Biology Reference
In-Depth Information
Colorado potato beetle,
Leptinotarsa decemlineata
; the American cockroach,
Periplaneta americana
; and the twospotted spider mite,
Tetranychus urticae
.
After amplification, the PCR products were sequenced, and only five of 60 clones
were not derived from
para
homologs. This study, and others, suggests that
degenerate primers derived from conserved segments of characterized
D. mela-
nogaster
genes can be used to clone genes from diverse arthropods. Interestingly,
intraspecific polymorphisms were found in the sequence from three moth spe-
cies, which could reflect the presence of duplicated genes or allelic variants in the
populations.
Similarly, primers for the conserved
Actin
gene(s) in insects were used to clone
these genes from the predatory mite
Metaseiulus occidentalis
, despite the long
evolutionary separation of insects and mites (
Hoy et al. 2000
). The rich source
of sequence information in GenBank for the complete
Drosophila
genome and
those of other species makes this approach increasingly feasible.
8.5.6 Detecting Gene Amplification
Sometimes it is important to determine whether a gene has been amplified
(increased in copy number), leading to increased levels of gene product. Some
insects are resistant to pesticides due to amplification of esterase genes. A method
called comparative PCR can be used to detect gene amplification (
Brass et al. 1998
).
8.5.7 Detecting Methylation of DNA
Genomic imprinting is often due to DNA methylation at several sites in the genome.
A methylation-specific PCR assay can be used to detect methylation of specific
genes more quickly than the use of Southern-blot assays (
Kubota et al. 1997
).
8.5.8 Detecting Pathogens in Vector Arthropods
Arthropod vectors such as ticks, fleas, and mosquitoes are involved in maintain-
ing and transmitting (vectoring) pathogens to humans and other vertebrates.
Aphids and leafhoppers transmit (vector) viruses and mycoplasma to plants.
The detection of pathogenic microorganisms within vector arthropods is
important in conducting epidemiological studies and developing control strat-
egies. Several antigen-detection techniques have been developed, including
direct or indirect immunofluorescence tests and enzyme-linked immunosor-
bent assays (ELISA) using polyclonal or monoclonal antibodies. Other techniques
involve recovery of the microorganisms from vectors by culture in embryonated
eggs or tissue culture cells or by experimental infections in laboratory ani-
mals. The recovery of pathogenic microorganisms by these methods requires
