Quick blots and nonradioactive detection systems : improvements on methods for DNA hybridizations using mosquitoes

QUICK BLOTS AND NONRADIOACTIVE DETECTION SYSTEMS;
IMPROVEMENTS ON METHODS FOR DNA HYBRIDIZATIONS

USING MOSQUITOES

By

DAVID WILLIAM JOHNSON

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

1990

ACKNOWLEDGEMENT

The author would like to acknowledge the one who makes
all things possible, "for from him and through him and to
him are all things. To him be the glory forever! Amen."
(Romans 11:36)

11

TABLE OF CONTENTS

page

ACKNOWLEDGEMENT ii

KEY TO ABBREVIATIONS AND SYMBOLS v

ABSTRACT vi

INTRODUCTION 1

MATERIALS AND METHODS 7

General Molecular Methods 7

Sources of Mosquitoes and Probes 10

Squash Blots and Dot Blots 11

Isolation of the Culex-specif ic Probe, pCxl 12

Isolation of Anopheles nuneztovari-specif ic Probes 13

DNA Sequencing 14

Nonradioactive Detection Systems 16

Overview of Nonradioactive Detection Systems 16

Preparation and Use of Biotinylated Probes 17

Preparation and Use of ECL Probes 18

Preparation and Use of Genius Probes 19

MOSQUITO SPECIES-SPECIFIC DNA PROBES 22

Isolation Methods and the Relevance of Genome

Organization 22

Isolation of Probe pCxl 24

Isolation of Anopheles nuneztovari-specif ic

Probes 26

Mapping and Sequencing of Anopheles quadrimaculatus-

and Anopheles freeborni-specif ic Probes 26

QUICK BLOTS 37

Experiments Leading to the Quick Blot Protocol 37

First Attempts at Making Quick Blots 41

Steps in the Quick Blot Protocol 47

Experiments to Optimize Use of Quick Blots with

Mosquito Species-specific Probes 52

111

SYNTHETIC OLIGONUCLEOTIDE PROBES 72

CONCLUSIONS AND SUMMARY 79

Discussion of the Efforts to Isolate a Culex-specif ic

Probe 79

Significance of Oligonucleotide Probes and

Characterization of Other Mosquito Species-specific
Probes 80

Significance of the Quick Blot Protocol and

Nonradioactive Detections 82

REFERENCES 85

BIOGRAPHICAL SKETCH 88

IV

KEY TO ABBREVIATIONS AND SYMBOLS

°C degrees Centigrade

DNA deoxyribonucleic acid

EDTA ethylene-diamine-tetraacetic acid

kbp kilobase pair(s)

LF LA FRANCE (Dial Corp.)

M molar

mg milligrainCs)

min minute (s)

ml milliliter (s)

mm millimeter

mM millimolar

jug microgram (s)

Ml microliter (s)

NFDM nonfat dry milk

ng nanogram (s)

nm nanometers

pg picograms

QB guick blot

RNA ribonucleic acid

s second (s)

SSC saline sodium citrate

SDS sodium dodecyl sulfate

V

Abstract of Dissertation Presented to the Graduate School

of the University of Florida in Partial Fulfillment of the

Requirements for the Degree of Doctor of Philosophy

QUICK BLOTS AND NONRADIOACTIVE DETECTION SYSTEMS:

IMPROVEMENTS ON METHODS FOR DNA HYBRIDIZATIONS

USING MOSQUITOES

By

David William Johnson

December, 1990

Chairman: Dr. Jack A. Seawright

Major Department: Entomology and Nematology

A DNA (deoxyribonucleic acid) probe was isolated which
exhibited specificity for two mosquito species, Culex
niqripalpus Theobald and Culex salinarius Coquillett. The
nucleotide sequence of another probe specific for Anopheles
guadrimaculatus Say species A was determined in order to
identify sequences which conferred specificity to the probe
and to assist in the production of synthetic oligonucleotide
probes. Probes exhibiting specificity for Anopheles
nuneztovari Gabaldon were isolated in a primary screening,
and other mosquito species-specific probes were partially
characterized .

