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1.
Journal of Forensic Medicine ; (6): 611-614, 2021.
Article in English | WPRIM | ID: wpr-984061

ABSTRACT

OBJECTIVES@#To identify the common sarcosaprophagous flies in the Yangtze River Delta based on mitochondrial cytochrome c oxidase subunit Ⅰ(COⅠ) gene sequence and verify the reliability of this method.@*METHODS@#Seven common genetically stable sarcosaprophagous flies in three families and six genera were collected from large domestic pig carcasses placed in the field and cultured in the laboratory for many generations. The whole genome DNA was extracted and the COⅠ gene fragment was amplified. The forward and reverse sequencing was followed by splicing. The base composition of the amplified fragment and the rate of interspecific evolutionary divergence were analyzed by software such as Mega 7.0.26. The phylogenetic tree of COⅠ gene sequence of common necrophagous flies in the Yangtze River Delta was established by neighbor joining (NJ) method and unweighted pair-group method with arithmetic means (UPGMA) method.@*RESULTS@#The average base composition of different flies was A(30.14%), T(38.23%), C(15.98%), G(15.65%). The rate of interspecific evolutionary divergence ranged from 2.2% to 15.3%, the lowest rate was between Chrysomya megacephala and Chrysomya pinguis, the highest rate was between Muscina stabulans and Boettcherisca peregrina.@*CONCLUSIONS@#COⅠ gene can be used to identify the common necrophagous flies in the Yangtze River Delta.


Subject(s)
Animals , Cadaver , Diptera/genetics , Phylogeny , Reproducibility of Results , Rivers
2.
Journal of Forensic Medicine ; (6): 749-754, 2020.
Article in Chinese | WPRIM | ID: wpr-985170

ABSTRACT

Objective To identify the species of common necrophagous flies in Fujian Province by gene fragment sequences of mitochondrial cytochrome c oxidase subunit Ⅰ (COⅠ) and 16S ribosomal deoxyribonucleic acid (16S rDNA), and to explore the identification efficacy of these two molecular markers. Methods In total 22 common necrophagous flies were collected from the death scenes in 9 different regions in Fujian Province and DNA was extracted from the flies after morphological identification. The gene fragments of COⅠ and 16S rDNA were amplified and sequenced. All the sequences were uploaded to GeneBank and BLAST and MEGA 10.0 software were used to perform sequence alignment, homology analysis and intraspecific and interspecific genetic distance analysis. The phylogenetic trees of DNA fragment sequences of COⅠ and 16S rDNA of common necrophagous flies in Fujian Province were established by unweighted pair-group method with arithmetic means (UPGMA), respectively. Results The flies were classified into 6 species, 5 genera and 3 families by morphological identification. The results of gene sequence analysis showed that the average number of interspecific and intraspecific genetic distance of 16S rDNA ranged from 1.8% to 8.9% and 0.0% to 2.4%, respectively. The average number of interspecific and intraspecific genetic distance of COⅠ ranged from 7.2% to 13.6% and 0.0% to 6.3%, respectively. Conclusion The gene sequences of COⅠ and 16S rDNA can accurately identify the species of different necrophagous flies, and 16S rDNA showed higher value in species identification of common calliphoridae necrophagous flies in Fujian Province.


Subject(s)
Animals , Humans , DNA, Ribosomal/genetics , Diptera/genetics , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Species Specificity
3.
Journal of Forensic Medicine ; (6): 181-186, 2019.
Article in English | WPRIM | ID: wpr-984995

