Contents - Faperta

478 Biotechnological Approaches for Pest Management and Ecological Sustainability
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Isozyme and protein profi les;
Immunodiagnostic methods; and
DNA-based methods.
Molecular Tools for Diagnosis of Insect Pests
Polymerase Chain Reaction
Current molecular systematics depends on polymerase chain reaction (PCR) amplifi cation
of a few “universal” genes to provide phylogenetic data. However, the need for sequencing
is increasing quite fast (Murphy et al., 2001; Wheeler et al., 2001; Philippe et al., 2004; Teeling
et al., 2005), and expanding PCR approaches to a wider selection of genes becomes diffi cult
because of the need to develop new degenerate primers for the amplifi cation of singlecopy
loci.
Random Amplified Polymorphic DNA
Random amplifi ed polymorphic DNA (RAPDs)-based DNA fi ngerprinting has been used
for insect diagnosis that does not require prior knowledge of DNA sequence. RAPDs provide
a rapid means of identifying genetic markers to distinguish closely related species
(Alvarez and Hoy, 2002). These markers are inexpensive, rapid, and easy to use when studies
involve many insects. However, results at times are diffi cult to reproduce (Ellsworth,
Rittenhouse, and Honeycut, 1993).
Expressed Sequence Tags
The expressed sequence tags (ESTs) provide a more readily available resource of genomic
data (Rudd, 2003). Most publicly available ESTs have been generated for gene discovery or
to complement genome sequencing efforts. However, species for EST analyses have rarely
been selected based on taxonomic criteria, which limits their use for phylogenetic analyses
and comparative genomics. A concerted effort to enlarge EST databases to encompass disparate
taxa may alleviate these problems (Bapteste et al., 2002; Theodorides et al., 2002).
The EST databases for the taxa specifi cally selected to obtain comprehensive coverage of
Coleoptera, a group that includes nearly one-third of all known species of animals, have
also been generated. A critical problem for comparative studies is that ESTs from different
taxa may not contain overlapping sets of genes. The challenge of matching orthologous
genes between taxa is amplifi ed by the low expression of many transcripts. Sequencing of
tens of thousands of ESTs in Drosophila melanogaster Meigen (Rubin et al., 2000) and Bombyx
mori L. (Mita et al., 2003) has fallen short of reaching a full complement of predicted genes.
The EST databases are growing rapidly, with approximately 27.6 million entries in the
GenBank as of June 2005 (http://www.ncbi.nlm.nih.gov/dbEST/). However, taxonomic
coverage of the Class Insecta has been limited to eight of the 25 insect orders. The EST representation
in insects has been severely biased towards Diptera, comprising 15 of 47 holometabolous
insects.