VI

A new technique for preparing targets for hybridization
of nucleic acid probes, called the quick blot protocol, was
developed. It allowed rapid preparation of multiple (10 or
more) equivalent sample-containing filters, called quick
blots. Samples were applied uniformly in an orderly
arrangement on the filters. The quick blot protocol was used
to prepare targets for hybridization with mosquito species-
specific DNA probes. Using quick blots, detection of
radiolabeled probes was compared with detection of probes
prepared with three nonradioactive detection systems. A
method was developed for the differential detection of two
nonradioactive probes hybridized simultaneously to a quick
blot. Finally, the use of synthetic oligonucleotide probes
with quick blots was demonstrated.

Vll

INTRODUCTION

The need for nonmorphological methods to identify
specimens occurs when specimens of related species cannot be
distinguished by morphology. These cryptic species probably
have arisen from recent speciation events and may therefore
provide valuable models for the study of evolutionary
processes.

In the case of mosquitoes, it is valuable to have
methods for readily identifying cryptic species which differ
in their abilities to serve as vectors for a parasite of
humans. An example is the Anopheles qambiae Giles species
complex, a group of at least six species which are
indistinguishable morphologically. These species differ in
their significance as vectors of malaria, and under certain
conditions can be distinguished by cytological (Coluzzi &
Sabatini, 1967) and isoenzyme (Hunt & Coetzee, 1986)
analyses. But perhaps the easiest way to distinguish three
members of this complex is with a DNA probe (Collins et al . ,
1988) . However, this single probe will not distinguish all
the known members of the complex.

There are two major areas of concern in the efforts to
make probe technologies useful. One is the isolation of
probes with the desired traits, and the other is the

2
development of methods that can best detect the hybridized
probe molecules.

The specificity of base pairing in the annealing, or
hybridization, of separated strands of nucleic acid has
allowed the development of DNA probe technologies. These
have proven invaluable for the detection of pathogens of
humans (Hyypia et al., 1989), viruses and viroids infecting
plants (Mclnnes & Symons, 1989a) , and human genetic
disorders (Sutherland & Mulley, 1989) . The specificity of
DNA probes has also been used to identify species of
mosquitoes (Cockburn et al., 1988; Cockburn, 1990). Methods
for using nucleic acid probes usually involve preparation of
a suitable target, in which the nucleic acid to be probed is
immobilized in a denatured form on a glass or membrane
filter support. Denatured probe is then given a chance to
anneal with the target nucleic acid in a hybridization step,
in conditions conducive to duplex formation. From this point
on, the focus will be on DNA probes, for even though RNA
probes could be of value in tissue- or age-specific
detection, they have not been as widely used as DNA probes
and are unstable.

The two main parameters that relate to the value of a
given DNA probe are specificity (selectivity) and
sensitivity. These parameters are determined in part by the

3
nucleotide sequence of the probe, but also depend greatly on
the conditions used to anneal the probe to the target, and
the characteristics of the detection system used to
visualize the hybridized (bound) probe.

There are several techniques used for preparing the
targets for nucleic acid probes. Nucleic acids may be
extracted from tissues, and applied to a filter to form a
slot blot (Wahl et al., 1987) or a dot blot (Costanzi &
Gillespie, 1987) . Or, an organism or isolated tissue may be
used directly to form a squash blot (Cockburn, 1990; Keating
et al., 1989; Kirkpatrick et al., 1987; Tchen et al . , 1985).
This is done by using enough force while squashing the
material against the filter that some DNA is freed from the
cells and becomes bound to the filter. The squash blot
method is useful in the preparation of two equivalent
filters containing the DNA of individual mosquitoes
(Cockburn, 1990) .

