Overexpression of Mutant Galactose Permease (ScGal2_N376F) Effective for Utilization of Glucose/Xylose or Glucose/ Galactose Mixture by Engineered Kluyveromyces marxianus.

Mutant sugar transporter ScGAL2-N376F was overexpressed in Kluyveromyces marxianus for efficient utilization of xylose, which is one of the main components of cellulosic biomass. K. marxianus ScGal2_N376F, the ScGAL2-N376F-overexpressing strain, exhibited 47.04 g/l of xylose consumption and 26.55 g/l of xylitol production, as compared to the parental strain (24.68 g/l and 7.03 g/l, respectively) when xylose was used as the sole carbon source. When a mixture of glucose and xylose was used as the carbon source, xylose consumption and xylitol production rates were improved by 195% and 360%, respectively, by K. marxianus ScGal2_N376F. Moreover, the glucose consumption rate was improved by 27% as compared to that in the parental strain. Overexpression of both wild-type ScGAL2 and mutant ScGAL2-N376F showed 48% and 52% enhanced sugar consumption and ethanol production rates, respectively, when a mixture of glucose and galactose was used as the carbon source, which is the main component of marine biomass. As shown in this study, ScGAL2-N376F overexpression can be applied for the efficient production of biofuels or biochemicals from cellulosic or marine biomass.

Monitoring of Pyrethroid Resistance Allele Frequency in the Common Bed Bug (Cimex lectularius) in the Republic of Korea.

Two-point mutations (V419L and L925I) on the voltage-sensitive sodium channel of bed bugs (Cimex lectularius) are known to confer pyrethroid resistance. To determine the status of pyrethroid resistance in bed bugs in Korea, resistance allele frequencies of bed bug strains collected from several US military installations in Korea and Mokpo, Jeollanamdo, from 2009-2019 were monitored using a quantitative sequencing. Most bed bugs were determined to have both of the point mutations except a few specimens, collected in 2009, 2012 and 2014, having only a single point mutation (L925I). No susceptible allele was observed in any of the bed bugs examined, suggesting that pyrethroid resistance in bed bug populations in Korea has reached a serious level. Large scale monitoring is required to increase our knowledge on the distribution and prevalence of pyrethroid resistance in bed bug populations in Korea. Based on present study, it is urgent to restrict the use of pyrethroids and to introduce effective alternative insecticides. A nation-wide monitoring program to determine the pyrethroid resistance level in bed bugs and to select alternative insecticides should be implemented.

Alleviation of catabolite repression in Kluyveromyces marxianus: the thermotolerant SBK1 mutant simultaneously coferments glucose and xylose.

BACKGROUND: Simultaneous cofermentation of glucose and xylose mixtures would be a cost-effective solution for the conversion of cellulosic biomass to high-value products. However, most yeasts ferment glucose and xylose sequentially due to glucose catabolite repression. A well known thermotolerant yeast, Kluyveromyces marxianus, was selected for this work because it possesses cost-effective advantages over Saccharomyces cerevisiae for biofuel production from cellulosic biomass. RESULTS: In the present study, we employed a directed evolutionary approach using 2-deoxyglucose to develop a thermotolerant mutant capable of simultaneous cofermentation of glucose and xylose by alleviating catabolite repression. The selected mutant, K. marxianus SBK1, simultaneously cofermented 40 g/L glucose and 28 g/L xylose to produce 23.82 g/L ethanol at 40 °C. This outcome corresponded to a yield of 0.35 g/g and productivity of 0.33 g/L h, representing an 84% and 129% improvement, respectively, over the parental strain. Interestingly, following mutagenesis the overall transcriptome of the glycolysis pathway was highly downregulated in K. marxianus SBK1, except for glucokinase-1 (GLK1) which was 21-fold upregulated. Amino acid sequence of GLK1 from K. marxianus SBK1 revealed three amino acid mutations which led to more than 22-fold lower enzymatic activity compared to the parental strain. CONCLUSIONS: We herein successfully demonstrated that the cofermentation of a sugar mixture is a promising strategy for the efficient utilization of cellulosic biomass by K. marxianus SBK1. Through introduction of additional biosynthetic pathways, K. marxianus SBK1 could become a chassis-type strain for the production of fuels and chemicals from cellulosic biomass.