ABSTRACT

Objective To assess the feasibility of using 28S ribosomal RNA (28S rRNA) and mitochondrial cytochrome c oxidase subunit Ⅰ (COⅠ) gene sequences of nine necrophagous Calliphorid flies for the identification of common necrophagous Calliphorid flies, and to provide technical support for postmortem interval (PMI) estimation. Methods Twenty-three Calliphorid flies were collected and identified morphologically, and DNA were extracted from legs. The gene fragments of 28S rRNA and COⅠ were amplified and sequenced, then the sequence alignment was performed with BLAST. The composition of obtained sequences was analyzed and evolutionary divergence rate between species and intraspecies were established. The phylogeny tree was constructed with neighbor-joining method. Results The 23 necrophagous Calliphorid flies were identified to 9 species of 5 genera. The 715 bp from 28S rRNA and 637 bp from COⅠ gene were obtained and the online BLAST result showed more than 99% of similarity. The phylogeny tree showed that the necrophagous flies could cluster well into 9 groups, which was consistent with morphological identification results. The intraspecific difference in 28S rRNA was 0 and the interspecific difference was 0.001-0.033. The intraspecific difference in COⅠ was 0-0.008 and the interspecific difference was 0.006-0.101. Conclusion Combined use of 28S rRNA and COⅠ gene sequence fragments can effectively identify the nine Calliphorid flies in this study. However, for closely related blowfly species, more genetic markers should be explored and used in combination in future.


Subject(s)
Animals , DNA, Mitochondrial/genetics , Diptera/genetics , Phylogeny , RNA, Ribosomal, 28S/genetics , Sequence Analysis, DNA , Species Specificity
4.
Electron. j. biotechnol ; 29: 47-56, sept. 2017. ilus, tab
Article in English | LILACS | ID: biblio-1017093

ABSTRACT

Background: The salivary glands of Lucilia sericata are the first organs to express specific endopeptidase enzymes. These enzymes play a central role in wound healing, and they have potential to be used therapeutically. Methods: Rapid amplification of cDNA ends and rapid amplification of genomic ends were used to identify the coding sequence of MMP-1 from L. sericata. Different segments of MMP1 gene, namely the middle part, 3' end, and 5' end, were cloned, sequenced, and analyzed using bioinformatics tools to determine the distinct features of MMP-1 protein. Results: Assembling the different segments revealed that the complete mRNA sequence of MMP-1 is 1932 bp long. CDS is 1212 bp long and is responsible for the production of MMP-1 of 404 amino acid residues with a predicted molecular weight of 45.1 kDa. The middle part, 3' end, and 5' end sequences were 933, 503, and 496 bp. In addition, it was revealed that the MMP-1 genomic sequence includes three exons and two introns. Furthermore, the three-dimensional structure of L. sericata MMP-1 protein was evaluated, and its alignment defined that it has high similarity to chain A of human MMP-2 with 100% confidence, 72% coverage, and 38% identity according to the SWISS-MODEL modeling analysis. Conclusions: MMP-1 of L. sericata has a close relationship with its homologs in invertebrates and other insects. The present study significantly contributes to understanding the function, classification, and evolution of the characterized MMP-1 from L. sericata and provides basic required information for the development of an effective medical bioproduct.


Subject(s)
Salivary Glands/enzymology , Matrix Metalloproteinase 1/genetics , Diptera/enzymology , Diptera/genetics , RNA, Messenger/genetics , Polymerase Chain Reaction , Sequence Analysis, RNA , DNA, Complementary/genetics , Computational Biology , Larva
5.
Braz. arch. biol. technol ; 60: e17160106, 2017. tab, graf
Article in Portuguese | LILACS | ID: biblio-1142496

ABSTRACT

ABSTRACT Accurate insect specimen identification is usually a crucial first step in a forensic entomological analysis. It is traditionally done by morphological classification using identification keys. However, due to sensibility limitations in the identification of animal species based only on their morphology, new methods have been developed, including species identification by DNA barcodes. The objective of this study was to identify forensically important species of Diptera in Espirito Santo state using DNA barcodes. For this, adult flies were collected in Espirito Santo, Southeast Region of Brazil. After DNA extraction, COI gene was amplified and sequenced. All sequences were matched to BOLD platform and alternatively to GenBank MegaBLAST. As result, 281 adult flies were collected and identified morphologically. From these, 36% of samples were classified as Calliphoridae, 34% of Muscidae and 30% of Sarcophagidae. Approximately 10% of all collected samples were analyzes by DNA. It was possible to identify only 35.7% of tested samples, probably due to lack of samples deposited in databases. Therefore, more efforts should be made to deposit a greater variety of dipterous in databases to allow the use of this technique in forensic routine, especially in BOLD.


