gb sequence reads from both paired-end and mate-pair libraries of the WGA gDNA were trimmed and then de novo assembled using the CLC Asembly Cell v.4.0 for contig assembly and SSPACE for scaffolding. The assembly generated approximately 6,545 scaffolds with N50 value of 92,945 and total size of ~193Mb, which was in a good agreement with our previous estimation. Repeat analysis showed that about 30% of genome (~58Mb) was masked as repeats, most of which were unclassified novel elements. For gene predictions, generated were the PASA models based on genomic alignments of RNA-seq reads from 4 different chigger mite samples (i.e. male, female, larva, and protonymph) and the GeneWise models based on genomic alignments of protein sequences from 4 closely related species with chigger mite. Independently, ab initio gene predictions were performed with AUGUSTUS and FgeneSH with custom trained matrices optimized for L. pallidum and GENEID with pre-trained matrix for Acyrthopsiphon pisum. By combining all together, 15,842 genes were predicted finally. Manual curation is in progress for various groups of genes, including chemosensory receptor genes, immune-related genes, acaricide target genes, etc.

Recently, the expression of acetylcholinesterase1 (AChE1) in honeybee worker has been found to be seasonally fluctuated. Seasonal investigation on the AChE1 expression profiles revealed that it is abundantly expressed in January but its expression was completely abolished in February in both head and abdomen. In an attempt to predict the physiological function of seasonally expressed AChE1, proteomic analysis of honeybee worker was conducted using the samples collected in January and February. Total protein samples separately extracted from the head and abdomen of honeybee forager were compared by 2-D electrophoresis (2-DE). More than 2-fold differences in expression patterns between the two different samples were observed in 50 and 85 protein spots in the head and abdomen, respectively. Among them, 20 protein spots showing >17-fold differences in expression between the two different samples of the head were identified by mass spectrometry. Most of the proteins were identified to be the major royal jelly protein (MRJP) families (e.g., MRJP, MRJP2 and MRJP3), which are known to be expressed in nurse bees during brooding season, and their expression was significantly higher in January than in February. This result was unexpected because brooding usually began in the study site apiary during February and the worker bees used for analysis were assumed to be foragers (old workers). Thus, current findings suggest, though speculative, that the workers collected in January may function as nurses despite their old ages in January or that MRJPs may have other not-yet-characterized functions, which is apart from the conventionally known roles. Finally, possible association of MRJPs with AChE1 was discussed.

Leptotrombidium pallidum and Leptotrombidium scutellare are the major vector mites for Orientia tsutsugamushi, the causative agent of scrub typhus. Before these organisms can be subjected to whole-genome sequencing, the genome sizes of L. pallidum and L. scutellare were estimated by a method based on quantitative real-time PCR. In addition, k-mer analysis of the genome sequences obtained from Illumina sequencing was conducted to verify the mutual compatibility and reliability of results. The genome sizes estimated by qPCR were 191.3±7 Mb for L. pallidum and 262.1±13 Mb for L. scutellare. The estimated genome sizes based on k-mer analysis were 175.5 Mb for L. pallidum and 286.6 Mb for L. scutellare. The estimates from two independent methods were mutually complementary and in a similar range to those of other Acariform mites. The relatively small genome size would facilitate genome analysis, which could contribute to understanding Arachnida genome evolution and mite vector competence and provide key information for scrub typhus prevention.

The chigger mite, Leptotrombidium pallidum, is widely distributed throughout South Korea and is a major vector for Orientia tsutsugamushi, the causative agent of scrub typhus. In this study, the genome size of the chigger mite was estimated to determine the necessary coverage level prior to whole genome sequencing. Cloning of EF1α and RpS3 as putative single copy reference genes were conducted and their partial sequences were determined. Using the serially diluted reference genes with known amount as standard templates, the weight of a single copy of the genome was predicted by a method based on quantitative real time PCR. The average genome length estimated from the weight using two methods was 191 ± 7 Mb. When the genome size of other arthropods (Drosophila melanogster, Apis mellifera and Tetranychus urticae), with their genome analysis completed, were estimated using the same method and compared with actual values, the estimation accuracy was 79.8-98.9%, suggesting our current estimation of L. pallidum genome size is reliable. The estimated L. pallidum genome size is in a similar range to other Acariform mites, such as the dust mite and scabie mite, but appoximately 10-fold smaller compared to the deer tick, which belongs to Parasitiform. Our finding provides key information for further genome sequencing and understanding of mite genome evolution.

The body and head lice (Pediculus humanus humanus and Pediculus humanus capitis, respectively) are hematophagous ectoparasites of humans and only the body louse between two is known to transmit three bacterial diseases through its feces. The proliferation profiles of Bartonella quintana, the causative agent of trench fever, inside the louse body and its excretion patterns were investigated in the two louse subspecies following oral challenge with B. quintana-infected blood meal. The initial density of B. quintana was sustained inside head lice without any noticeable proliferation for the entire period after infection. In contrast, B. quintana proliferated rapidly inside body lice and the maximum density reached at 10 days post-infection. The numbers of bacteria detected in feces from infected lice were almost the same and steadily decreased over time in both body and head lice. Nevertheless, the viability of the bacteria, as determined by fluorescence, was significantly higher in body louse feces, especially at 1 day post-infection and this tendency lasted for 11 days. These findings suggest that excretion of feces containing more viable B. quintana that is proliferated inside body lice following ingestion of infected blood meal is responsible for the higher vector competence of body lice.