Ethanol production from xylose is highly increased by the Kluyveromyces marxianus mutant 17694-DH1.

Directed evolutionary approach and random mutagenesis were performed on thermotolerant yeast Kluyveromyces marxianus KCTC17694 for isolating a yeast strain producing ethanol from xylose efficiently. The isolated mutant strain, K. marxianus 17694-DH1, showed 290% and 131% improvement in ethanol concentration and ethanol production yield from xylose, respectively, as compared with the parental strain. Sequencing of the KmXYL1 gene of K. marxianus 17694-DH1 revealed substitutions of arginine and tryptophan with lysine and leucine at positions 25 and 202, respectively, as compared to the parental strain. In addition, sequencing of the KmXYL2 gene uncovered a substitution of glutamate with leucine at position 232. When enzymatic assays of xylose reductase (XR) and xylitol dehydrogenase (XDH) from the parental strain and K. marxianus 17694-DH1 were performed, XR activities were not significantly different whereas XDH activities were significantly improved in the mutant strain up to 50 °C of reaction temperatures. RNA-Seq based transcriptome analysis showed that alcohol dehydrogenases and glucose transporters were up-regulated while TCA cycle involved enzymes were down-regulated in K. marxianus 17694-DH1.

An investigation of the molecular and biochemical basis underlying chlorantraniliprole-resistant Drosophila strains and their cross-resistance to other insecticides.

Chlorantraniliprole is an anthranilic diamide insecticide that binds to the insect ryanodine receptor (RyR) and induces an uncontrolled release of Ca2+ , resulting in paralysis and ultimately death of the target insects. Recently, it was reported that chlorantraniliprole-resistant diamondback moths, Plutella xylostella Linnaeus, have mutations in their RyR. In this study, we developed two different chlorantraniliprole-resistant Drosophila melanogaster strain. The resistance ratio (RR) of the low-concentration chlorantraniliprole-treated resistant (Low-Res) strain was 2.3, while that of the high-concentration chlorantraniliprole-treated resistant (High-Res) strain was 21.3. The LC 50 of the untreated control (Con) strain was 23.8~25.9 ppm, which was significantly higher than that reported for the susceptible diamondback moth (0.03~0.51 ppm). The high LC 50 of the Con may be because the helix S2 amino acid sequence of D. melanogaster RyR ( DmRyR) is identical to the I4790M mutation of the chlorantraniliprole-resistant diamondback moths, resulting in a lower binding affinity of DmRyR for chlorantraniliprole. Among the tested detoxification enzymes, the activity of esterase was significantly increased in the two Res strains, but glutathione S-transferases and acetylcholinesterase were significantly decreased in the two Res strains. The cross-resistance of the High-Res strain to other insecticides with different modes of actions (MoAs) revealed that the RRs of the neuronal acetylcholine receptor allosteric and competitive modulators were significantly increased, while those of the Na 2+ channel modulators were significantly reduced. Our studies showed that RRs against the same insecticide vary with the treatment concentration, and that RRs against other insecticides with different MoAs can be altered.

Transcriptome-based identification and characterization of genes commonly responding to five different insecticides in the diamondback moth, Plutella xylostella.

When the 3rd instar larvae of the diamondback moth (DBM), Plutella xylostella, were pretreated with sublethal doses (LC10) and then subsequently exposed to lethal doses (LC50) of chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad via leaf dipping, their tolerance to insecticides was significantly enhanced. To identify genes that commonly respond to the treatment of different insecticides and are responsible for the tolerance enhancement, transcriptomic profiles of larvae treated with sublethal doses of the five insecticides were compared with that of untreated control. A total of 117,181 transcripts with a mean length of 662bp were generated by de novo assembly, of which 35,329 transcripts were annotated. Among them, 125, 143, 182, 215 and 149 transcripts were determined to be up-regulated whereas 67, 45, 60, 60 and 38 genes were down-regulated following treatments with chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad, respectively. Gene ontology (GO) analysis of differentially expressed genes (DEGs) revealed little differences in their GO profiles between treatments with different insecticides except for spinosad. Finally, the DEGs commonly responding to all insecticides were selected for further characterization, and some of their over-transcription levels were confirmed by quantitative PCR. The most notable examples of commonly responding over-transcribed genes were two cytochrome P450 genes (Cyp301a1 and Cyp9e2) and nine cuticular protein genes. In contrast, several genes composing the mitochondrial energy generation system were significantly down-regulated in all treated larvae. Considering the distinct structure and mode of action of the five insecticides tested, the differentially expressed genes identified in this study appear to be involved in general chemical defense at the initial stage of intoxication. Their possible roles in the tolerance/resistance development were discussed.