Subject(s)
Animals , Diptera/classification , DNA Barcoding, Taxonomic , Brazil , Databases, Nucleic Acid , Diptera/anatomy & histology , Diptera/genetics , Forensic Entomology
6.
Mem. Inst. Oswaldo Cruz ; 108(6): 671-678, set. 2013. tab, graf
Article in English | LILACS | ID: lil-685492

ABSTRACT

Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.


Subject(s)
Animals , Female , Male , Gastrointestinal Tract/enzymology , Insect Vectors/parasitology , Phlebotomus/enzymology , Serine Proteinase Inhibitors/isolation & purification , CHO Cells , Cricetulus , Chymotrypsin/metabolism , Diptera/genetics , Gene Expression , Leishmaniasis/prevention & control , Life Cycle Stages/genetics , Psychodidae/parasitology , Regression Analysis , Reverse Transcriptase Polymerase Chain Reaction , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Analysis , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/metabolism , Thrombin/metabolism , Trypsin/metabolism
7.
Braz. j. microbiol ; 44(1): 325-328, 2013.
Article in English | LILACS | ID: lil-676913

ABSTRACT

Bacteria associated with the parthenogenetic troglobiont sand fly Deanemyia maruaga were characterized by sequencing cloned 16S rDNA PCR products. Eleven novel partial 16S rDNA sequences, with varying degrees of similarity to Actinobacteria, were identified. None of the sequences identified had homology to those known from parthenogenesis-inducing bacteria.


Subject(s)
Actinobacteria/genetics , Actinobacteria/isolation & purification , Base Sequence , Cloning, Molecular , Diptera/genetics , In Vitro Techniques , Industrial Microbiology , Polymerase Chain Reaction , Chiroptera , Methods , Virulence
8.
Bol. malariol. salud ambient ; 52(1): 46-65, jun. 2012. ilus, tab
Article in Spanish | LILACS | ID: lil-659199

ABSTRACT

Culex quinquefasciatus Say (Diptera; Culicidae) es un mosquito perteneciente al complejo Pipiens con distribución amplia en el mundo y en Venezuela tanto en zonas urbanas como rurales; es acentuadamente antropofílico y vector de varios virus y parásitos mantenidos en la naturaleza en un ciclo enzoótico ave-mosquito-ave. Dicha distribución y ocupación de hábitats larvales de origen antropogénico podría sugerir la presencia de subpoblaciones geográficas que pudieran participar en forma diferencial en la transmisión de patógenos. Los análisis filogenéticos y redes de haplotipos de éste trabajo, con secuencias de los genes mitocondriales (Subunidad I del Citocromo oxidasa y Subunidad 5 de la NADH deshidrogenasa) de poblaciones de Cx. quinquefasciatus colectadas en nueve localidades (cementerios) de Venezuela sugieren alta homogeneidad genética inter-poblacional y una sola entidad filogenética (monofilia). Se demostró que el fragmento del gen COI tiene mayor resolución en la definición de la filogenia de especies cercanas y a nivel de géneros y ajustado con la clasificación actual. El gen ND5 con alta variación es más útil para estudios poblacionales, sin embargo muestra parafilia entre Cx. corniger y Cx. quinquefasciatus, que representa una evidencia de posible homogenización por entrecruzamiento, introgresión o infección por Wolbachia. Las redes de haplotipos sugieren poblaciones en expansión con alta variabilidad haplotípica y heterogeneidad genética intra poblacional y homogeneidad inter poblacional, con implicaciones evolutivas en la dispersión de sus poblaciones y el éxito en áreas urbanas, así como evidencia de posible cuello de botella en poblaciones producto de marcadas campañas de aplicación de insecticidas, información útil en la planificación de futuras estrategias de control sanitario.


