Kentaro Itokawa

Mechanisms of Pyrethroid Resistance in the Dengue Mosquito Vector, Aedes aegypti: Target Site Insensitivity, Penetration, and Metabolism

Aedes aegypti is the major vector of yellow and dengue fevers. After 10 generations of adult selection, an A. aegypti strain (SP) developed 1650-fold resistance to permethrin, which is one of the most widely used pyrethroid insecticides for mosquito control. SP larvae also developed 8790-fold resistance following selection of the adults. Prior to the selections, the frequencies of V1016G and F1534C mutations in domains II and III, respectively, of voltage-sensitive sodium channel (Vssc, the target site of pyrethroid insecticide) were 0.44 and 0.56, respectively. In contrast, only G1016 alleles were present after two permethrin selections, indicating that G1016 can more contribute to the insensitivity of Vssc than C1534. In vivo metabolism studies showed that the SP strain excreted permethrin metabolites more rapidly than a susceptible SMK strain. Pretreatment with piperonyl butoxide caused strong inhibition of excretion of permethrin metabolites, suggesting that cytochrome P450 monooxygenases (P450s) play an important role in resistance development. In vitro metabolism studies also indicated an association of P450s with resistance. Microarray analysis showed that multiple P450 genes were over expressed during the larval and adult stages in the SP strain. Following quantitative real time PCR, we focused on two P450 isoforms, CYP9M6 and CYP6BB2. Transcription levels of these P450s were well correlated with the rate of permethrin excretion and they were certainly capable of detoxifying permethrin to 4′-HO-permethrin. Over expression of CYP9M6 was partially due to gene amplification. There was no significant difference in the rate of permethrin reduction from cuticle between SP and SMK strains.

Genomic structures of Cyp9m10 in pyrethroid resistant and susceptible strains of Culex quinquefasciatus.

Development of insecticide resistance reduces the efficacy of controlling measures against the medical and agricultural insect pests. Cytochrome P450s are one of the major detoxification enzymes involved in insecticide metabolisms. Previously, we have reported that the P450 gene Cyp9m10 is about 260-fold overexpressed in a pyrethroid-resistant strain of Culex quinquefasciatus compared to a susceptible strain. In this study, we obtained direct evidence that the Cyp9m10 overexpression is caused by a cis-acting mutation. Additionally, a region of approximately 100 kb in length including the Cyp9m10 locus was specifically duplicated in the resistant strain. The two duplicated Cyp9m10 copies shared a completely identical sequence within the transcribed region and the flanking region up to the breakpoint located 1.1 kb upstream of the transcriptional start site. A Miniature Inverted-repeat Transposable Element (MITE)-like element was specifically inserted 0.2 kb upstream of both Cyp9m10 copies in the resistant strain. In backcross experiment, a haplotype containing the two duplicated Cyp9m10 copies was strongly associated with the pyrethroid resistance.

A Single Crossing-Over Event in Voltage-Sensitive Na + Channel Genes May Cause Critical Failure of Dengue Mosquito Control by Insecticides

The voltage-sensitive sodium (Na+) channel (Vssc) is the target site of pyrethroid insecticides. Pest insects develop resistance to this class of insecticide by acquisition of one or multiple amino acid substitution(s) in this channel. In Southeast Asia, two major Vssc types confer pyrethroid resistance in the dengue mosquito vector Aedes aegypti, namely, S989P+V1016G and F1534C. We expressed several types of Vssc in Xenopus oocytes and examined the effect of amino acid substitutions in Vssc on pyrethroid susceptibilities. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to permethrin by 100- and 25-fold, respectively, while S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to permethrin by 1100-fold. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to deltamethrin by 10- and 1-fold (no reduction), respectively, but S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to deltamethrin by 90-fold. These results imply that pyrethroid insecticides are highly likely to lose their effectiveness against A. aegypti if such a Vssc haplotype emerges as the result of a single crossing-over event; thus, this may cause failure to control this key mosquito vector. Here, we strongly emphasize the importance of monitoring the occurrence of triple mutations in Vssc in the field population of A. aegypti.

Biting density and distribution of Aedes albopictus during the September 2014 outbreak of dengue fever in Yoyogi Park and the vicinity in Tokyo Metropolis, Japan.

