Introduction
ABSTRACT
Braconidae and Ichneumonidae are two parasitic wasps with different hosts. Distinguishing these two different subfamilies of wasps by morphological features is difficult. This sequence analysis of the mitochondrial 16S rDNA gene has been used for molecular taxonomy in many insects. In this paper I analyzed a 552 bp region of the mitochondrial 16S rRNA gene in the two subfamilies of wasps. The results showed that molecular technique combined with morphological characters could be useful in identification of subfamilies.
Key words: 16S rDNA, Braconidae, Ichneumonidae
INTRODUCTION
Braconidae and ichneumonidae wasps have been extensively studied due to their importance as biological control agents. Both taxonomic groups are typically small (<15mm), dark in coloration, with two sets of wings and thin pedicel. Ichneumonidae and Braconidae are two subfamily of family Ichneumonidae. Morphological analyses suggested that two subfamilies are closely affiliated (Quicke, 1990). Precise identification of specimens is very difficult - involving very close examination of microscopic features. Molecular methods are expected to provide new and more precise means in distinguishing different subfamilies. The mitochondrial 16S ribosomal DNA gene was chosen because it has been used in many insect phylogenetic studies, at both lower and higher taxonomic levels, as well as in studies of diverse Hymenopteran groups (Simon, 1994; Dowton, 1994; Dowton, 1998).
MATERIALS AND METHODS
Wasp discrimination by morphological character
Wasps were kindly offered by Kevin Cox. They were preserved in 70% ethanol. Microscopic analysis of wing veination was used to morphologically distinguish Braconidae and Ichneumonidae.
DNA extraction
Abdomens were removed from Ichneumonidae samples and used for extraction while the abdomen and thorax were used for Braconidae samples because Ichneumonidae is bigger than Braconidae. The head region was excluded as it frequently contained PCR inhibitors. (Dowton, 1998). With each sample I, respectively: (a) removed ethanol by washing three times (15 min each) in 10 mM Tris-Hcl (pH 8) containing 100 mM mM NaCl and 1mM MgCl2, (b) homogenized tissue using a disposable minipestle, (c) added 25 ul of 50 mM DTT (Dithiothreitol), (d) added 25 ul of 0.5 M KOH, (e) spinned for 5 s at 13, 000 rpm, (f) incubated in a heat block for 5 min at 65C, (g) vortexed for 10 s, (h) incubated for 5 min at 65C, (i) spin for 10 s at 13,000 rpm, (j) add 25 ul of 0.5 M HCl, (k) add 8.4 ul of 0.5 M Tris-HCl pH. 9.0, (l) spin for 10 s at 13,000 rpm, (m) mix by hand agitation, (n) spin for 1 min at 13,000 rpm, (o) transfer 50 ul of the supernatant into a new tube (Starks, 2002).
Primers
16S rDNA primers were synthesized in an oligonucleotide synthesizer in our laboratory. The forward primer is 5-CACCTGTTTATCAAAAACAT-3 and reverse primer 5-CTTATTCAACATCGAGGTC-3 (Crozier, 1993). These primers were designed to generate a 552 bp DNA fragment.
PCR
PCR was performed in a 25 ul reaction containing 2.5 ml 10x PCR buffer, 2.0 ml 2.5mM dNTPs, 0.75 ml 50 mM MgCl2, 0.5 ml 10 pmol/ul forward primer, 0.5 ml 10 pmol/ul reverse primer, 0.175 ml Taq DNA polymerase (5 U/ ul), 87.5~175.0 ng DNA template. Tubes were placed in a Perkins-Elmer Thermal Cycler. Cycling conditions were as follows: (a) denaturation at 94C for 30 sec, (b) 50 cycles of 1min denaturation at 94C, 1 min annealing at 55C, 1 min extension at 72C, and (c) 8 min extension at 72C. (Patrick, 1999). Products were stained with ethidium bromide and visualized under UV light.
PCR purification
The PCR product was purified using Rapid PCR Purification Systems (Marligen Bioscience). For each 25 ml PCR reaction, I (a) added 400 ml of Binding solution (H1) and mix, (b) placed a cartridge in a 2ml collection tube and loaded the sample from step (a) into the cartridge, (c) centrifuged at 13000*g for 60 sec, (d) discarded flow-through and place column back into the same tube, (e) added 700 ul Wash buffer (H2) to column and centrifuge at 13000*g for 2 min, (f) discarded flow-through, placed column into tube, and centrifuge column for an additional 2 min at 13000*g speed, (g) placed column in a clean 1.5 ml microfuge tube, (h) added 50 ul dH2O to the center of column, allowed for 1 min, and then centrifuged at 13000g for 2 min.
Sequencing
Sequencing by Sanger dideoxy method was performed using AmpliCycle Sequencing kit( Perkin Elmer, Norwalk, CT). For each reaction, 4 ul of 10X buffer, 2 ul of reverse primer, 50 fmol of DNA, and 0.25 ul aP33 (dATP) was used. 6 ul of this mixture was added to each of four 200 ul Eppendorf tubes, each containing either 2 ul of ddGTP, ddATP, ddTTP, or ddCTP. Each tube was overlaid with mineral oil and placed in a Perkin-Elmer Thermal Cycler for the reaction. Cycling conditions were: 94C for 2 min, 35 cycles of 94C for 30 sec, 58C for 30 sec, 72C for 1 min. Following the sequencing reactions, 4 ul of stop solution was added to each tube and samples were boiled for 3 min prior to loading and electrophoresis on a sequencing gel.
Sequence alignment
Sequences were read into a computer from radiographs and aligned using MacVector 6.5.3.