Agroinfiltration-based expression of hairpin RNA in soybean plants for RNA interference against Tetranychus urticae.

The coatomer subunit alpha (COPA) and aquaporin 9 (AQ9) genes from the two-spotted spider mite, Tetranychus urticae, were previously determined to exhibit RNA interference (RNAi)-based lethality when their double-stranded RNAs were systemically delivered via multi-unit chambers (Kwon et al., 2016 [8]). In current study, the hairpin RNAs of the COPA and AQ9 were transiently expressed in soybean plants by agroinfiltration. When T. urticae was fed with the soybean plants agroinfiltrated with the COPA and AQ9 hairpin RNA cassettes, the cumulative mortality increased significantly at 6days post-infestation. Quantitative PCR analysis revealed that the transcript level of both COPA and AQ9 was significantly reduced in T. urticae after 2days post-infestation, thereby confirming that the significant increases in mortality resulted from the knockdown of COPA and AQ9 transcripts. Our findings demonstrate the utility of COPA and AQ9 as potential genes for plant host-mediated RNAi control of T. urticae. In addition, we proved the usefulness of agroinfiltration as a rapid validation tool for confirming the RNAi-based lethality of target genes against arthropod pests before producing transgenic plants as agroinfiltration requires less time and skill to validate transgene function. Furthermore, these findings prove the concept that hairpin RNA expressed in plant hosts can also induce RNAi and eventually kill T. urticae, a sap-sucking pest.

Screening of target genes for RNAi in Tetranychus urticae and RNAi toxicity enhancement by chimeric genes.

Due to its rapid development of resistance to nearly all arrays of acaricide, Tetranychus urticae is extremely hard to control using conventional acaricides. As an alternative control measure of acaricide-resistant mites, RNA interference (RNAi)-based method has recently been suggested. A double-stranded RNA (dsRNA) delivery method using multi-unit chambers was established and employed to screen the RNAi toxicity of 42 T. urticae genes. Among them, the dsRNA treatment of coatomer I (COPI) genes, such as coatomer subunit epsilon (COPE) and beta 2 (COPB2), resulted in high mortality [median lethal time (LT50)=89.7 and 120.3h, respectively]. The transcript level of the COPE gene was significantly (F3,9=16.2, P=0.001) reduced by up to 24% following dsRNA treatment, suggesting that the toxicity was likely mediated by the RNAi of the target gene. As a toxicity enhancement strategy, the recombinant dsRNA was generated by reciprocally recombining half-divided fragments of COPE and COPB2. The two recombinant dsRNAs exhibited higher toxicity than the respective single dsRNA treatments as determined by LT50 values (79.2 and 81.5h, respectively). This finding indicates that the recombination of different genes can enhance RNAi toxicity and be utilized to generate synthetic dsRNA with improved RNAi efficacy.

Phenotypic- and Genotypic-Resistance Detection for Adaptive Resistance Management in Tetranychus urticae Koch.

