Biology Reference
In-Depth Information
rearing is required to obtain accurate identifications, which can cause a signifi-
cant delay in a quarantine or eradication program.
Using RAPD-PCR,
Sonvico et al. (1996)
found that it is possible to discriminate
between immature stages of Medflies and
Anastrepha fraterculus
.
Haymer et al.
(1994)
identified unique repetitive DNA probes that they used in slot blots or
squash blots to discriminate between eggs or larvae of three tephritid species.
The squash-blot procedure used a nonradioactive hybridization and detection
method, making it simple and rapid to carry out and potentially allowing rapid
identification of infested fruits at the earliest stage during quarantine and erad-
ication procedures.
AFLP-PCR was used by
Kakouli-Durate et al. (2001)
to discriminate between
C. capitata
and
C. rosa
. A species-specific repetitive marker was cloned and used
as a probe for genomic dot-blot hybridizations; the probes were sequenced and
primers were developed. AFLP-PCR products from
C. capitata
electrophoresed on
polyacrylamide gel revealed diagnostic bands after silver staining (
Kakouli-Durate
et al. 2001
). However, the probe produced faint bands with DNA from
Bactrocera
cucurbitae
and
B. oleae
, suggesting that the repetitive DNA fragment exists in low-
copy number in them. The number of “outgroup” tephritids tested was limited, so
the probe may only be useful to discriminate among a few species. However, the
authors did investigate the method's sensitivity and found that, because the probe
sequences are found in multiple copies in the genome, sufficient DNA could be
extracted from wings or legs of
C. capitata
, yet yield positive results.
Ribosomal ITS1 polymorphisms were investigated in
C. capitata
and
C. rosa
to
provide species-specific probes and to investigate the differences in size among
different populations of each species (
Douglas and Haymer 2001
). Recall that
insects have multiple ribosomal genes (rDNA) and that the noncoding ITS region
tends to vary sufficiently to allow discrimination of lower-level taxonomic
groups. ITS1 sequences were isolated from a
C. capitata
genomic library and
sequenced; sequences from
C. capitata
and from
Drosophila melanogaster
were
aligned to identify conserved sequences and primers were designed to amplify
across the variable ITS1 regions. No obvious size variability in the PCR products
was found among
C. capitata
from Hawaii, Guatemala, Spain, Greece, Costa
Rica, and Peru, and sequencing of some of these ITS1 regions revealed only sin-
gle nucleotide changes, with length variation ranging from 829 to 832 bp. Thus,
ITS1 sequence data would not allow a researcher to discriminate among these
populations, but they do allow
C. capitata
to be identified. The same primers
amplified ITS1 sequences from two populations of
C. rosa
and produced prod-
ucts that were 717bp and 930bp long, which might be used to distinguish