This study focused on the isolation, characterization,
and use of mosquito species-specific DNA probes. The
specific objectives were: (1) isolation of new DNA probes
showing specificity for certain Culex and Anot3heles species
(especially the vector of St. Louis encephalitis virus,
Culex niqripalpus) , (2) characterization of the nucleotide
sequence (s) conferring specificity in one or more probes,

4
(3) development of a fast, reliable system for the
preparation of multiple targets for mosquito DNA probes
using individual mosquitoes, (4) assessment of the
usefulness of commercially available nonradioactive
detection systems when applied to systems for hybridization
of mosquito DNA probes (including a comparison to
radioactive detection methods) , (5) assessment of the value
of synthetic oligonucleotides as mosquito species-specific
probes.

Central to this study were repetitive DNA probes shown
previously to exhibit specificity for the four known members
of the A. quadrimaculatus species complex (Cockburn, 1990) .
These probes will be useful in assessing the potential of
the members of the complex to serve as vectors of malaria.
Probes Arp2 , Brpl, and Crpl hybridized primarily to DNA from
A. cruadrimaculatus species A, B, and C, respectively.
However, Arp2 hybridized slightly to species B DNA. This
probe was chosen as a model for the characterization of
mosquito species-specific probes, in part because of the
possibility of using its nucleotide sequence to prepare
oligonucleotide probes which might exhibit improved
specificity. Also, restriction analysis of probe Arp2
indicated that it was probably composed of multiple
identical (or very similar) repeat sequences (personal

5

communication, A. F. Cockburn, United States Department of
Agriculture (USDA) ) which could be detected by nucleotide
sequencing of suitable subclones.

A new method, called the quick blot (QB) protocol, is
described for preparing hybridization targets using
mosquitoes. The QB protocol was used to produce ten
equivalent sets of nucleic acid targets on filters, called
quick blots (QBs) , for use in nucleic acid hybridization
assays. It has been found useful in the analysis of
individual mosquitoes, with up to 96 individuals per filter.
The filters were used successfully as DNA hybridization
targets for mosquito species-specific DNA probes. The main
advantages that the QB protocol offers over previous methods
include: the uniformity of sample application, the orderly
arrangement of samples on the filters, the ability to
produce multiple identical sample-containing filters, and
the rapidity with which numerous samples can be processed.

Specific detection of DNA probes hybridized to QBs was
achieved with nonradioactive labeling and detection systems.
These results were compared to those obtained with
radiolabeled probes.

Nucleotide sequence data were obtained from plasmids
containing mosquito species-specific DNA and used to specify
the synthesis of oligonucleotides. These oligonucleotides

6
were tested for their usefulness as species-specific probes
to QBs, and advantages of these synthetic probes were
demonstrated. Thus, QBs may be used as targets for
hybridization of nucleic acid probes; nonradioactive
detection systems may be used to advantage with QBs in some
situations; and synthetic DNA probes can offer advantages
over conventional genomic clones.

MATERIALS AND METHODS
General Molecular Methods

Gels were 0.5-1.0% agarose (Sigma), buffered and run in
IX TBE (89inM Tris-borate, 89inM boric acid, 2itiM EDTA) at less
than 5.5 volts per centimeter. Fragments were sized using a
Hind III digest of bacteriophage lambda or 1 kbp ladder
fragments (Bethesda Research Laboratories, Life
Technologies, Inc. (BRL) ) as markers.

Plasmids were prepared by a modification of the
alkaline-lysis method of Birnboim & Doly (1979) and cesium
chloride purification, or by the boiling method (Holmes St
Quigley, 1981) . Insect genomic DNA was prepared by the
method of Cockburn & Seawright (1988) . Standard methods were
used for restriction analysis of plasmid and genomic DNA,
except that restriction enzymes were used in excess of the
manufacturer's (BRL) recommendations. Nucleic acids were
quantified by ultraviolet absorption at 260 nm.

