Sleep and APOE-ε4 have a synergistic effect on plasma biomarkers and longitudinal cognitive decline in older adults.

BACKGROUND: Sleep disorders are prevalent among patients with Alzheimer's disease (AD), and the APOE ε4 genotype is a key genetic risk factor for sporadic AD. However, the combined effect of the genotype and sleep disorders on cognitive decline remains uncertain. METHODS: A total of 972 participants were drawn from the SILCODE cohort, comprising 655 without the ε4 allele (APOE-) and 317 with ε4 allele (APOE+). Data were collected, including neuropsychological assessments, sleep measurements, plasma biomarkers, and PET imaging. A Sleep Composite Index (SCI) was created, categorizing participants into high risk (Sleep+) and low risk (Sleep-). RESULTS: Significant predictions of dementia risk associated with plasma p-tau181, neurofilament light chain (NfL), and SCI. Individuals with both Sleep+ and APOE+ had a higher risk of dementia compared to those with Sleep-. The Sleep+/APOE+ group had higher plasma NfL levels than the Sleep-/APOE- group. Similar trends emerged in plasma NfL levels among the Aß PET-positive subgroup. Plasma NfL levels explained 23% of the relationship between SCI and cognitive impairment. CONCLUSION: Our study highlights sleep disorder was associated with cognitive decline, with plasma NfL playing a partial mediating role. These findings explain how sleep disorders affect cognitive function and emphasize the importance of healthy sleep for older adults.

AsOBP1 is required for bioallethrin repellency in the malaria vector mosquito Anopheles sinensis.

The use of insecticides, primarily pyrethroids, is a pivotal strategy for mosquito control globally. Bioallethrin, the first commercially available volatile pyrethroid, can elicit spatial (i.e., noncontact) repellency to mosquitoes through the coactivation of olfactory receptor neurons and sodium channels. However, the olfactory mechanism of the repellency elicited by bioallethrin in mosquitoes is still unclear. Here, we demonstrated the involvement of AsOBP1 in the bioallethrin repellency in Anopheles sinensis, one of the main vectors of vivax malaria in China and other Southeast Asian countries. The behavioral and electrophysiological analyses in AsOrco-/- mutant found that the spatial repellency elicited by bioallethrin depended on the odorant receptor (OR)-mediated olfactory pathway. Furthermore, the repellency was reduced in the AsOBP1-/- mutant and a pyrethroid-resistant strain, in which the expression of AsOBP1 was significantly decreased. Moreover, recombinant AsOBP1 protein bound to bioallethrin in an in vitro competition assay. These results indicate that activation of the AsOBP1-mediated olfactory pathway is an important component of bioallethrin repellency. Our research lays the foundation for further elucidation into the olfactory mechanism of bioallethrin repellency and the behavioral modifications of pyrethroid-resistant mosquitoes.

Glutathione S-transferase (GST) genes and their function associated with pyrethroid resistance in the malaria vector Anopheles sinensis.

BACKGROUND: Glutathione S-transferases (GSTs), a multifunctional protein family, are involved in insecticide resistance. However, a systematic analysis of GSTs in Anopheles sinensis, an important vector for malaria transmission, is lacking. In this study, we investigated the diversity and characteristics of GST genes, and analyzed their expression patterns and functions associated with insecticide resistance in this species. RESULTS: We identified 32 putative cytosolic and three putative microsomal GST genes in the An. sinensis genome. Transcriptome analysis showed that GSTs were highly expressed in larvae, and mainly expressed in the antennae, midgut and Malpighian tubules of adults. In addition, we found that GSTd2 and GSTe2 were significantly upregulated in four An. sinensis pyrethroid-resistant field populations. Furthermore, silencing of GSTd2 and GSTe2 significantly increased the susceptibility of An. sinensis to deltamethrin, and recombinant GSTd2 and GSTe2 exhibited high enzymatic activity in the metabolism of 1-chloro-2,4-dinitrobenzene and dichlorodiphenyltrichloroethane (DDT). CONCLUSION: These results showed that GSTs are involved in the development of insecticide resistance in An. sinensis through transcriptional overexpression and enzymatic metabolization, facilitating our understanding of insecticide resistance in insects. © 2022 Society of Chemical Industry.