Culex quinquefasciatus Say (Diptera, Culicidae) is a mosquito belonging to the Pipiens complex with wide distribution in the world and in Venezuela, both urban and rural. Besides, it is a markedly anthropophilic vector of several viruses and parasites maintained in nature in a cycle enzootic bird-mosquito-bird. The wide distribution and occupation of larval habitats of anthropogenic origin may suggest the presence of geographical subpopulations, which may differentially participate in the transmission of pathogens. Phylogenetic analysis and haplotype networks with sequences of mitochondrial genes (cytochrome oxidase subunit I and subunit 5 of NADH dehydrogenase) of populations collected in nine locations (cemeteries) of Venezuela showed high interpopulation genetic homogeneity and a single phylogenetic entity (monophyly). It demostrated that the COI gene fragment had a higher resolution in the definition of the phylogeny of closely related species and genera level and correlated with the current classification. The ND5 gene variation is highly useful for population studies. However, this gene showed paraphyly between Cx. corniger and Cx. quinquefasciatus as an evidence of possible homogenization by inbreeding, introgression or infection with Wolbachia. The haplotype networks suggest expanding populations with high haplotype variability and genetic heterogeneity occurred within populations. Moreover, the analysis, showed homogeneity among populations with evolutionary implications in the dispersion of their populations and successful occupation in urban areas, as well as evidence of possible population bottleneck as consequence of insecticide control campaigns.


Subject(s)
Animals , Culex , Culicidae , Culex/anatomy & histology , Culex/genetics , Genetic Vectors , Viruses , Diptera , Diptera/genetics
9.
Journal of Forensic Medicine ; (6): 281-286, 2012.
Article in Chinese | WPRIM | ID: wpr-983750

ABSTRACT

OBJECTIVE@#To explore the application of a 289bp fragment of the 16S rDNA gene to identify various species of sarcosaphagous Calliphorid flies.@*METHODS@#Twenty-six Calliphorid flies were collected from 14 Chinese provinces. All specimens were properly assigned into three genera and six species. The DNA of the pectoralis was extracted using CTAB method. Then PCR amplification was done for the 289 bp fragment of the 16S rDNA gene. The PCR products were then purified and sequenced, and the obtained sequences were uploaded to GenBank. The phylogenetic tree was built by the neighbor-joining method and intraspecific and interspecific divergences were calculated by sequence analysis.@*RESULTS@#The above 26 sarcosaphagous flies could be well clustered according to different genera and species. The evolutional intraspecific values were all zero, the evolutional interspecific variations varied from 0.3% to 6.5%.@*CONCLUSION@#The 289 bp fragment of the 16S rDNA of sarcosaphagous flies can be effectively used to identify most of the flies at species level. This method appears to be fast and low dissipative, which might be used to estimate postmortem interval by sarcosaphagous flies.


Subject(s)
Animals , Rabbits , DNA Primers , DNA, Mitochondrial/genetics , DNA, Ribosomal/genetics , Diptera/genetics , Entomology , Forensic Medicine/methods , Phylogeny , Polymerase Chain Reaction/methods , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Species Specificity
10.
Journal of Forensic Medicine ; (6): 265-270, 2011.
Article in Chinese | WPRIM | ID: wpr-983662

ABSTRACT

OBJECTIVE@#To compare effects of three different methods for mtDNA extraction from common sarcosaphagous insects including cetyl trimethyl ammonium bromide (CTAB) method, sodium dodecyl sulfate-potassium acetate (SDS-KAc) method and sodium dodecyl sulfate-proteinase K (SDS-PK) method.@*METHODS@#Seventy-two insects from four species [Chrysomya megacephala (Fabricius, 1784), Eusilpha bicolor (Fairmaire, 1896), Paraeutrichopus pecoudi (Mateu, 1954), Vespa velutina (Lepeletier, 1836)] were collected from the corpses of the rabbits in Changsha district. The total DNA of above samples was extracted by CTAB, SDS-Kac and SDS-PK methods. The purity and concentration of DNA were examined by protein-nucleic acid spectrophotometry, and mtDNA were amplified by specific primers and PCR products were detected by agarose gel electrophoresis. Then PCR products were sequenced and subsequently up-loaded to GenBank.@*RESULTS@#mtDNA was successfully extracted with three methods from most of the samples. The SDS-PK method was better in DNA purity compared to other methods and the CTAB method was superior in extracting DNA from old samples, while SDS-KAc method showed no significant difference for extraction effects of different samples.@*CONCLUSION@#The most appropriate method should be chosen depending on different situations. SDS-PK method is expected to obtain high-quality DNA, while CTAB method is preferred in extracting obsolete samples. SDS-KAc method is low cost and can be used in various kinds of preliminary experiments.


