Genome-wide profiling and functional analysis of miR-310 cluster associated with DDT resistance in Drosophila melanogaster

Keon Mook Seong,Barry Pittendrigh 한국응용곤충학회 2019 한국응용곤충학회 학술대회논문집 Vol.2019 No.10

Molecular Mechanism of ABC Transporter Mdr49A Associated with a Positive Cross-Resistance in Transgenic Drosophila

Keon Mook Seong,Barry R. Pittendrigh 한국응용곤충학회 2020 한국응용곤충학회지 Vol.59 No.4

ATP-binding cassette (ABC) transporter는 다양한 기질을 세포 밖과 세포 안으로 수송하는 대표적인 수송단백질이다. 곤충에서 ABC transporter는 살충제에 대한 저항성을 발달시키는 중요한 역할을 한다. 현재까지 모델곤충인 초파리를 대상으로 ABC transporter의 살충제 교차저항성에 관한 연구는 많이 수행되어오지 않았다. 본 연구에서는 ABC transporter에 속하는 Mdr49A 유전자가 여섯 종류의 살충제에 보이는 교차저항성 기작을 형질전환 초파리를 이용하여 구명하였다. 초파리 91-R과 91-C 계통은 공통된 조상으로부터 유래되었으며 91-R은 60년 이상 DDT에 노출되었지만 91-C는 어떠한 살충제에도 노출되지 않고 유지되어 왔다. 91-R 계통의 MDR49A 단백질에서 유래된 3개의 아미노산 돌연변이를 형질전환 초파리에 과발현 시켰을 때 carbofuran에 대해서 2.0~6.7배 그리고 permethrin에 대해서 2.5~10.5배의 교차저항성을 나타 낸 반면 다른 약제, abamectin, imidacloprid, methoxychlor, prothiofos에 대해서는 어떠한 교차저항성도 나타내지 않았다. 이상의 결과는 Mdr49A 유전자의 과발현과 더불어 3개의 아미노산 돌연변이는 두 개 약제, carbofuran과 permethrin에 대해 교차저항성 기능을 한다고 제시하고 있다 The ATP-binding cassette (ABC) transporter superfamily represents the largest transmembrane protein that transports a variety of substrates across extra- and intra-cellular membranes. In insects, the ABC transporter proteins play crucial roles in insecticide resistance. To date, no studies have investigated the involvement of ABC transporter gene for cross-resistance to insecticide chemistries. Here, we studied such possible mechanisms against six conventional insecticides using transgenic Drosophila melanogaster strains carrying Mdr49 transcript variant A. For the 91-R and 91-C strains of Drosophila melanogaster, although they have a common origin, 91-R has been intensely selected with DDT for over 60 years, while 91-C has received no insecticide selection. Our transgenic analyses showed that overexpression of 91-R-MDR49 transcript variant A along with three amino acid variations can yield a relatively low degree of cross-resistance to carbofuran (2.0~6.7-fold) and permethrin (2.5~10.5-fold) but did not show cross-resistance to abamectin, imidacloprid, methoxychlor, and prothiofos as compared to the Gal4-driver control strain without transgene expression. These results indicate that the overexpression of Mdr49A in itself leads to a cross-resistance and three amino acid changes have additional effects on positive cross-resistance to carbofuran and permethrin.

Genomics of Body/Head Lice and Its Application

Si Hyeock Lee,Ju Hyeon Kim,J. Marshall Clark,Barry B. Pittendrigh 한국응용곤충학회 2014 한국응용곤충학회 학술대회논문집 Vol.2014 No.04

Both the body louse (Pediculus humanus humanus) and the head louse (P. humanus capitis) are obligatory human ectoparasites. The body louse is a serious public health threat because it transmits a variety of human diseases whereas the head lice causes one of the most prevalent human infestations, pediculosis. Recent genome analysis revealed that both body and head lice have one of the smallest insect genomes (~108 Mb). Comparison of transcriptional profiles uncovered that almost the same number of genes was annotated both in the head louse (10,770 genes) and the body louse (10,771 genes) among 10,775 protein-coding genes predicted from the body louse genome. Despite their similar genetic background, body and head lice have several differences in their biological features, such as choice of habitat on human host, body size and vector competence. Annotation of major detoxification genes revealed that they are dramatically reduced in human lice compared to other insects except for the honey bee and that, despite the overall reduction in number, human lice retain at least a minimum repertoire of genes known to confer metabolic or toxicokinetic resistance to insecticides, suggesting their high potential for resistance development. Comparison of insecticide target site gene sequences and transcription levels of detoxification genes enabled the identification of toxicodynamic and metabolic factors of insecticide resistance and further allowed the development of molecular markers for resistance detection. Transcriptional profiling during tolerance was used to identify ivermectinmetabolizing detoxification genes, indicating that such an approach may allow proactive resistance management. Comparison of genomes and transcriptomes between body and head lice suggested that vector competence difference is not attributed to the difference in the composition of immune related genes but rather to their transcriptional regulation and/or not-yet-identified epigenetic factors.