Genome-wide identification and expression profiling of odorant receptor genes in the malaria vector Anopheles sinensis.

BACKGROUND: The olfactory system plays a crucial role in regulating insect behaviors. The detection of odorants is mainly mediated by various odorant receptors (ORs) that are expressed in the dendrites of olfactory neurons of chemosensilla. Anopheles sinensis is a major malaria vector in Eastern Asia and its genome has recently been successfully sequenced and annotated. In this study, we present genome-wide identification and expression profiling of OR genes in different chemosensory tissues of An. sinensis. METHODS: The OR genes were identified using the available genome sequences of An. sinensis. A series of bioinformatics analyses were conducted to investigate the structure, genome distribution, selective pressure and phylogenetic relationships of OR genes, the conserved domains and specific functional sites in the OR amino acid sequences. The expression levels of OR genes were analyzed from transcriptomic data from An. sinensis antennae, proboscis and maxillary palps of both sexes. RESULTS: A total of 59 putative OR genes have been identified and characterized in An. sinensis. This number is significantly less than that in An. gambiae. Whether this difference is caused by the contraction or expansion of OR genes after divergence of the two species remains unknown. The RNA-seq analysis showed that AsORs have obvious tissue- and sex-specific expression patterns. Most AsORs are highly expressed in the antennae and the expression pattern and number of AsORs expressed in antennae are similar in males and females. However, the relative levels of AsOR transcripts are much higher in female antennae than in male antennae, which indicates that the odor sensitivity is likely to be increased in female mosquitoes. Based on the expression patterns and previous studies, we have speculated on the functions of some OR genes but this needs to be validated by further behavioral, molecular and electrophysiological studies. Further studies are necessary to compare the olfactory-driven behaviors and identify receptors that respond strongly to components of human odors that may act in the process of human recognition. CONCLUSIONS: This is the first genome-wide analysis of the entire repertoire of OR genes in An. sinensis. Characterized features and profiled expression patterns of ORs suggest their involvement in the odorous reception of this species. Our findings provide a basis for further research on the functions of OR genes and additional genetic and behavioral targets for more sustainable management of An. sinensis in the future.

CRISPR/Cas9 mediates efficient site-specific mutagenesis of the odorant receptor co-receptor (Orco) in the malaria vector Anopheles sinensis.

BACKGROUND: Anopheles sinensis is the most widely distributed mosquito species and is the main transmitter of Plasmodium vivax malaria in China. Most previous research has focused on the mechanistic understanding of biological processes in An. sinensis and novel ways of interrupting malaria transmission. However, the development of functional genomics and genetics-based vector control strategies against An. sinensis remain limited because of insufficient site-specific genome editing tools. RESULTS: We report the first successful application of the CRISPR/Cas9 mediated knock-in for highly efficient, site-specific mutagenesis in An. sinensis. The EGFP marker gene driven by the 3 × P3 promoter was precisely integrated into the odorant receptor co-receptor (Orco) by direct injections of Cas9 protein, double-stranded DNA donor, and Orco-gRNA. We achieved a mutation rate of 3.77%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed by polymerase chain reaction (PCR) amplification and sequencing. The Orco mutation severely impaired mosquito sensitivity to some odors and their ability to locate and discriminate a human host. CONCLUSION: Orco was confirmed as a key mediator of multiple olfactory-driven behaviors in the An. sinensis life cycle, highlighting the importance of Orco as a key molecular target for malaria control. The results also demonstrated that CRISPR/Cas9 was a simple and highly efficient genome editing technique for An. sinensis and could be used to develop genetic control tools for this vector. © 2022 Society of Chemical Industry.

Electroporation-mediated nucleic acid delivery during non-embryonic stages for gene-function analysis in Anopheles sinensis.