Subject(s)
Animals , Rabbits , Coleoptera/genetics , DNA Primers , DNA, Mitochondrial/isolation & purification , Diptera/genetics , Electrophoresis, Agar Gel , Entomology , Forensic Medicine/methods , Gene Amplification , Insecta/genetics , Polymerase Chain Reaction/methods , Quaternary Ammonium Compounds/chemistry , Reproducibility of Results , Sequence Analysis, DNA , Sodium Dodecyl Sulfate/chemistry
11.
Journal of Forensic Medicine ; (6): 133-138, 2011.
Article in Chinese | WPRIM | ID: wpr-983640

ABSTRACT

Species identification of sarcosaphagous insects is one of the important steps in forensic research based on the knowledge of entomology. Recent studies reveal that the application of molecular biology, especially the mtDNA sequences analysis, works well in the species identification of sarcosaphagous insects. The molecular biology characteristics, structures, polymorphism of mtDNA of sarcosaphagous insects, and the recent studies in species identification of sarcosaphagous insects are reviewed in this article.


Subject(s)
Animals , Amino Acid Sequence , Base Sequence , DNA, Mitochondrial/genetics , Diptera/genetics , Electron Transport Complex IV/genetics , Entomology , Forensic Medicine/methods , Genes, Mitochondrial/genetics , Insecta/genetics , Polymerase Chain Reaction , Polymorphism, Genetic , RNA, Ribosomal/genetics , Sequence Analysis, DNA , Species Specificity
12.
Braz. j. med. biol. res ; 43(12): 1143-1152, Dec. 2010. ilus
Article in English | LILACS | ID: lil-569006

ABSTRACT

5-Bromo-2’-deoxyuridine (BrdUrd) has long been known to interfere with cell differentiation. We found that treatment ofBradysia hygida larvae with BrdUrd during DNA puff anlage formation in the polytene chromosomes of the salivary gland S1 region noticeably affects anlage morphology. However, it does not affect subsequent metamorphosis to the adult stage. The chromatin of the chromosomal sites that would normally form DNA puffs remains very compact and DNA puff expansion does not occur with administration of 4 to 8 mM BrdUrd. Injection of BrdUrd at different ages provoked a gradient of compaction of the DNA puff chromatin, leading to the formation of very small to almost normal puffs. By immunodetection, we show that the analogue is preferentially incorporated into the DNA puff anlages. When BrdUrd is injected in a mixture with thymidine, it is not incorporated into the DNA, and normal DNA puffs form. Therefore, incorporation of this analogue into the amplified DNA seems to be the cause of this extreme compaction. Autoradiographic experiments and silver grains counting showed that this treatment decreases the efficiency of RNA synthesis at DNA puff anlages.


Subject(s)
Animals , Bromodeoxyuridine/pharmacology , DNA , Diptera/genetics , Insect Proteins/drug effects , Salivary Glands/chemistry , Salivary Proteins and Peptides/drug effects , Autoradiography , Cell Differentiation , Insect Proteins/genetics , Larva/drug effects , Salivary Glands/drug effects , Salivary Proteins and Peptides/genetics
13.
Braz. j. med. biol. res ; 43(5): 437-444, May 2010. ilus
Article in English | LILACS | ID: lil-546328

