Shinji Kasai

Genome of the house fly, Musca domestica L., a global vector of diseases with adaptations to a septic environment

BackgroundAdult house flies, Musca domestica L., are mechanical vectors of more than 100 devastating diseases that have severe consequences for human and animal health. House fly larvae play a vital role as decomposers of animal wastes, and thus live in intimate association with many animal pathogens.ResultsWe have sequenced and analyzed the genome of the house fly using DNA from female flies. The sequenced genome is 691 Mb. Compared with Drosophila melanogaster, the genome contains a rich resource of shared and novel protein coding genes, a significantly higher amount of repetitive elements, and substantial increases in copy number and diversity of both the recognition and effector components of the immune system, consistent with life in a pathogen-rich environment. There are 146 P450 genes, plus 11 pseudogenes, in M. domestica, representing a significant increase relative to D. melanogaster and suggesting the presence of enhanced detoxification in house flies. Relative to D. melanogaster, M. domestica has also evolved an expanded repertoire of chemoreceptors and odorant binding proteins, many associated with gustation.ConclusionsThis represents the first genome sequence of an insect that lives in intimate association with abundant animal pathogens. The house fly genome provides a rich resource for enabling work on innovative methods of insect control, for understanding the mechanisms of insecticide resistance, genetic adaptation to high pathogen loads, and for exploring the basic biology of this important pest. The genome of this species will also serve as a close out-group to Drosophila in comparative genomic studies.

Overexpression of cytochrome P450 genes in pyrethroid-resistant Culex quinquefasciatus.

JPal-per strain of Culex quinquefasciatus exhibits extremely high resistance against pyrethroids in larvae, though the resistance is greatly lower in adults. Increased microsome monooxygenase metabolism is one of the major factors of the larval resistance in this strain. We cloned 46 novel cytochrome P450 cDNAs from JPal-per strain. An oligonucleotide microarray was designed for the novel 46 genes plus 16 previously reported P450 genes along with other non-P450 gene probes. Of these, five P450 genes were upregulated (>2.5-fold) in JPal-per larvae as compared with a susceptible strain. The expression ratios for the highest three among the five P450 genes screened in the microarray analysis, CYP9M10, CYP4H34 and CYP6Z10, were further validated by qPCR as 264-, 8.3-, and 3.9-fold, respectively. In JPal-per, the transcription levels of CYP9M10 and CYP4H34 showed a similar stage-dependent pattern as a high expression level during the larvfrom Ogasawara Islands in Japanal stage dramatically decreases in the adult stage. This larval specific overexpression manner of the two genes was consistent with the characteristic of stage-dependent resistance of JPal-per strain previously reported, suggesting that the two P450s, CYP9M10 and CYP4H34, are involved in pyrethroid detoxification in JPal-per strain.

Multimodal Pyrethroid Resistance in Malaria Vectors, Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. in Western Kenya

Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. are the most important species for malaria transmission. Pyrethroid resistance of these vector mosquitoes is one of the main obstacles against effective vector control. The objective of the present study was to monitor the pyrethroid susceptibility in the 3 major malaria vectors in a highly malaria endemic area in western Kenya and to elucidate the mechanisms of pyrethroid resistance in these species. Gembe East and West, Mbita Division, and 4 main western islands in the Suba district of the Nyanza province in western Kenya were used as the study area. Larval and adult collection and bioassay were conducted, as well as the detection of point mutation in the voltage-gated sodium channel (1014L) by using direct DNA sequencing. A high level of pyrethroid resistance caused by the high frequency of point mutations (L1014S) was detected in An. gambiae s.s. In contrast, P450-related pyrethroid resistance seemed to be widespread in both An. arabiensis and An. funestus s.s. Not a single L1014S mutation was detected in these 2 species. A lack of cross-resistance between DDT and permethrin was also found in An. arabiensis and An. funestus s.s., while An. gambiae s.s. was resistant to both insecticides. It is noteworthy that the above species in the same area are found to be resistant to pyrethroids by their unique resistance mechanisms. Furthermore, it is interesting that 2 different resistance mechanisms have developed in the 2 sibling species in the same area individually. The cross resistance between permethrin and DDT in An. gambiae s.s. may be attributed to the high frequency of kdr mutation, which might be selected by the frequent exposure to ITNs. Similarly, the metabolic pyrethroid resistance in An. arabiensis and An. funestus s.s. is thought to develop without strong selection by DDT.

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.

Symbiotic bacteria associated with stomach discs of human lice

ABSTRACT The symbiotic bacteria associated with the stomach disc, a large aggregate of bacteriocytes on the ventral side of the midgut, of human body and head lice were characterized. Molecular phylogenetic analysis of 16S rRNA gene sequences showed that the symbionts formed a distinct and well-defined clade in the Gammaproteobacteria. The sequences exhibited AT-biased nucleotide composition and accelerated molecular evolution. In situ hybridization revealed that in nymphs and adult males, the symbiont was localized in the stomach disc, while in adult females, the symbiont was not in the stomach disc but in the lateral oviducts and the posterior pole of the oocytes due to female-specific symbiont migration. We propose the designation “Candidatus Riesia pediculicola” for the louse symbionts.

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.