The delivery of exogenous nucleic acids to eggs or non-embryonic individuals by microinjection is a vital reverse genetics technique used to determine gene function in mosquitoes. However, DNA delivery to eggs is complex and time-consuming, and conventional, non-embryonic-injection techniques may result in unobvious phenotypes caused by insufficient absorption of nucleic acid fragments by cells at target body parts or tissues. In this study, we developed a set of electroporation-mediated non-embryonic microinjections for the delivery of exogenous nucleic acids in Anopheles sinensis. Gene silencing using this method led to down-regulation of target gene expression (AsCPR128) by 77% in targeted body parts, compared with only 10% in non-targeted body parts, thus increasing the defect-phenotype rate in the target area by 5.3-fold, compared with non-shock injected controls. Electroporation-mediated somatic transgenesis resulted in stable phenotypic characteristics of the reporter gene at the shocked body parts during the pupal-adult stages in about 69% of individuals. Furthermore, injecting plasmid DNA near the ovaries of female mosquitoes after a blood meal followed by electric shock produced three germline G1 transgenic lines, with a transformation rate of about 11.1% (calculated from ovulatory G0 females). Among the positive G1 lines, 42%, 40%, and 31% of individuals emitted red fluorescence in the larval stage. When the red fluorescent larvae developed into adults, green fluorescence was emitted from the ovaries of the females upon feeding. These results suggest that electroporation-mediated non-embryonic microinjection can be an efficient, rapid, and simple technique for analyzing gene function in non-model mosquitoes or other small insects.

Modification of contact avoidance behaviour associated with pyrethroid resistance in Anopheles sinensis (Diptera: Culicidae).

BACKGROUND: Anopheles sinensis is the primary vector of vivax malaria in China and its control is under great threat as the development of insecticide resistance. In contrast to physiological resistance, there is no report of behavioural modifications of resistant An. sinensis after long-term insecticide use, despite their huge potential impact on malaria transmission. METHODS: Larvae or pupae of An. sinensis were collected from Yuanyang, Bishan, and Wuhe counties from southwestern to eastern China. Resistance to deltamethrin was assayed using the standard World Health Organization (WHO) susceptibility test. The frequency distribution of the kdr allele of the para-type sodium channel gene was determined by polymerase chain reaction (PCR) amplification and DNA sequencing. Contact repellency to deltamethrin-impregnated bed nets was evaluated using a modified WHO cone bioassay. RESULTS: All contemporary field populations for all three geographic locations were resistant to deltamethrin, with mortality ranging from 6.00 to 26.79%. Three kdr genotypes with either an L1014F or L1014C substitution with frequencies of 76.10-100% were identified in the Bishan and Wuhe populations, but no kdr mutations were detected in the Yuanyang samples despite high phenotypic resistance. The susceptible mosquitoes exhibited significantly longer flying time and more takeoffs on deltamethrin-treated bed nets (DTN) than on untreated bed nets (UTN), suggestive of robust avoidance behaviour. However, no significant increases in the frequency of takeoffs or flying time were observed in deltamethrin-resistant An. sinensis populations when exposed on DTNs, regardless of the presence of a kdr mutation. Moreover, the first takeoff from DTNs by resistant mosquitoes significantly lagged behind compared to susceptible mosquitoes. CONCLUSION: The An. sinensis populations were highly resistant to deltamethrin and exhibited decreased avoidance behaviour. Behavioural modification significantly associated with deltamethrin resistance, but not directly related to the presence of kdr mutations, indicating that there are additional factors contributing to the changes.

Genome-wide and expression-profiling analyses suggest the main cytochrome P450 genes related to pyrethroid resistance in the malaria vector, Anopheles sinensis (Diptera Culicidae).

BACKGROUND: Anopheles sinensis is one of the major malaria vectors. However, pyrethroid resistance in An. sinensis is threatening malaria control. Cytochrome P450-mediated detoxification is an important pyrethroid resistance mechanism that has been unexplored in An. sinensis. In this study, we performed a comprehensive analysis of the An. sinensis P450 gene superfamily with special attention to their role in pyrethroid resistance using bioinformatics and molecular approaches. RESULTS: Our data revealed the presence of 112 individual P450 genes in An. sinensis, which were classified into four major clans (mitochondrial, CYP2, CYP3 and CYP4), 18 families and 50 subfamilies. Sixty-seven genes formed nine gene clusters, and genes within the same cluster and the same gene family had a similar gene structure. Phylogenetic analysis showed that most of An. sinensis P450s (82/112) had very close 1: 1 orthology with Anopheles gambiae P450s. Five genes (AsCYP6Z2, AsCYP6P3v1, AsCYP6P3v2, AsCYP9J5 and AsCYP306A1) were significantly upregulated in three pyrethroid-resistant populations in both RNA-seq and RT-qPCR analyses, suggesting that they could be the most important P450 genes involved in pyrethroid resistance in An. sinensis. CONCLUSION: Our study provides insight on the diversity of An. sinensis P450 superfamily and basis for further elucidating pyrethroid resistance mechanism in this mosquito species. © 2018 Society of Chemical Industry.