Double-stranded DNA was radiolabeled by nick
translation (Nick Translation System, BRL) with ^^P-dCTP,
and unincorporated label was removed by size exclusion
chromatography (using Bio-Gel P-60, BioRad) .
Oligonucleotides were radiolabeled with ^^P-ATP and T4
polynucleotide kinase. Unincorporated nucleotides were

8
removed by size exclusion chromatography (using Bio-Rad Bio-
Spin 30 columns) .

Unless otherwise noted, filters were subjected to the
following treatments after application of the target DNA.
Prior to prehybridization, nitrocellulose filters were baked
for 20-45 min at 80 °C under vacuum (vacuum-baked) , and nylon
filters were subjected to treatment with 300 nm ultraviolet
(UV) light (1-2 min on the glass surface of a Chromato-Vue
Transilluminator, Ultraviolet Products, Model TM-36) .
Filters were prehybridized in 1% NFDM (nonfat dry milk) ,
0.2% SDS at 55 °C for at least 30 min, and hybridized with
(denatured) probe in 30% formamide, 5X SSPE (20X SSPE is
3.6M NaCl, 0.2M NaH2P0^ pH 7.4, 20mM EDTA) , 1% NFDM, 0.2%
SDS at 55 °C overnight.

Prehybridization of blots for oligonucleotide probes
was in buffer (6X SSPE, 0.3% SDS, 1.0% NFDM) for one hour at
65 °C. Hybridization with labeled oligonucleotide probe was
performed by adding the probe samples to the bags containing
the filters and prehybridization buffer, resealing, and
incubating for 24 hours at 37 °C. Following hybridization,
the filters were washed four times for 15 min each wash in
4X SSPE at 65 °C. All films used for autoradiography and
chemiluminescent detection (see below) were Kodak X-AR with
Kodak intensifying screens.

9
Excess probe annealing to highly repetitive DNA can
provide a higher sensitivity than a probe annealing to
moderately repetitive or single-copy sequences, if whole
genomic DNA serves as the target. In this study, excess
probe (0.5-1.0 ixq per filter) was used in each hybridization
experiment to ensure that detection of bound probe was not
limited by probe concentration in the hybridization step.

Two distinct terms are used to describe spurious
detection: background and nonspecific detection. The term
background is used to refer to apparent signal development
in areas of the target filter (or its image on film) not
corresponding to locations where nucleic acid was applied.
The term nonspecific detection (or nonspecific signal) is
used to denote the appearance on the filter (or film) of
signal in areas where nucleic acid was applied but where no
probe was expected to be localized (based on the known
specificity of the probe) .

The DH5-a and JM103 strains of Escherichia coli were
the hosts for all plasmids, and the DH5-a and HBlOl strains
were the hosts for all transformations. Bacteria were grown
on Luria-Bertani culture medium with 3 0 /xg/ml kanamycin or
50 jLtg/ml ampicillin. Bacteria were transformed by standard
methods (Hanahan, 198 3) and screened for plasmids of
appropriate size, using agarose gel electrophoresis.

10
Oligonucleotide probes were synthesized at the ICBR DNA
Synthesis Facility, Gainesville, Florida.

Sources of Mosquitoes and Probes
Specimens of the following mosquito species used in
this study were supplied by the mosquito rearing facility at
the Medical and Veterinary Entomology Research Laboratory,
USDA, Gainesville, Florida: Aedes taeniorhynchus
(Wiedemann) , Anopheles albimanus Wiedemann, A.
quadrimaculatus species A (ORLANDO strain) , Culex
quinquefasciatus Say, and C. salinarius Coquillett.
Specimens of Anopheles crucians Wiedemann, Coquillettidea
perturbans (Walker) , and Culex niqripalpus Theobald were
supplied by Mr. O. R. Willis (USDA) , and were collected in
Alachua County, Florida. A. quadrimaculatus species B, C,
and D mosquitoes were supplied by P. E. Kaiser and S. E.
Mitchell (USDA) .