Comparison of the immune response in alimentary tract tissues from body versus head lice following Escherichia coli oral infection

Ju Hyeon Kim,Kyong Sup Yoon,Domenic J. Previte,Barry R. Pittendrigh,J. Marshall Clark,이시혁 한국응용곤충학회 2012 Journal of Asia-Pacific Entomology Vol.15 No.3

Human body and head lice have been hematophagous ectoparasites of humans for thousands of years. Although both body and head lice belong to a single species, Pediculus humanus, only body lice are known to transmit several bacterial diseases to humans. This difference in vector competence is assumed to be due to their different immune responses. Here, the immune reactions in the alimentary tract were investigated in both body and head lice following oral challenge of Escherichia coli as a model Gram-negative bacterium. In proliferation assay, head lice suppressed the growth of E. coli effectively at the early stage of infection,resulting in gradual reduction of E. coli number in alimentary tract tissues. In contrast, the number of E. coli steadily increased in alimentary tract tissues of body lice. No apparent alteration of transcription was observed following E. coli challenge in three important genes for the humoral immune responses, peptidoglycan recognition protein as a recognition gene and defensin 1 and defensin 2 as effector genes. Nevertheless, the basal transcription levels of these genes were higher in the gut tissues of body versus head lice. Considering that there is no cellular immune reaction in gut tissues, these findings suggest that the higher constitutive transcription levels of major immune genes in head lice can contribute to their rapid defense and enhanced immune capacity against intestinal bacterial infection.

Comparative response of two seasonal spotted wing drosophila (Drosophila suzukii) morphs to different classes of insecticides

Seong Keon Mook,Sun Weilin,Huang Juan,Gut Larry,Kim Young Ho,Pittendrigh Barry R. 한국곤충학회 2022 Entomological Research Vol.52 No.11

Drosophila suzukii Matsumura (Drosophilidae) is a devasting invasive pest affecting berry crops and cherry production throughout North America, South America, and Europe. This species has two different morph phenotypes, winter and summer morphs, which are temperature dependent. Chemical control is the most widely used management approach for managing the spotted wing drosophila, D. suzukii. Little is known regarding the differential response of the two seasonal morphs to insecticides. In this study, we identified a high number of differentially expressed genes likely involved in phase I, II, and III of detoxification pathways and other cuticular proteins in winter morphs as compared to summer morphs. Specifically, several detoxification genes (phase I: Cyp4e3, Cyp4s3, Cyp6d5, Cyp49a1, Cyp318a1; phase II: GstD10, Ugt35Bb, Ugt37b1, Ugt58Fa; phase III: Mdr65) were overexpressed more than two-fold in winter morph. Additionally, we determined the median lethal concentration, LC50, values of Malathion, Cyantraniliprole, Imidan, Zeta- cypermethrin, and Spinetoram insecticides to compare the insecticide susceptibility against two seasonal morphs of D. suzukii. For most of the pesticides tested, there were no differences between the LC50 values, between summer and winter morphs, however, we found that winter morph exhibited an LC50 value of Spinetoram 3.7- fold, significantly higher than that of the summer morph. Overall, we demonstrated that seasonally induced different morphological phenotypes may result in different transcriptional response of phases I, II, and III of the detoxification pathways and other cuticular proteins. However, we found different responses to at least one insecticide, Spinetoram. It remains to be determined what are underlying physiological differences that lead to these changes in response to Spinetoram.

Comparison of the genome profiles between head and body lice

강재순,조용준,김주현,김상현,유승일,노승재,박준형,윤경섭,J. Marshall Clark,Barry R. Pittendrigh,천종식,이시혁 한국응용곤충학회 2015 Journal of Asia-Pacific Entomology Vol.18 No.3