Genome-wide identification, characterization and classification of ionotropic glutamate receptor genes (iGluRs) in the malaria vector Anopheles sinensis (Diptera: Culicidae).

BACKGROUND: Ionotropic glutamate receptors (iGluRs) are conserved ligand-gated ion channel receptors, and ionotropic receptors (IRs) were revealed as a new family of iGluRs. Their subdivision was unsettled, and their characteristics are little known. Anopheles sinensis is a major malaria vector in eastern Asia, and its genome was recently well sequenced and annotated. METHODS: We identified iGluR genes in the An. sinensis genome, analyzed their characteristics including gene structure, genome distribution, domains and specific sites by bioinformatic methods, and deduced phylogenetic relationships of all iGluRs in An. sinensis, Anopheles gambiae and Drosophila melanogaster. Based on the characteristics and phylogenetics, we generated the classification of iGluRs, and comparatively analyzed the intron number and selective pressure of three iGluRs subdivisions, iGluR group, Antenna IR and Divergent IR subfamily. RESULTS: A total of 56 iGluR genes were identified and named in the whole-genome of An. sinensis. These genes were located on 18 scaffolds, and 31 of them (29 being IRs) are distributed into 10 clusters that are suggested to form mainly from recent gene duplication. These iGluRs can be divided into four groups: NMDA, non-NMDA, Antenna IR and Divergent IR based on feature comparison and phylogenetic analysis. IR8a and IR25a were suggested to be monophyletic, named as Putative in the study, and moved from the Antenna subfamily in the IR family to the non-NMDA group as a sister of traditional non-NMDA. The generated iGluRs of genes (including NMDA and regenerated non-NMDA) are relatively conserved, and have a more complicated gene structure, smaller ω values and some specific functional sites. The iGluR genes in An. sinensis, An. gambiae and D. melanogaster have amino-terminal domain (ATD), ligand binding domain (LBD) and Lig_Chan domains, except for IR8a that only has the LBD and Lig_Chan domains. However, the new concept IR family of genes (including regenerated Antenna IR, and Divergent IR), especially for Divergent IR are more variable, have a simpler gene structure (intron loss phenomenon) and larger ω values, and lack specific functional sites. These IR genes have no other domains except for Antenna IRs that only have the Lig_Chan domain. CONCLUSIONS: This study provides a comprehensive information framework for iGluR genes in An. sinensis, and generated the classification of iGluRs by feature and bioinformatics analyses. The work lays the foundation for further functional study of these genes.

Comparative genomics of chemosensory protein genes (CSPs) in twenty-two mosquito species (Diptera: Culicidae): Identification, characterization, and evolution.

Chemosensory proteins (CSP) are soluble carrier proteins that may function in odorant reception in insects. CSPs have not been thoroughly studied at whole-genome level, despite the availability of insect genomes. Here, we identified/reidentified 283 CSP genes in the genomes of 22 mosquitoes. All 283 CSP genes possess a highly conserved OS-D domain. We comprehensively analyzed these CSP genes and determined their conserved domains, structure, genomic distribution, phylogeny, and evolutionary patterns. We found an average of seven CSP genes in each of 19 Anopheles genomes, 27 CSP genes in Cx. quinquefasciatus, 43 in Ae. aegypti, and 83 in Ae. albopictus. The Anopheles CSP genes had a simple genomic organization with a relatively consistent gene distribution, while most of the Culicinae CSP genes were distributed in clusters on the scaffolds. Our phylogenetic analysis clustered the CSPs into two major groups: CSP1-8 and CSE1-3. The CSP1-8 groups were all monophyletic with good bootstrap support. The CSE1-3 groups were an expansion of the CSP family of genes specific to the three Culicinae species. The Ka/Ks ratios indicated that the CSP genes had been subject to purifying selection with relatively slow evolution. Our results provide a comprehensive framework for the study of the CSP gene family in these 22 mosquito species, laying a foundation for future work on CSP function in the detection of chemical cues in the surrounding environment.