Probes pA2 , pBrpl-Sl, pCrpl-Sl, pCrp-S2, and pCrp-S3
were supplied by A. F. Cockburn and were derived by
subcloning of phage Arp2 , Brpl, and Crpl (Cockburn, 1990)
Sal I fragments into plasmid pK19. It was demonstrated
previously (Cockburn, 1990) that the probes Arp2 , Brpl, and
Crpl probes exhibited sufficient specificity for A.
quadrimaculatus species A, B, and C, respectively, to allow
differentiation of specimens of all four members of the A.

11

guadrimaculatus species complex. The insert in pA2 was 2.8
kbp, much smaller than the 12 kbp sai I fragment in phage
Arp2 . The sequence organization of this clone (see below)
suggests that a deletion occurred by recombination between
one or more internal repeats. Probe pKA2 was derived by
transferring the (sai I) insert of pA2 into pK19 using Hind
III and EcoR I. Probe pAfl-Sl was prepared by subcloning a
3 . 4 kbp Sal I fragment from an Anopheles f reeborni Aitken-
specific phage probe (A. F. Cockburn, USDA) into pK19 . The
derivation of other probes and subclones is detailed in the
appropriate sections below. All plasmid probes contained
vector pK19 unless otherwise indicated.

Squash Blots and Dot Blots
Squash blots for hybridization of mosquito species-
specific DNA probes were prepared as described previously
(Cockburn, 1990) . A damp blotting filter was covered with
mosquitoes arranged in a grid pattern. A second filter was
placed on top of the mosquitoes, and a metal rod was rolled
over the filters to thoroughly squash the mosquitoes in
between. The resulting sandwiches were laid on filter paper
soaked with denaturing solution (0.5M NaOH, 1 . 5M NaCl) for
about 5 min per side, then transferred to paper saturated
with neutralizing solution (1.5M NaCl, IM Tris, pH 8.0) for
5 min per side. The two filters were separated, and

12
subjected to either the UV fixation (nylon filters) or
vacuum-baking (nitrocellulose filters) steps as described
above.

Dot blots were prepared using standard methods
(Costanzi & Gillespie, 1987) . The dot blot is a way to
prepare hybridization targets using purified DNA. The DNA
can be diluted serially and applied to a blotting filter to
provide spots containing different amounts of bound DNA.
Application of sample solutions to the filter is simple when
using an apparatus called the dot blot manifold (Table 2) .
Isolation of the Culex-specif ic probe, pCxl
Attempts were made to isolate a DNA probe specific for
C. niqripalpus by the method of Cockburn & Mitchell (1989) .
Two variations of this approach were tried, using a phage
vector and a plasmid vector.

Recombinant phage was prepared by ligation of C.
niqripalpus DNA cut with ecoR I and xba I with LambdaGEM-4
EcoR I -Xba I Arms (Promega) . Ligated DNA was packaged
(Gigapack Gold, Stratagene) for screening. Library screening
was performed according to Cockburn (1990), using duplicate
plaque lifts hybridized separately to C. niqripalpus and C.
salinarius genomic DNA. Phage were grown on E. coli strain
P2392.

13
Plasmid subclones were obtained from recombinant
LambdaGEM-4 according to the directions supplied by the
manufacturer (Promega) . Inserts in the LambdaGEM-4 vector
were contained within the pGEM-4 plasmid which is included
as part of the vector; plasmid subclones were easily derived
by cutting the purified recombinant LambdaGEM-4 DNA with spe
I, ligating, and transforming suitable host bacteria.

Plasmid libraries containing C. niqripalpus genomic DNA
in pK19 were prepared using double digests with Hind III and
xba I, EcoR I and Pst I, or ecoR I and Hind III. A plasmid
library was also prepared with C. salinarius DNA in pK19
using a Hind III and ecoR I double digest. Transformants from
the ligation mixtures were grown on kanamycin-containing
medium. Colony lifts were prepared according to the method
of Buluwela et al. (1989), and served as targets for
differential hybridization to C. niqripalpus. C. salinarius,
and C. quinquefasciatus genomic DNA.

