ABSTRACT
α-Amylases are common enzymes responsible for hydrolyzing starch. Insect-pests, whose larvae develop in seeds, rely obligatorily on α-amylase activity to digest starch, as their major food source. Considering the relevance of insect α-amylases and the natural α-amylase inhibitors present in seeds to protect from insect damage, we report here the molecular cloning and nucleotide sequence of the full-length AmyHha cDNA of the coffee berry borer, Hypothenemus hampei, a major insect-pest of coffee crops. The AmyHha sequence has 1879 bp, containing a 1458 bp open reading frame, which encodes a predicted protein with 485 amino acid residues, with a predicted molecular mass of 51.2 kDa. The deduced protein showed 55-79% identity to other insect α-amylases, including Anthonomus grandis, Ips typographus and Sitophilus oryzae α-amylases. In depth analysis revealed that the highly conserved three amino acid residues (Asp184, Glu220, and Asp285), which compose the catalytic site are also presented in AmyHha amylase. The AmyHha gene seems to be a single copy in the haploid genome and AmyHha transcription levels were found higher in L2 larvae and adult insects, both corresponding to major feeding phases. Modeling of the AmyHha predicted protein uncovered striking structural similarities to the Tenebrio molitor α-amylase also displaying the same amino acid residues involved in enzyme catalysis (Asp184, Glu220 and Asp285). Since AmyHha gene was mostly transcribed in the intestinal tract of H. hampei larvae, the cognate α-amylase could be considered a high valuable target to coffee bean insect control by biotechnological strategies.
Subject(s)
Coleoptera/physiology , DNA, Complementary/genetics , Feeding Behavior , alpha-Amylases/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Coleoptera/classification , Coleoptera/enzymology , Models, Molecular , Molecular Sequence Data , Open Reading Frames , Phylogeny , Real-Time Polymerase Chain Reaction , Sequence Homology, Amino Acid , alpha-Amylases/chemistryABSTRACT
Twenty-five RAPD loci and 6 isozyme loci were studied to characterize the genetic variability of natural populations of Anthonomus grandis from two agroecosystems of Brazil. The random-amplified polymorphic DNA data disclosed a polymorphism that varied from 52 to 84% and a heterozygosity of 0.189 to 0.347. The index of genetic differentiation (GST) among the six populations was 0.258. The analysis of isozymes showed a polymorphism and a heterozygosity ranging from 25 to 100% and 0.174 to 0.277, respectively. The genetic differentiation (FST) among the populations obtained by isozyme data was 0.544. It was possible to observe rare alleles in the populations from the Northeast region. The markers examined allowed us to distinguish populations from large-scale, intensive farming region (cotton belts) versus populations from areas of small-scale farming
Subject(s)
Weevils/genetics , Animals , Brazil , Genetic Variation , Isoenzymes/genetics , Random Amplified Polymorphic DNA Technique , Weevils/enzymologyABSTRACT
Twenty-five RAPD loci and 6 isozyme loci were studied to characterize the genetic variability of natural populations of Anthonomus grandis from two agroecosystems of Brazil. The random-amplified polymorphic DNA data disclosed a polymorphism thatvaried from 52 to 84% and a heterozygosity of 0.189 to 0.347. The index of genetic differentiation (GST) among the six populations was 0.258. The analysis of isozymes showed a polymorphism and a heterozygosity ranging from 25 to 100% and 0.174 to 0.277, respectively. The genetic differentiation (FST) among the populations obtained by isozyme data was 0.544. It was possible to observe rare alleles in the populations fromthe Northeast region. The markers examined allowed us to distinguish populations from large-scale, intensive farming region (cotton belts) versus populations from areas of small-scale farming.
Subject(s)
Animals , Weevils/genetics , Genetic Variation , Brazil , Isoenzymes/genetics , Random Amplified Polymorphic DNA Technique , Weevils/enzymologyABSTRACT
A sensitive and specific polymerase chain reaction (PCR) based on a highly repeated deoxyribonucleic acid (DNA) sequence (188 bp; SspI repeat) was tested for the detection of Wuchereria bancrofti DNA in blood and urine samples collected during the day from individuals in Coque, Recife, Brazil, an endemic area for W. bancrofti. All microfilaraemic individuals were also positive by PCR, irrespective of the samples used. The PCR system was capable of detecting W. bancrofti DNA in amicrofilaraemic individuals: c. 93% were positive by PCR when day blood samples were used and 59.7% when urine samples collected at 07:00 were used. Thus, nocturnally periodic W. bancrofti infection can be detected in blood samples collected during the day, which is convenient for large-scale screening. In addition, non-invasive urine collection provided suitable samples for PCR, which is clearly advantageous for preliminary mass diagnosis.