Rapid resistance detection is necessary for the adaptive management of acaricide-resistant populations of Tetranychus urticae. Detection of phenotypic and genotypic resistance was conducted by employing residual contact vial bioassay (RCV) and quantitative sequencing (QS) methods, respectively. RCV was useful for detecting the acaricide resistance levels of T. urticae, particularly for on-site resistance detection; however, it was only applicable for rapid-acting acaricides (12 out of 19 tested acaricides). QS was effective for determining the frequencies of resistance alleles on a population basis, which corresponded to 12 nonsynonymous point mutations associated with target-site resistance to five types of acaricides [organophosphates (monocrotophos, pirimiphos-methyl, dimethoate and chlorpyrifos), pyrethroids (fenpropathrin and bifenthrin), abamectin, bifenazate and etoxazole]. Most field-collected mites exhibited high levels of multiple resistance, as determined by RCV and QS data, suggesting the seriousness of their current acaricide resistance status in rose cultivation areas in Korea. The correlation analyses revealed moderate to high levels of positive relationships between the resistance allele frequencies and the actual resistance levels in only five of the acaricides evaluated, which limits the general application of allele frequency as a direct indicator for estimating actual resistance levels. Nevertheless, the resistance allele frequency data alone allowed for the evaluation of the genetic resistance potential and background of test mite populations. The combined use of RCV and QS provides basic information on resistance levels, which is essential for choosing appropriate acaricides for the management of resistant T. urticae.

Toxicodynamic mechanisms and monitoring of acaricide resistance in the two-spotted spider mite.

The two-spotted spider (Tetranychus urticae) is one of the most serious pests world-wide and has developed resistance to many types of acaricides. Various mutations on acaricide target site genes have been determined to be responsible for toxicodynamic resistance, and the genotyping and frequency prediction of these mutations can be employed as an alternative resistance monitoring strategy. A quantitative sequencing (QS) protocol was reported as a population-based genotyping technique, and applied for the determination of resistance allele frequencies in T. urticae field populations. In addition, a modified glass vial bioassay method (residual contact vial bioassay, RCV) was implemented as a rapid on-site resistance monitoring tool. The QS protocol, together with the RCV, would greatly facilitate monitoring of T. urticae resistance. Recent completion of T. urticae genome analysis should facilitate the identification of additional resistance genetic markers that can be employed for molecular resistance monitoring.

Mutation and duplication of arthropod acetylcholinesterase: Implications for pesticide resistance and tolerance.

A series of common/shared point mutations in acetylcholinesterase (AChE) confers resistance to organophosphorus and carbamate insecticides in most arthropod pests. However, the mutations associated with reduced sensitivity to insecticides usually results in the reduction of catalytic efficiency and leads to a fitness disadvantage. To compensate for the reduced catalytic activity, overexpression of neuronal AChE appears to be necessary, which is achieved by a relatively recent duplication of the AChE gene (ace) as observed in the two-spotted spider mite and other insects. Unlike the cases with overexpression of neuronal AChE, the extensive generation of soluble AChE is observed in some insects either from a distinct non-neuronal ace locus or from a single ace locus via alternative splicing. The production of soluble AChE in the fruit fly is induced by chemical stress. Soluble AChE acts as a potential bioscavenger and provides tolerance to xenobiotics, suggesting its role in chemical adaptation during evolution.

Identification and characterization of an esterase involved in malathion resistance in the head louse Pediculus humanus capitis.

Enhanced malathion carboxylesterase (MCE) activity was previously reported to be involved in malathion resistance in the head louse Pediculus humanus capitis (Gao et al., 2006 [8]). To identify MCE, the transcriptional profiles of all five esterases that had been annotated to be catalytically active were determined and compared between the malathion-resistant (BR-HL) and malathion-susceptible (KR-HL) strains of head lice. An esterase gene, designated HLCbE3, exhibited approximately 5.4-fold higher transcription levels, whereas remaining four esterases did not exhibit a significant increase in their transcription in BR-HL, indicating that HLCbE3 may be the putative MCE. Comparison of the entire cDNA sequences of HLCbE3 revealed no sequence differences between the BR-HL and KR-HL strains and suggested that no single nucleotide polymorphism is associated with enhanced MCE activity. Two copies of the HLCbE3 gene were observed in BR-HL, implying that the over-transcription of HLCbE3 is due to the combination of a gene duplication and up-regulated transcription. Knockdown of HLCbE3 expression by RNA interference in the BR-HL strain led to increases in malathion susceptibility, confirming the identity of HLCbE3 as a MCE responsible for malathion resistance in the head louse. Phylogenetic analysis suggested that HLCbE3 is a typical dietary esterase and belongs to a clade containing various MCEs involved in malathion resistance.