Isolation of Anopheles nuneztovari-specif ic Probes
Isolation of Anopheles nuneztovari-specif ic probes was
accomplished according to the methods of Cockburn & Mitchell
(1989) and Cockburn (1990) , using an A. nuneztovari library
in phage EMBL 3 A supplied by A. F. Cockburn. Genomic DNAs
from A. nuneztovari and Anopheles oswaldoi (Peryassu) ,

14

supplied by L. P. Lounibos and J. Conn, were used for the
primary differential hybridization screening.

DNA Sequencing

Subcloning strategies were designed for the selection
of deletion subclones generated by restriction enzymes, and
to allow the use of the standard universal (forward) and
reverse primers.

Sequencing of pKA2 was aided by subcloning of nsI I
fragments from the insert. The recipient vector (pK19) DNA
was cut with Pst I and phosphatased, and pKA2 DNA was cut
with Nsi I. These two digests were ligated, and
transformants were selected and used for sequencing. Using
this approach, each subclone was expected to contain a
single Nsi I fragment from the insert. The subclones were
called PKA2-N1, pKA2-N2, etc.

Nucleotide sequence data were also obtained from other
plasmid clones. These were the A. quadrimaculatus species B-
specific probe pBrpl-Sl, the A. freeborni -specific probe
pAfl-Sl, the Culex-specif ic probe pCxl, and three plasmid
subclones of the A. quadrimaculatus species C-specific probe
(pCrpl-Sl, pCrpl-S2, and pCrpl-S3, comprising the total
insert in the parental recombinant phage probe) .
Sequencing of the unrearranged phage Arp2 insert was

15
accomplished by preparing wsi I subclones in pK19. These
were designated pArp2-Nl, pArp2-N2, etc.

Sequencing reactions were performed on boiling-method
preparations of 1-5 ^,q of plasmid DNA extracted from 2-ml
bacterial cultures grown up overnight. Primer annealing was
performed on alkali-denatured plasmid DNA. Sequenase version
2.0 (U.S. Biochemical Corp.) was used for sequencing by the
chain-terminating method (Sanger, 1977) with manufacturer-
supplied reaction solutions and procedures. Reaction
products were labeled with ^^S dATP in buffers containing
Mg""* ions. Sequencing reactions were run on 0.2-0.9 mm wedge
gels (4% acrylamide [19:1 linear to bis, LKB] , 8M urea, IX
THE) at 55 °C, 1750 volts on a Macrophor (LKB) or Sequigen
(BioRad) electrophoresis unit. Gels were rinsed for 10-20
min in 10% acetic acid before drying in a forced-air oven at
80 °C. Gels run on the Macrophor were bonded to the running
plate, and others were transferred to filter paper prior to
drying and autoradiography.

Sequence analysis was done on the Multiple Sequence
Editor (Massachusetts Institute of Technology) and the
Genetics Computer Group Software Package (Devereux et al.,
1984) version 6.1, both running on a MicroVAX II computer.
Nucleotide sequence searches were performed using the
European Molecular Biology Laboratory (EMBL) version 22

16

(modified; February, 1990) and Genbank version 63 (March,
1990) databases.

Nonradioactive Detection Systems
Overview of Nonradioactive Detection Systems

Three different nonradioactive labeling and detection
methods were used in this study: the SA-AP (streptavidin-
alkaline phosphatase) method (GENE-TECT protocol, Clontech
Laboratories, with BRL reagents) ; the ECL (enhanced
chemiluminescence) method (ECL kit, Amersham Corporation) ;
and the Genius method (Genius Nonradioactive DNA Labeling
and Detection Kit, Boehringer Mannheim Biochemicals) .