Genome-wide identification and characterization of odorant-binding protein (OBP) genes in the malaria vector Anopheles sinensis (Diptera: Culicidae).

Anopheles sinensis is a major malaria vector. Insect odorant-binding proteins (OBPs) may function in the reception of odorants in the olfactory system. The classification and characterization of the An. sinensis OBP genes have not been systematically studied. In this study, 64 putative OBP genes were identified at the whole-genome level of An. sinensis based on the comparison between OBP conserved motifs, PBP_GOBP, and phylogenetic analysis with An. gambiae OBPs. The characterization of An. sinensis OBPs, including the motif's conservation, gene structure, genomic organization and classification, were investigated. A new gene, AsOBP73, belonging to the Plus-C subfamily, was identified with the support of transcript and conservative motifs. These An. sinensis OBP genes were classified into three subfamilies with 37, 15 and 12 genes in the subfamily Classic, Atypical and Plus-C, respectively. The genomic organization of An. sinensis OBPs suggests a clustered distribution across nine different scaffolds. Eight genes (OBP23-28, OBP63-64) might originate from a single gene through a series of historic duplication events at least before divergence of Anopheles, Culex and Aedes. The microsynteny analyses indicate a very high synteny between An. sinensis and An. gambiae OBPs. OBP70 and OBP71 earlier classified under Plus-C in An. gambiae are recognized as belonging to the group Obp59a of the Classic subfamily, and OBP69 earlier classified under Plus-C has been moved to the Atypical subfamily in this study. The study established a basic information frame for further study of the OBP genes in insects as well as in An. sinensis.

Insight into the possible mechanism of the summer diapause of Delia antiqua (Diptera: Anthomyiidae) through digital gene expression analysis.

The onion fly, Delia antiqua, is a major underground agricultural pest that can enter pupal diapause in the summer and winter seasons. However, little is known about its molecular regulation due to the lack of genomic resources. To gain insight into the possible mechanism of summer diapause (SD), high-throughput RNA-Seq data were generated from non-diapause (ND) and SD (initial, maintenance and quiescence phase) pupae. Three pair-wise comparisons were performed and identified, 1380, 1471 and 435, and were significantly regulated transcripts. Further analysis revealed that the enrichment of several functional terms related to juvenile hormone regulation, cell cycle, carbon hydrate and lipid metabolism, innate immune and stress responses, various signalling transductions, ubiquitin-dependent proteosome, and variation in cuticular and cytoskeleton components were found between ND and SD and between different phases of SD. Global characterization of transcriptome profiling between SD and ND contributes to the in-depth elucidation of the molecular mechanism of SD. Our results also offer insights into the evolution of insect diapause and support the importance of using the onion fly as a model to compare the molecular regulation events of summer and winter diapauses.

De novo transcriptome sequencing and sequence analysis of the malaria vector Anopheles sinensis (Diptera: Culicidae).