ABSTRACT

Elongation factor 1A is a highly conserved protein that participates in translation. We report the occurrence of two genes homologous to the eukaryotic Elongation Factor 1A in Bradysia hygida and describe the partial cloning and characterization of the B. hygida eukaryotic Elongation Factor 1A-F1 (BheEF1A-F1) gene. The pattern of BheEF1A-F1 expression in the salivary gland at the end of the fourth larval instar was investigated using real-time PCR. The results showed that BheEF1A-F1 expression levels are relatively constant at the time when rapid changes in protein synthesis occur in this tissue. In situ hybridization experiments coupled to Southern blot analyses showed that the BheEF1A-F1 gene is located at position 3d of the A chromosome and a second gene homologous to eEF1A is located at position 6a of the X chromosome. Southern blot analyses showed that both the BheEF1A-F1 gene and the second gene homologous to eEF1A constitute non-amplified genes. The present results contribute to the molecular characterization of a sciarid eEF1A gene.


Subject(s)
Animals , Diptera/genetics , Genes, Insect/genetics , Peptide Elongation Factor 1/genetics , Base Sequence , Blotting, Southern , Larva/genetics , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , RNA, Messenger/genetics
14.
Journal of Forensic Medicine ; (6): 336-339, 2010.
Article in Chinese | WPRIM | ID: wpr-983590

ABSTRACT

OBJECTIVE@#To explore mitochondrial DNA (mtDNA) extraction effects of different parts from sarcosaphagous insects using improved cetyltriethylammnonium bromide (CTAB) method.@*METHODS@#Thirteen Lucilia sericata (Meigen) and 13 Nicrophorus fossor (Erichson) were collected from the corpses of rabbits placed on the outdoor lawn in Huhehot district. Four parts (head, chest muscle, legs and wings) of insect were collected, and the mtDNA of all samples were extracted using CTAB method. The purity and concentration were tested using protein and nucleic acid spectrophotometry. The integrity of the extracted mtDNA and PCR products were checked by agarose gel electrophoresis. The PCR products were sequenced and the obtained sequences were imputed into GenBank for comparison.@*RESULTS@#mtDNA were successfully extracted from 10 head samples, 6 legs samples, 4 wing samples and 13 chest muscle samples of the Lucilia sericata (Meigen). Also, mtDNA were successfully extracted from 5 head samples, 8 legs samples, 3 wing samples and 13 chest muscle samples of the Nicrophorus fossor (Erichson).@*CONCLUSION@#mtDNA can be obtained from chest muscle and other parts of sarcosaphagous insects using the improved CTAB method.


Subject(s)
Animals , Rabbits , Coleoptera/genetics , DNA, Mitochondrial/isolation & purification , Diptera/genetics , Electron Transport Complex IV/genetics , Electrophoresis, Agar Gel , Entomology , Forensic Medicine/methods , Polymerase Chain Reaction/methods , Sequence Analysis, DNA , Species Specificity
15.
Journal of Forensic Medicine ; (6): 104-107, 2010.
Article in Chinese | WPRIM | ID: wpr-983549

ABSTRACT

OBJECTIVE@#Using CO II sequences to identify common species of carrion-breeding flies and larvae.@*METHODS@#flies and larvae were collected on the corpses of rats in Zhengzhou district, DNA was extracted, CO II sequences were amplified and sequenced. Clustalx and MEGA 4.0 software were used to analyze the gene sequences and to construct the phylogenetic trees.@*RESULTS@#There was no significant gene difference between adults and larvae. COII gene sequences could be used to identify Boettcherisca peregrina, Aldrichina grahami and Lucilia illustris but they could not distinguish Lucilia cuprina from the Lucilia sericata because of their close evolutionary distance and single nucleotide polymorphisms in aldrichina grahami and Lucilia illustris populations were found.@*CONCLUSION@#CO II sequence of mtDNA in Zhengzhou district can be used effectively to identify some common species of carrion-breeding fly. The method is simple and accurate.