A total of 160 autochthonous dengue cases transmitted by Aedes albopictus were reported between August and October of 2014 in Tokyo Metropolis, Japan. Ae. albopictus is a medically important vector of dengue virus, which has expanded its geographic distribution in temperate regions. Understanding the distribution and biting density of Ae. albopictus during the 2014 dengue outbreak in Tokyo is important to evaluate the epidemic risks of dengue fever in other highly populated cities in Europe and Asia. Of the 160 patients, 134 visited the same park (Yoyogi Park) located in central Tokyo. Mosquitoes infected with dengue virus were collected from this park, suggesting that it was the exclusive location for the transmission of dengue. This study aimed to collect referential data to estimate the transmission threshold of dengue virus in terms of biting density of Ae. albopictus and determined high transmission risk areas of dengue virus in Yoyogi Park and its vicinity. The overall mean density of biting Ae. albopictus (7.13/man/8 min) was sufficiently high for successful transmission of dengue virus, and areas with biting densities higher than the overall mean density were classified as high risk areas for the transmission of dengue virus in Yoyogi Park.

Cis-acting mutation and duplication: History of molecular evolution in a P450 haplotype responsible for insecticide resistance in Culex quinquefasciatus

A cytochrome P450 gene, Cyp9m10, is more than 200-fold overexpressed in a pyrethroid resistant strain of Culex quinquefasciatus, JPal-per. The haplotype of this strain contains two copies of Cyp9m10 resulted from recent tandem duplication. In this study, we discovered and isolated a Cyp9m10 haplotype closely related to this duplicated Cyp9m10 haplotype from JHB, a strain used for the recent genome project for this mosquito species. The isolated haplotype (JHB-NIID-B haplotype) shared the same insertion of a transposable element upstream of the coding region with JPal-per strain but not duplicated. The JHB-NIID-B haplotype was considered to have diverged from the JPal-per lineage just before the duplication event. Cyp9m10 was moderately overexpressed in larvae with the JHB-NIID-B haplotype. The overexpressions in JHB-NIID-B and JPal-per haplotypes were developmentally regulated in similar pattern indicating both haplotypes share a common cis-acting mutation responsible for the overexpressions. The isolated moderately overexpressed haplotype conferred resistance, however, its efficacy was relatively small. We hypothesized that the first cis-acting mutation modified the consequence of the subsequent duplication in JPal-per lineage to confer stronger phenotypic effect than that if it occurred before the first cis-acting mutation.

Testing the causality between CYP9M10 and pyrethroid resistance using the TALEN and CRISPR/Cas9 technologies

Recently-emerging genome editing technologies have enabled targeted gene knockout experiments even in non-model insect species. For studies on insecticide resistance, genome editing technologies offer some advantages over the conventional reverse genetic technique, RNA interference, for testing causal relationships between genes of detoxifying enzymes and resistance phenotypes. There were relatively abundant evidences indicating that the overexpression of a cytochrome P450 gene CYP9M10 confers strong pyrethroid resistance in larvae of the southern house mosquito Culex quinquefasciatus. However, reverse genetic verification has not yet been obtained because of the technical difficulty of microinjection into larvae. Here, we tested two genome editing technologies, transcription activator-like effector nucleases (TALEN)s and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), to disrupt CYP9M10 in a resistant strain of C. quinquefasciatus. Additionally, we developed a novel, effective approach to construct a TALE using the chemical cleavage of phosphorothioate inter-nucleotide linkages in the level 1 assembly. Both TALEN and CRISPR/Cas9 induced frame-shifting mutations in one or all copies of CYP9M10 in a pyrethroid-resistant strain. A line fixed with a completely disrupted CYP9M10 haplotype showed more than 100-fold reduction in pyrethroid resistance in the larval stage.

Global spread and genetic variants of the two CYP9M10 haplotype forms associated with insecticide resistance in Culex quinquefasciatus Say.

Insecticide resistance develops as a genetic factor (allele) conferring lower susceptibility to insecticides proliferates within a target insect population under strong positive selection. Intriguingly, a resistance allele pre-existing in a population often bears a series of further adaptive allelic variants through new mutations. This phenomenon occasionally results in replacement of the predominating resistance allele by fitter new derivatives, and consequently, development of greater resistance at the population level. The overexpression of the cytochrome P450 gene CYP9M10 is associated with pyrethroid resistance in the southern house mosquito Culex quinquefasciatus. Previously, we have found two genealogically related overexpressing CYP9M10 haplotypes, which differ in gene copy number (duplicated and non-duplicated). The duplicated haplotype was derived from the non-duplicated overproducer probably recently. In the present study, we investigated allelic series of CYP9M10 involved in three C. quinquefasciatus laboratory colonies recently collected from three different localities. Duplicated and non-duplicated overproducing haplotypes coexisted in African and Asian colonies indicating a global distribution of both haplotype lineages. The duplicated haplotypes both in the Asian and African colonies were associated with higher expression levels and stronger resistance than non-duplicated overproducing haplotypes. There were slight variation in expression level among the non-duplicated overproducing haplotypes. The nucleotide sequences in coding and upstream regions among members of this group also showed a little diversity. Non-duplicated overproducing haplotypes with relatively higher expression were genealogically closer to the duplicated haplotypes than the other non-duplicated overproducing haplotypes, suggesting multiple cis-acting mutations before duplication.