BACKGROUND: Anopheles sinensis is the major malaria vector in China and Southeast Asia. Vector control is one of the most effective measures to prevent malaria transmission. However, there is little transcriptome information available for the malaria vector. To better understand the biological basis of malaria transmission and to develop novel and effective means of vector control, there is a need to build a transcriptome dataset for functional genomics analysis by large-scale RNA sequencing (RNA-seq). METHODS: To provide a more comprehensive and complete transcriptome of An. sinensis, eggs, larvae, pupae, male adults and female adults RNA were pooled together for cDNA preparation, sequenced using the Illumina paired-end sequencing technology and assembled into unigenes. These unigenes were then analyzed in their genome mapping, functional annotation, homology, codon usage bias and simple sequence repeats (SSRs). RESULTS: Approximately 51.6 million clean reads were obtained, trimmed, and assembled into 38,504 unigenes with an average length of 571 bp, an N50 of 711 bp, and an average GC content 51.26%. Among them, 98.4% of unigenes could be mapped onto the reference genome, and 69% of unigenes could be annotated with known biological functions. Homology analysis identified certain numbers of An. sinensis unigenes that showed homology or being putative 1:1 orthologues with genomes of other Dipteran species. Codon usage bias was analyzed and 1,904 SSRs were detected, which will provide effective molecular markers for the population genetics of this species. CONCLUSIONS: Our data and analysis provide the most comprehensive transcriptomic resource and characteristics currently available for An. sinensis, and will facilitate genetic, genomic studies, and further vector control of An. sinensis.

The Anopheles community and the role of Anopheles minimus on malaria transmission on the China-Myanmar border.

BACKGROUND: Malaria around the China-Myanmar border is a serious health problem in the countries of South-East Asia. An. minimus is a principle malaria vector with a wide geographic distribution in this area. Malaria is endemic along the boundary between Yunnan province in China and the Kachin State of Myanmar where the local Anopheles community (species composition) and the malaria transmission vectors have never been clarified. METHODS: Adult Anopheles specimens were collected using CDC light traps in four villages along the border of China and Myanmar from May 2012 to April 2013. Morphological and molecular identification of mosquito adults confirmed the species of Anopheles. Blood-meal identification using the female abdomens was conducted using multiplex PCR. For sporozoite detection in An. minimus, sets of 10 female salivary glands were pooled and identified with SSU rDNA using nested PCR. Monthly abundance of An. minimus populations during the year was documented. The diversity of Anopheles and the role of An. minimus on malaria transmission in this border area were analyzed. RESULTS: 4,833 adult mosquitoes in the genus Anopheles were collected and morphologically identified to species or species complex. The Anopheles community is comprised of 13 species, and 78.83% of our total specimens belonged to An. minimus s.l., followed by An. maculatus (5.55%) and the An. culicifacies complex (4.03%). The quantity of trapped An. minimus in the rainy season of malaria transmission was greater than during the non-malarial dry season, and a peak was found in May 2012. An. minimus fed on the blood of four animals: humans (79.8%), cattle (10.6%), pigs (5.8%) and dogs (3.8%). 1,500 females of An. minimus were pooled into 150 samples and tested for sporozoites: only 1 pooled sample was found to have sporozoites of Plasmodium vivax. CONCLUSION: Anopheles is abundant with An. minimus being the dominant species and having a high human blood index along the China-Myanmar border. The sporozoites in An. minimus were determined to be Plasmodium vivax with a 0.07-0.7% infection rate.

Identification and characterization of a gene encoding a UBX domain-containing protein in the migratory locust, Locusta migratoria manilensis.

Ubiquitin regulatory X (UBX) domain-containing proteins are believed to function as cofactors for p97/CDC48, an adenosine triphosphatase shown to be involved in multiple cellular processes. In the present study, a full-length complementary DNA (cDNA) of UBX domain-containing gene, termed LmUBX1, was cloned from Locusta migratoria manilensis and characterized, using random amplification of cDNA ends polymerase chain reaction (RACE PCR), sequence analysis and quantitative real-time PCR. LmUBX1, 1 600 bp in length, is predicted to encode a 446-amino acid protein with a predicted molecular weight of 51.18 kDa that contains a central PUB domain and a carboxy-terminal UBX domain. Homology analysis revealed that LmUBX1 has higher similarity to the known UBX domain-containing proteins from insects than from other species. Moreover, based on sequence characteristics and phylogenetic relationships, it is suggested that LmUBX1 can be classified into the UBXD1 subfamily. Expression analysis founded that LmUBX1 exhibited significant expression variations at different developmental stages and in different tissues, suggesting that the expression of LmUBX1 was highly regulated. Interestingly, its messenger RNA transcript was more abundant in ovary and testis than in other tissues examined, suggesting that it may have more important roles in the reproductive system. In addition, LmUBX1 was differentially expressed in gregarious and solitary locusts and was significantly up-regulated in third and fifth instars of gregarious locusts, implying that LmUBX1 was also likely involved in the phase polyphenisms in L. migratoria manilensis. To our knowledge, this is the first report of cloning of a full-length cDNA of UBX domain-containing gene from L. migratoria manilensis.