Subject(s)
Animals , Rats , Base Sequence , China , DNA Primers , DNA, Mitochondrial/genetics , Diptera/genetics , Electron Transport Complex IV/genetics , Entomology , Forensic Medicine/methods , Genes, Insect , Larva/genetics , Phylogeny , Polymerase Chain Reaction/methods , Sequence Analysis, DNA , Species Specificity
16.
Mem. Inst. Oswaldo Cruz ; 104(7): 1047-1050, Nov. 2009. tab
Article in English | LILACS | ID: lil-534175

ABSTRACT

The investigation of the genetic variation and population structure of Chrysomya species is of great interest for both basic and applied research. However, very limited genetic information is available for this genus across its geographical distribution. Here, we describe 12 polymorphic microsatellite loci isolated from Chrysomya putoria with expected heterozygosities ranging from 0.1402-0.8312. These markers are of potential applied interest for forensic entomologists and for the characterisation of the genetic structure of C. putoria from recently colonised regions, with great promise for understanding the colonisation dynamics and spread of the genus Chrysomya in the New World.


Subject(s)
Animals , Diptera/genetics , Microsatellite Repeats/genetics , Polymorphism, Genetic/genetics , Brazil , Diptera/classification , Genetics, Population , Gene Frequency/genetics , Genetic Markers/genetics , Heterozygote , Sequence Analysis, DNA
17.
Journal of Forensic Medicine ; (6): 421-424, 2009.
Article in Chinese | WPRIM | ID: wpr-983516

ABSTRACT

OBJECTIVE@#To establish an effective phenol-chloroform method coupled with paramagnetic particle method for human DNA extraction from maggot crop contents in STR genotyping.@*METHODS@#Human DNA was extracted from the maggot crop contents using phenol-chloroform method and purified by paramagnetic particle method. DNA was quantified by PCR with Quantifiler Human DNA Quantification Kit using 7500 real-time fluorescence quantitative PCR instrument. PCR products were genotyped by AmpFlSTR Identifiler PCR Amplification Kit using 3130XL-Avant genetic analyzer.@*RESULTS@#The template DNA yield by the method described were increased at least 2 times than the phenol-chloroform extraction method alone. All of the full 16 STR profiles could be obtained with the samples extracted by this method when the DNA yield reached (0.218 +/- 0.041) ng/microL.@*CONCLUSION@#Phenol-chloroform method coupled with paramagnetic particle method can effectively increase the sensitivity of STR analysis of human DNA recovered from maggot crop contents and is a valuable tool for forensic entomology.


Subject(s)
Animals , Humans , Cadaver , Chloroform/chemistry , DNA/isolation & purification , DNA Fingerprinting/methods , Diptera/genetics , Entomology/methods , Forensic Sciences/methods , Gastrointestinal Contents , Larva/genetics , Phenol/chemistry , Polymerase Chain Reaction/methods , Sensitivity and Specificity , Tandem Repeat Sequences
18.
Genet. mol. biol ; 32(1): 177-185, 2009. graf, tab
Article in English | LILACS | ID: lil-505766

ABSTRACT

The ADH (alcohol dehydrogenase) system is one of the earliest known models of molecular evolution, and is still the most studied in Drosophila. Herein, we studied this model in the genus Anastrepha (Diptera, Tephritidae). Due to the remarkable advantages it presents, it is possible to cross species with different Adh genotypes and with different phenotype traits related to ethanol tolerance. The two species studied here each have a different number of Adh gene copies, whereby crosses generate polymorphisms in gene number and in composition of the genetic background. We measured certain traits related to ethanol metabolism and tolerance. ADH specific enzyme activity presented gene by environment interactions, and the larval protein content showed an additive pattern of inheritance, whilst ADH enzyme activity per larva presented a complex behavior that may be explained by epistatic effects. Regression models suggest that there are heritable factors acting on ethanol tolerance, which may be related to enzymatic activity of the ADHs and to larval mass, although a pronounced environmental effect on ethanol tolerance was also observed. By using these data, we speculated on the mechanisms of ethanol tolerance and its inheritance as well as of associated traits.