Isolation and characterization of a new iflavirus from Armigeres spp. mosquitoes in the Philippines

During an entomological surveillance for arthropod-borne viruses in the Philippines, we isolated a previously unrecognized virus from female Armigeres spp. mosquitoes. Whole-genome sequencing, genetic characterization and phylogenetic analysis revealed that the isolated virus, designated Armigeres iflavirus (ArIFV), is a novel member of the iflaviruses (genus Iflavirus, family Iflaviridae) and phylogenetically related to Moku virus, Hubei odonate virus 4, slow bee paralysis virus and Graminella nigrifrons virus 1. To our knowledge, this is the first successful isolation of iflavirus from a dipteran insect. Spherical ArIFV particles of approximately 30 nm in diameter contained at least three major structural proteins. ArIFV multiplied to high titres (~109 p.f.u. ml-1) and formed clear plaques in a mosquito cell line, C6/36. Our findings provide new insights into the infection mechanism, genetic diversity and evolution of the Iflaviridae family.

A summary of the imported cases of Chikungunya fever in Japan from 2006 to June 2016

Background Due to the huge 2-way human traffic between Japan and Chikungunya (CHIK) fever-endemic regions, 89 imported cases of CHIK fever were confirmed in Japan from January 2006 to June 2016. Fifty-four of 89 cases were confirmed virologically and serologically at the National Institute of Infectious Diseases, Japan and we present the demographic profiles of the patients and the phylogenetic features of 14 CHIK virus (CHIKV) isolates. Methods Patients were diagnosed with CHIK fever by a combination of virus isolation, viral RNA amplification, IgM antibody-, IgG antibody-, and/or neutralizing antibody detection. The whole-genome sequences of the CHIKV isolates were determined by next-generation sequencing. Results Prior to 2014, the source countries of the imported CHIK fever cases were limited to South and Southeast Asian countries. After 2014, when outbreaks occurred in the Pacific and Caribbean Islands and Latin American countries, there was an increase in the number of imported cases from these regions. A phylogenetic analysis of 14 isolates revealed that four isolates recovered from three patients who returned from Sri Lanka, Malaysia and Angola, belonged to the East/Central/South African genotype, while 10 isolates from 10 patients who returned from Indonesia, the Philippines, Tonga, the Commonwealth of Dominica, Colombia and Cuba, belonged to the Asian genotype. Conclusion Through the phylogenetic analysis of the isolates, we could predict the situations of the CHIK fever epidemics in Indonesia, Angola and Cuba. Although Japan has not yet experienced an autochthonous outbreak of CHIK fever, the possibility of the future introduction of CHIKV through an imported case and subsequent local transmission should be considered, especially during the mosquito-active season. The monitoring and reporting of imported cases will be useful to understand the situation of the global epidemic, to increase awareness of and facilitate the diagnosis of CHIK fever, and to identify a future CHIK fever outbreak in Japan.

Dengue Virus Infection in Aedes albopictus during the 2014 Autochthonous Dengue Outbreak in Tokyo Metropolis, Japan

In 2014 in Japan, 162 autochthonous dengue cases were reported for the first time in nearly 70 years. Here, we report the results of the detection and isolation of dengue virus (DENV) from mosquitoes collected in Tokyo Metropolis in 2014 and 2015. The phylogenetic relationship among DENV isolates from mosquitoes and from patients based on both the entire envelope gene and whole coding sequences was evaluated. Herein, 2,298 female and 956 male Aedes albopictus mosquitoes were collected at six suspected locations of DENV infection in Tokyo Metropolis from August to October in 2014 and grouped into 124 and 35 pools, respectively, for viral genome detection and DENV isolation. Dengue virus RNA was detected using reverse transcription polymerase chain reaction and TaqMan assays from 49 female pools; 16 isolates were obtained using C6/36 and Vero cells. High minimum infection rates (11.2-66.7) persisted until mid-September. All DENV isolates belonged to the genotype I in serotype 1 (DENV-1), and its sequences demonstrated > 99% homology to the sequence of the DENV isolated from a patient in the vicinity of Tokyo Metropolis in 2014. Therefore, Ae. albopictus was a major DENV vector, and a single DENV-1 strain circulated in Tokyo Metropolis in 2014. Dengue virus was not detected from male mosquitoes in 2014 and wild larvae in April 2015. Thus, the possibility of both vertical transmission and overwintering of DENV was extremely low, even in dengue-epidemic areas. This study reports the first entomological information on a dengue outbreak in a temperate region, where no Aedes aegypti mosquitoes are distributed.