Estimation of the genome sizes of the chigger mites Leptotrombidium pallidum and Leptotrombidium scutellare based on quantitative PCR and k-mer analysis.

BACKGROUND: 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, it is necessary to estimate their genome sizes to obtain basic information for establishing the strategies that should be used for genome sequencing and assembly. METHOD: The genome sizes of L. pallidum and L. scutellare were estimated by a method based on quantitative real-time PCR. In addition, a k-mer analysis of the whole-genome sequences obtained through Illumina sequencing was conducted to verify the mutual compatibility and reliability of the results. RESULTS: The genome sizes estimated using qPCR were 191 ± 7 Mb for L. pallidum and 262 ± 13 Mb for L. scutellare. The k-mer analysis-based genome lengths were estimated to be 175 Mb for L. pallidum and 286 Mb for L. scutellare. The estimates from these two independent methods were mutually complementary and within a similar range to those of other Acariform mites. CONCLUSIONS: The estimation method based on qPCR appears to be a useful alternative when the standard methods, such as flow cytometry, are impractical. The relatively small estimated genome sizes should facilitate whole-genome analysis, which could contribute to our understanding of Arachnida genome evolution and provide key information for scrub typhus prevention and mite vector competence.

Knockdown resistance allele frequencies in North American head louse (Anoplura: Pediculidae) populations.

The study examines the extent and frequency of a knockdown-type resistance allele (kdr type) in North American populations of human head lice. Lice were collected from 32 locations in Canada and the United States. DNA was extracted from individual lice and used to determine their zygosity using the serial invasive signal amplification technique to detect the kdr-type T917I (TI) mutation, which is most responsible for nerve insensitivity that results in the kdr phenotype and permethrin resistance. Previously sampled sites were resampled to determine if the frequency of the TI mutation was changing. The TI frequency was also reevaluated using a quantitative sequencing method on pooled DNA samples from selected sites to validate this population genotyping method. Genotyping substantiated that TI occurs at high levels in North American lice (88.4%). Overall, the TI frequency in U.S. lice was 84.4% from 1999 to 2009, increased to 99.6% from 2007 to 2009, and was 97.1% in Canadian lice in 2008. Genotyping results using the serial invasive signal amplification reaction (99.54%) and quantitative sequencing (99.45%) techniques were highly correlated. Thus, the frequencies of TI in North American head louse populations were found to be uniformly high, which may be due to the high selection pressure from the intensive and widespread use of the pyrethrins- or pyrethroid-based pediculicides over many years, and is likely a main cause of increased pediculosis and failure of pyrethrins- or permethrin-based products in Canada and the United States. Alternative approaches to treatment of head lice infestations are critically needed.

Biochemical and toxicological properties of two acetylcholinesterases from the common bed bug, Cimex lectularius.

We examined the molecular and enzymatic properties of two acetylcholinesterases (AChEs; ClAChE1 and ClAChE2) from the common bed bug, Cimex lectularius. Native polyacrylamide gel electrophoresis followed by activity staining and Western blotting revealed that ClAChE1 is the main catalytic enzyme and is abundantly expressed in various tissues. Both ClAChEs existed in dimeric form connected by a disulfide bridge and were attached to the membrane via a glycophosphatidylinositol anchor. To determine their kinetic and inhibitory properties, both ClAChE1 and ClAChE2 were in vitro expressed in Sf9 cells using a baculovirus expression system. ClAChE1 showed higher catalytic efficiency toward acetylcholine, supporting the hypothesis that ClAChE1 plays a major role in postsynaptic transmission. An inhibition assay revealed that ClAChE1 is generally more sensitive to organophosphates and carbamates examined although ClAChE2 was >4000-fold more sensitive to malaoxon than ClAChE1. The relatively higher correlation between the in vitro ClAChE1 inhibition and the in vivo toxicity suggested that ClAChE1 is the more relevant toxicological target for organophosphates and carbamates. Although the physiological function of ClAChE2 remains to be elucidated, ClAChE2 also appears to have neuronal functions, as judged by its tissue distribution and molecular and kinetic properties. Our findings help expand our knowledge on insect AChEs and their toxicological properties.