Subject(s)
Animals , Alcohol Dehydrogenase/metabolism , Drug Tolerance , Diptera/genetics , Enzyme Induction , Ethanol , Hybridization, Genetic , Phenotype , Polymorphism, Genetic , Data Interpretation, Statistical
19.
São Paulo; s.n; 16 dez. 2008. 148[15] p. ilus, graf, tab.
Thesis in Portuguese | LILACS | ID: lil-508068

ABSTRACT

Lisozimas são enzimas que fazem parte do mecanismo de defesa contra bactérias, no entanto lisozimas com função digestiva também são encontradas no trato digestivo de vertebrados e no intestino médio de insetos. As Iisozimas digestivas de insetos são do tipo "c" e assim compartilham semelhanças estruturais e mecanísticas com a lisozima da clara de ovo de galinha (HEWL). Entretanto, para desempenhar sua função digestiva, as lisozimas de insetos apresentam algumas propriedades particulares entre as quais se destaca um pH ótimo mais ácido em relação às lisozimas não-digestivas. Para elucidar as bases moleculares dessa diferença no pH ótimo, duas lisozimas digestivas (lisozima 1 — AAQ20048 e lisozima 2 — AAQ20047) da larva de Musca domestica (mosca — Diptera Cyclorrhapha), clonadas em Pichia pastoris e purificadas, foram caracterizadas estruturalmente e cineticamente com o substrato sintético (MUQ3) e natural (cápsulas de Micrococcus lysodeikticus). Foi observado que o efeito do pH na atividade das lisozimas 1 e 2 sobre o MUQ3 é uma curva com formato de sino e pH ótimo mais ácido que o da HEWL. Essas curvas foram reflexos da diminuição simultânea dos valores de p’K IND.a’s do nucleófilo e do doador de prótons...


Subject(s)
Animals , Digestion/physiology , Diptera/enzymology , Diptera/genetics , Enzymes , Molecular Biology , Muramidase/physiology , Muramidase/isolation & purification , Pichia/enzymology , Pichia/isolation & purification , Recombinant Proteins/isolation & purification , Cell Culture Techniques , Electrophoresis, Agar Gel , Polymerase Chain Reaction
20.
Braz. j. biol ; 67(4,supl): 945-950, Dec. 2007. ilus, tab
Article in English | LILACS | ID: lil-474236

ABSTRACT

The chromosome modal number in Muscoidea Diptera is 2n = 12, including five pairs of autosomes and one sex chromosome pair. Nevertheless, some species with 2n = 10 chromosomes have been described, all of them from the Muscidae family. We analyzed the karyotype of some Muscidae species from different subfamilies and compared the obtained data with the karyotypes of some species of the families Calliphoridae and Sarcophagidae. Comparisons of these species with other Muscidae species revealed a considerable variation among their sex chromosomes. This variation in the length of the sex chromosomes suggests that parts of these chromosomes were lost or fused with autosomes. The constitutive heterochromatic regions and the nucleolar organizer regions (NORs) were also analyzed and some aspects about the relationship between these regions and the sex chromosomes are discussed.


O número modal de cromossomos dos Dípteros Muscóideos é 2n = 12, incluindo cinco pares de autossomos e um par de cromossomos sexuais. No entanto, algumas espécies com 2n = 10 cromossomos já foram descritas, sendo todas pertencentes à família Muscidae. No presente trabalho, foram analisados os cariótipos de algumas espécies de Muscidae de diferentes subfamílias e os dados obtidos foram comparados com os cariótipos de algumas espécies das famílias Calliphoridae e Sarcophagidae. Comparações destas espécies com outras da família Muscidae revelaram uma considerável variação entre seus cromossomos sexuais. Esta variação no tamanho dos cromossomos sexuais sugere que parte destes cromossomos foram perdidos ou sofreram fusão com autossomos. As regiões de heterocromatina constitutiva e as regiões organizadoras de nucléolos (RONs) foram também analisadas e alguns aspectos sobre a relação destas com os cromossomos sexuais são discutidos.


Subject(s)
Animals , Female , Male , Chromosome Banding/methods , Diptera/genetics , Heterochromatin/genetics , Sex Chromosomes/genetics , Diptera/classification , In Situ Hybridization, Fluorescence , Nucleolus Organizer Region/genetics
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