Host-Feeding Habits of Culex pipiens and Aedes albopictus (Diptera: Culicidae) Collected at the Urban and Suburban Residential Areas of Japan

ABSTRACT To evaluate the vectorial capacity of mosquitoes for viruses in Japan, the host-feeding habits of the mosquitoes were analyzed by sequencing polymerase chain reaction-amplified fragments of the cytochrome b and 16S ribosomal RNA regions of the mitochondrial DNA of 516 mosquitoes of 15 species from seven genera that were collected from residential areas during 2003–2006. Culex pipiens L. and Aedes albopictus Skuse were the most commonly collected species in urban and suburban residential areas. Anautogenous Culex pipiens pallens Coquillett was distinguished from the autogenous Cx. pipiens form molestus Forskal using a polymerase chain reaction-based identification method. Both Cx. p. pollens and Cx. p. form molestus exhibited similar host-feeding habits, broadly preferring avian (50.0 and 42.5% of avian, respectively) and mammalian (38.6 and 45.0% of avian, respectively) hosts, such as tree sparrows, ducks, and humans. Conversely, Ae. albopictus exhibited a highly mammalophilic and anthropophilic feeding pattern, with 84.2% feeding on mammalian hosts and 68.5% of these on humans. We concluded that in Japan, Cx. pipiens might play a significant role in the avian-to-mammal transmission of viruses, such as West Nile virus, whereas Ae. albopictus might play a role in the human-human transmission of dengue and Chikungunya viruses.

Genetics and mechanisms of permethrin resistance in the YPER strain of house fly

Abstract A strain of house fly was collected from the Third Yumenoshima Island in Japan and selected with the pyrethroid insecticide permethrin. Resistance to permethrin evolved to >18,400-fold in the selected (YPER) strain, and the mechanisms and genetics of resistance in this strain were examined. Permethrin resistance was decreased by pretreatment with piperonyl butoxide or 2-propynyl 2,3,6-trichlorophenyl ether, but not S , S , S -tributylphosphorotrithioate or diethylmaleate. The level of total cytochromes P450 was 2.7-fold increased, and the level of cytochrome b 5 was 1.5-fold increased, in YPER compared to the susceptible CS strain. These results suggest P450 monooxygenases, but not hydrolases or glutathione S -transferase, are a mechanism of resistance in the YPER strain. Analysis of the para -homologous sodium channel α-subunit gene in YPER indicates this strain has the super-kdr allele. Permethrin resistance in the YPER strain was inherited as a multigenic and incompletely recessive trait. A factorial analysis of resistance in the YPER strain indicated the relative contribution to resistance by the different autosomes was 2>3>5⩾1. Comparison of YPER with three other strains of house fly having high levels of permethrin resistance (LPR, NG98, and ALHF) indicates the genetic basis of this trait is variable between populations. This indicates there may be a greater difficulty for the development of diagnostic tools that could be used reliably over large areas to monitor pyrethroid resistance in house flies than was previously thought.

Pyrethroid resistance in Aedes aegypti and Aedes albopictus: Important mosquito vectors of human diseases

Aedes aegypti and A. albopictus mosquitoes are vectors of important human disease viruses, including dengue, yellow fever, chikungunya and Zika. Pyrethroid insecticides are widely used to control adult Aedes mosquitoes, especially during disease outbreaks. Herein, we review the status of pyrethroid resistance in A. aegypti and A. albopictus, mechanisms of resistance, fitness costs associated with resistance alleles and provide suggestions for future research. The widespread use of pyrethroids has given rise to many populations with varying levels of resistance worldwide, albeit with substantial geographical variation. In adult A. aegypti and A. albopictus, resistance levels are generally lower in Asia, Africa and the USA, and higher in Latin America, although there are exceptions. Susceptible populations still exist in several areas of the world, particularly in Asia and South America. Resistance to pyrethroids in larvae is also geographically widespread. The two major mechanisms of pyrethroid resistance are increased detoxification due to P450-monooxygenases, and mutations in the voltage sensitive sodium channel (Vssc) gene. Several P450s have been putatively associated with insecticide resistance, but the specific P450s involved are not fully elucidated. Pyrethroid resistance can be due to single mutations or combinations of mutations in Vssc. The presence of multiple Vssc mutations can lead to extremely high levels of resistance. Suggestions for future research needs are presented.

Use of isogenic strains indicates CYP9M10 is linked to permethrin resistance in Culex pipiens quinquefasciatus.

Previous studies on a strain of Culex pipiens quinquefasciatus from Saudi Arabia indicated permethrin resistance was a result of cytochrome P450 mediated detoxification and kdr. The P450 detoxification was found to be larval specific and associated with a fitness cost in certain environments. The P450 responsible for resistance (and the fitness cost) has not been identified, but recently two candidate P450s (CYP4H34 and CYP9M10) have been found. We measured cytochrome P450 and cytochrome b5 content as well as the expression levels of CYP4H34 and CYP9M10 in a susceptible (SLAB) and two isogenic strains (isolated by repeated backcrossing and selection) of mosquito (ISOP450 and ISOJPAL) resistant to permethrin. Cytochrome P450 protein levels of the resistant strains were significantly higher (1.5‐fold) than SLAB, but were not significantly different from one another. Expression of CYP4H34 in the larvae and adults of the resistant (ISOP450 and ISOJPAL) and susceptible (SLAB) strains were not statistically different. CYP9M10 was found to be significantly over‐expressed in larvae of both permethrin‐resistant isogenic strains (1800‐fold in ISOP450 and 870‐fold in ISOJPAL) when compared to SLAB. Partial sequence analysis of CYP9M10 revealed eight polymorphic sites that distinguished the susceptible allele from the resistant allele. We conclude that CYP9M10 is linked to permethrin resistance in these strains of C. p. quinquefasciatus, and is likely to be the P450 gene responsible for resistance in these strains.