Induction of soluble AChE expression via alternative splicing by chemical stress in Drosophila melanogaster.

Various molecular forms of acetylcholinesterase (AChE) have been characterized in insects. Post-translational modification is known to be a major mechanism for the molecular diversity of insect AChE. However, multiple forms of Drosophila melanogaster AChE (DmAChE) were recently suggested to be generated via alternative splicing (Kim and Lee, 2013). To confirm alternative splicing as the mechanism for generating the soluble form of DmAChE, we generated a transgenic fly strain carrying the cDNA of DmAChE gene (Dm_ace) that predominantly expressed a single transcript variant encoding the membrane-anchored dimer. 3' RACE (rapid amplification of cDNA ends) and western blotting were performed to compare Dm_ace transcript variants and DmAChE forms between wild-type and transgenic strains. Various Dm_ace transcripts and DmAChE molecular forms were observed in wild-type flies, whereas the transgenic fly predominantly expressed Dm_ace transcript variant encoding the membrane-anchored dimer. This supports alternative splicing as the major determinant in the generation of multiple forms of DmAChE. In addition, treatment with DDVP as a chemical stress induced the expression of the Dm_ace splice variant without the glycosylphosphatidylinositol anchor site in a dose-dependent manner and, accordingly, the soluble form of DmAChE in wild-type flies. In contrast, little soluble DmAChE was expressed in the transgenic fly upon exposure to DDVP. DDVP bioassays revealed that transgenic flies, which were unable to express a sufficient amount of soluble monomeric DmAChE, were more sensitive to DDVP compared to wild-type flies, suggesting that the soluble monomer may exert non-neuronal functions, such as chemical defense against xenobiotics.

Screening of lethal genes for feeding RNAi by leaf disc-mediated systematic delivery of dsRNA in Tetranychus urticae.

To identify genes that kill Tetranychus urticae when knocked down via RNA interference (RNAi), several lethal genes were screened by the systemic delivery of dsRNA via leaf disc feeding. Four candidate genes (ß subunit of coatomer protein complex, T-COPB2; M1 metalloprotease, T-M1MP; Ribosomal protein S4, T-RPS4; A subunit of V-ATPase, T-VATPase) and a control gene (EGFP) were tested for RNAi. All dsRNAs that permeated the leaf disc (ca. 15-mm diameter) were detected at 12h post-treatment, indicating that dsRNA could move through vascular tissues. To evaluate RNAi toxicity, mortalities were assessed for 120h following treatment with dsRNA. Treatment with T-COPB2, T-M1MP, T-RPS4 and T-VATPase dsRNAs caused 65.4%, 15.9%, 36.1% and 21.1% mortalities at 120h post-treatment, respectively. Reduction of all target gene transcripts following dsRNA treatment was confirmed by quantitative PCR, demonstrating that dsRNA feeding-based RNAi could indeed kill T. urticae. In summary, dsRNA delivery via leaf disc is an effective system to screen for lethal genes. Furthermore, some genes, such as T-COPB2, T-M1MP, T-RPS4 and T-VATPase, can be used to establish an RNAi-based control system against T. urticae.

The overexpression of acetylcholinesterase compensates for the reduced catalytic activity caused by resistance-conferring mutations in Tetranychus urticae.