Relationship between knockdown resistance, metabolic detoxification and organismal resistance to pyrethroids in Anopheles sinensis.

Anopheles sinensis is the most important vector of malaria in Southeast Asia, including China. Currently, the most effective measure to prevent malaria transmission relies on vector control through the use of insecticides, primarily pyrethroids. Extensive use of insecticides poses strong selection pressure on mosquito populations for resistance. Resistance to insecticides can arise due to mutations in the insecticide target site (target site resistance), which in the case of pyrethroids is the para-type sodium channel gene, and/or the catabolism of the insecticide by detoxification enzymes before it reaches its target (metabolic detoxification resistance). In this study, we examined deltamethrin resistance in An. sinensis from China and investigated the relative importance of target site versus metabolic detoxification mechanisms in resistance. A high frequency (>85%) of nonsynonymous mutations in the para gene was found in populations from central China, but not in populations from southern China. Metabolic detoxification as measured by the activity of monooxygenases and glutathione S-transferases (GSTs) was detected in populations from both central and southern China. Monooxygenase activity levels were significantly higher in the resistant than the susceptible mosquitoes, independently of their geographic origin. Stepwise multiple regression analyses in mosquito populations from central China found that both knockdown resistance (kdr) mutations and monooxygenase activity were significantly associated with deltamethrin resistance, with monooxygenase activity playing a stronger role. These results demonstrate the importance of metabolic detoxification in pyrethroid resistance in An. sinensis, and suggest that different mechanisms of resistance could evolve in geographically different populations.

Differential gene expression between summer and winter diapause pupae of the onion maggot Delia antiqua, detected by suppressive subtractive hybridization.

To gain a better understanding of the molecular mechanisms regulating pupal diapause of the onion maggot Delia antiqua, PCR-based suppressive subtractive hybridization was performed to identify genes involved in summer and/or winter diapause. A total of 209 unique sequences were obtained including 89 in forward library for winter diapausing pupae and 120 in the reverse library for summer diapausing pupae. 76.4% (68/89) and 68.3% (82/120) unique sequences had significant hits to non-redundant proteins database. Gene functional annotation showed these non-redundant sequences are involved in stress response and innate immunity, metabolism and energy, information processing and regulation, binding, food storage, morphogenesis and development, cell skeleton and cycle, protein synthesis and folding. Approximately 28.2% (59/209) transcripts showed no significant similarity to any other sequence in the public databases, probably representing unique genes of the onion maggot. Semi-quantitative RT-PCR revealed that the relative expression levels of 18 genes were comparable between summer and winter diapause. This study elucidates the temporal expression of diapause-related genes in onion maggot, also provides new insights into the differences in the physiological changes in summer and winter pupae. Functional characterization of some candidate genes will further enhance the understanding of the generating, maintaining, and breaking mechanism of diapause.

Population genetics of the malaria vector Anopheles aconitus in China and Southeast Asia.

Anopheles aconitus is a well-known vector of malaria and is broadly distributed in the Oriental Region, yet there is no information on its population genetic characteristics. In this study, the genetic differentiation among populations was examined using 140 mtDNA COII sequences from 21 sites throughout Southern China, Myanmar, Vietnam, Thailand, Laos and Sri Lanka. The population in Sri Lanka has characteristic rDNA D3 and ITS2, mtDNA COII and ND5 haplotypes, and may be considered a distinct subspecies. Clear genetic structure was observed with highly significant genetic variation present among population groups in Southeast Asia. The greatest genetic diversity exists in Yunnan and Myanmar population groups. All population groups are significantly different from one another in pairwise Fst values, except Northern Thailand with Central Thailand. Mismatch distributions and extremely significant F(s) values suggest that the populations passed through a recent demographic expansion. These patterns are discussed in relation to the likely biogeographic history of the region and compared to other Anopheles species.