The body louse (Pediculus humanus humanus) is known to have diverged fromthe head louse (P. humanus capitis) but genomic differences between these two subspecies still remain unexplored. To compare genomic profiles between head and body lice, whole genome sequences of head lice were determined by next generation sequencing methods based on both Illumina Genome analyzer and Roche GS FLX pyrosequencing and compared with the reference genome sequences of the body louse. Total consensuses generated by mapping to the body louse genome in conjunction with de novo assembly of head louse genome sequences revealed a head louse genome size of 110 Mbp with a 96% coverage of the body louse genome sequences. A total of 12,651 genes were predicted from the head louse genome sequences although more precise assembly and functional annotation of the genome is required for amore accurate gene count. Among the 873 genes thatwere putatively specific to the head louse, 15 genes were confirmed to be transcribed in both head and body lice, suggesting the previously estimated gene number of the body louse was likely underestimated. The single nucleotide polymorphism analysis showed that the nucleotide diversity of genome between head and body lice was 2.2%, whichwas larger than that of the transcriptome between head and body lice. An endosymbiont genome analysis showed that the composition of endosymbionts in head licewas similar to that of body lice and Candidatus Riesia pediculicola was the primary endosymbiont in both head and body lice.

Comparison of the genome profiles between head and body lice

Kang, J.S.,Cho, Y.J.,Kim, J.H.,Kim, S.H.,Yoo, S.,Noh, S.J.,Park, J.,Yoon, K.S.,Marshall Clark, J.,Pittendrigh, B.R.,Chun, J.,Lee, S.H. 한국응용곤충학회 2015 Journal of Asia-Pacific Entomology Vol.20 No.3

The body louse (Pediculus humanus humanus) is known to have diverged from the head louse (P. humanus capitis) but genomic differences between these two subspecies still remain unexplored. To compare genomic profiles between head and body lice, whole genome sequences of head lice were determined by next generation sequencing methods based on both Illumina Genome analyzer and Roche GS FLX pyrosequencing and compared with the reference genome sequences of the body louse. Total consensuses generated by mapping to the body louse genome in conjunction with de novo assembly of head louse genome sequences revealed a head louse genome size of 110Mbp with a 96% coverage of the body louse genome sequences. A total of 12,651 genes were predicted from the head louse genome sequences although more precise assembly and functional annotation of the genome is required for a more accurate gene count. Among the 873 genes that were putatively specific to the head louse, 15 genes were confirmed to be transcribed in both head and body lice, suggesting the previously estimated gene number of the body louse was likely underestimated. The single nucleotide polymorphism analysis showed that the nucleotide diversity of genome between head and body lice was 2.2%, which was larger than that of the transcriptome between head and body lice. An endosymbiont genome analysis showed that the composition of endosymbionts in head lice was similar to that of body lice and Candidatus Riesia pediculicola was the primary endosymbiont in both head and body lice.

Cowpea bruchid midgut transcriptome response to a soybean cystatin – costs and benefits of counter-defence

Chi, Y. H.,Salzman, R. A.,Balfe, S.,Ahn, J.-E.,Sun, W.,Moon, J.,Yun, D.-J.,Lee, S. Y.,Higgins, T. J. V.,Pittendrigh, B.,Murdock, L. L.,Zhu-Salzman, K. Blackwell Publishing Ltd 2009 INSECT MOLECULAR BIOLOGY Vol.18 No.1

<P>Abstract</P><P>The insect digestive system is the first line of defence protecting cells and tissues of the body from a broad spectrum of toxins and antinutritional factors in its food. To gain insight into the nature and breadth of genes involved in adaptation to dietary challenge, a collection of 20 352 cDNAs was prepared from the midgut tissue of cowpea bruchid larvae (<I>Callosobruchus maculatus</I>) fed on regular diet and diets containing antinutritional compounds. Transcript responses of the larvae to dietary soybean cystatin (scN) were analysed using cDNA microarrays, followed by quantitative real-time PCR (RT-PCR) confirmation with selected genes. The midgut transcript profile of insects fed a sustained sublethal scN dose over the larval life was compared with that of insects treated with an acute high dose of scN for 24 h. A total of 1756 scN-responsive cDNAs was sequenced; these clustered into 967 contigs, of which 653 were singletons. Many contigs (451) did not show homology with known genes, or had homology only with genes of unknown function in a B<SMALL>LAST</SMALL> search. The identified differentially regulated sequences encoded proteins presumptively involved in metabolism, structure, development, signalling, defence and stress response. Expression patterns of some scN-responsive genes were consistent in each larval stage, whereas others exhibited developmental stage-specificity. Acute (24 h), high level exposure to dietary scN caused altered expression of a set of genes partially overlapping with the transcript profile seen under chronic lower level exposure. Protein and carbohydrate hydrolases were generally up-regulated by scN whereas structural, defence and stress-related genes were largely down-regulated. These results show that insects actively mobilize genomic resources in the alimentary tract to mitigate the impact of a digestive protease inhibitor. The enhanced or restored digestibility that may result is possibly crucial for insect survival, yet may be bought at the cost of weakened response to other stresses.</P>