The mutations (G228S, A391T and F439W) and duplication of the acetylcholinesterase (AChE) gene (Tuace) are involved in monocrotophos resistance in the two-spotted spider mites, Tetranychus urticae (Kwon et al., 2010a, b). The overexpression of T. urticae AChE (TuAChE) as a result of Tuace duplication was confirmed in several field-collected populations by Western blotting using an AChE-specific antibody. To investigate the effects of each mutation on the insensitivity and fitness cost of AChE, eight variants of TuAChE were expressed in vitro using the baculovirus expression system. Kinetic analysis revealed that the G228S and F439W mutations confer approximately 26-fold and 99-fold increases in the insensitivity to monocrotophos, respectively, whereas the insensitivity increased over 1165-fold in the AChE with double mutations. Nevertheless, the presence of these mutations reduced the catalytic efficiency of AChE significantly. In particular, the TuAChE having both mutations together exhibited a 17.8∼27.1-fold reduced catalytic efficiency, suggesting an apparent fitness cost in the monocrotophos-resistant mites. The A391T mutation did not change the kinetic properties of either the substrate or inhibitor when present alone but mitigated the negative impacts of the F439 mutation. To simulate the catalytic activity of the overexpressed TuAChE in two T. urticae strains (approximately 6 copies for AD strain vs. 2 copies for PyriF strain), appropriate TuAChE variants were combined to make up the desired AChE copies and mutation frequencies, and their enzyme kinetics were determined. The reconstituted 6-copy and 2-copy TuAChEs exhibited catalytic efficiency levels comparable to those of a single-copy wildtype TuAChE, suggesting that, if mutations are present, multiple copies of AChE are required to restore a normal level of catalytic activity in the monocrotophos-resistant mites. In summary, the present study provides clear evidence that Tuace duplication resulted in the proportional overexpression of AChE, which was necessary to compensate for the reduced catalytic activity of AChE caused by mutations.

Comparison of the humoral and cellular immune responses between body and head lice following bacterial challenge.

The differences in the immune response between body lice, Pediculus humanus humanus, and head lice, Pediculus humanus capitis, were investigated initially by measuring the proliferation rates of two model bacteria, a Gram-positive Staphylococcus aureus and a Gram-negative Escherichia coli, following challenge by injection. Body lice showed a significantly reduced immune response compared to head lice particularly to E. coli at the early stage of the immune challenge. Annotation of the body louse genome identified substantially fewer immune-related genes compared with other insects. Nevertheless, all required genetic components of the major immune pathways, except for the immune deficiency (Imd) pathway, are still retained in the body louse genome. Transcriptional profiling of representative genes involved in the humoral immune response, following bacterial challenge, revealed that both body and head lice, regardless of their developmental stages, exhibited an increased immune response to S. aureus but little to E. coli. Head lice, however, exhibited a significantly higher phagocytotic activity against E. coli than body lice, whereas the phagocytosis against S. aureus differed only slightly between body and head lice. These findings suggest that the greater immune response in head lice against E. coli is largely due to enhanced phagocytosis and not due to differences in the humoral immune response. The reduced phagocytotic activity in body lice could be responsible, in part, for their increased vector competence.

Establishment of quantitative sequencing and filter contact vial bioassay for monitoring pyrethroid resistance in the common bed bug, Cimex lectularius.

Two point mutations (V419L and L925I) in the voltage-sensitive sodium channel alpha-subunit gene have been identified in deltamethrin-resistant bed bugs. A quantitative sequencing (QS) protocol was developed to establish a population-based genotyping method as a molecular resistance-monitoring tool based on the frequency of the two mutations. The nucleotide signal ratio at each mutation site was generated from sequencing chromatograms and plotted against the corresponding resistance allele frequency. Frequency prediction equations were generated from the plots by linear regression, and the signal ratios were shown to highly correlate with resistance allele frequencies (r2 > 0.9928). As determined by QS, neither mutation was found in a bed bug population collected in 1993. Populations collected in recent years (2007-2009), however, exhibited completely or nearly saturating L925I mutation frequencies and highly variable frequencies of the V419L mutation. In addition to QS, the filter contact vial bioassay (FCVB) method was established and used to determine the baseline susceptibility and resistance of bed bugs to deltamethrin and lambda-cyhalothrin. A pyrethroid-resistant strain showed >9,375- and 6,990-fold resistance to deltamethrin and lambda-cyhalothrin, respectively. Resistance allele frequencies in different bed bug populations predicted by QS correlated well with the FCVB results, confirming the roles of the two mutations in pyrethroid resistance. Taken together, employment of QS in conjunction with FCVB should greatly facilitate the detection and monitoring of pyrethroid-resistant bed bugs in the field. The advantages of FCVB as an on-site resistance-monitoring tool are discussed.