The nonradioactive labeling and detection kits were
used essentially as recommended by the suppliers, except
where otherwise noted. The SA-AP kit used biotinylation of
probe DNA via nick translation, and detection of hybridized
probe by binding of streptavidin-alkaline phosphatase,
followed by an enzyme-catalyzed color reaction. The ECL
probes were prepared by covalent binding of peroxidase to
the DNA, and detection of hybridized ECL probes was achieved
by a chemiluminescent reaction using X-ray film. The Genius
kit used random primed incorporation of the steriodal hapten
digoxigenin into probe DNA. Following hybridization. Genius
probes were detected by enzyme-linked immunoassay using an

17
antibody conjugate (a-digoxygenin-alkaline phosphatase
conjugate) , and the same color reaction used with the
SA-AP method.
Preparation and Use of Biotinylated Probes

The preparation of biotinylated probes was achieved by
nick translation of double-stranded template DNA for the
incorporation of biotinylated nucleotides. The BRL Nick
Translation System (BRL) reagents were used, according to
the recommendations for the Biotin-21-dUTP Labeling System
(Clontech Laboratories) . Unincorporated nucleotides were
removed by gel exclusion chromatography (using BIO-GEL P-60,
BioRad) .

Unless otherwise noted, prehybridization and
hybridization conditions for use of biotinylated probes were
as described in the section on general molecular methods,
above .

Detection of hybridized biotinylated probes was
accomplished according to the directions in the GENE-TECT
protocol (GENE-TECT Detection System, Clontech
Laboratories) . All detection steps were performed at room
temperature. Filters were first washed 30 min in 3% NFDM
(blocking step) . Then they were incubated for 25 min with
SA-AP (streptavidin-alkaline phosphatase) , in a solution
made by adding 2 . 5 jul SA-AP conjugate (BRL) per ml Buffer A

18

(0.2M NaCl, 0.05% Triton-X-100 , O.IM Tris, pH 7.5). The
filters were then washed 3 times with Buffer A, 10 min each
wash, then once for 10 min with Buffer C (O.IM NaCl, 50mM
MgClg, O.IM Tris, pH 9.5). Then the filters were incubated
in the color solution (Buffer C with chromogenic substrates)
in reduced illumination until signals were developed
properly. The color reaction was terminated with ImM EDTA.
Preparation and Use of ECL Probes

The directions supplied by the manufacturer of the ECL
kit (ECL Version 2, Amersham) were followed in the
preparation and use of ECL probes, including the
prehybridization and hybridization steps, except that SSPE
was substituted for SSC in the wash solutions (see below) .
Double-stranded DNA to be labeled was precipitated and
resuspended in deionized water at a concentration of 10
ng/ml . The DNA was boiled for 5 min, then immediately cooled
on ice for 5 min. An equivalent amount of DNA labeling
reagent and then glutaraldehyde solution were added to the
DNA and mixed thoroughly. The solution was consolidated by
spinning briefly (5s) in a microcentrifuge, then incubated
for 10 min at 37 "C. The labeled probe was stored in 50%
glycerol at -20°C until used.

19

The supplied hybridization buffer was used for both
prehybridization (at least 10 min at 40-42 "C) and
hybridization (overnight at 40-42 °C) after adding NaCl to
0.5M.

Following hybridization of probes according to the ECL
protocol, filters were removed from the hybridization medium
and washed twice (20 min each wash) at 40-42 °C with primary
wash buffer (6M urea, 14mM SDS, 0.5X SSPE) . Then the filters
were washed twice (5 min each wash) at room temperature with
2X SSPE. Equal volumes of detection solutions 1 and 2 were
mixed, and the filters were incubated in this detection
buffer 1 min at room temperature. Filters were wrapped in
plastic wrap and exposed to x-ray film in the dark, with the
side of the filter which received the DNA during application
of target DNA facing the film. The film was developed after
a 1 min exposure, followed by longer exposures as needed.
Preparation and Use of Genius Probes

The directions supplied by the manufacturer of the
Genius kit (Genius Nonradioactive DNA Labeling and Detection
Kit, Boehringer Mannheim Biochemicals) were followed in the
preparation and use of Genius probes, except that labeled
probes were precipitated with NaCl rather than LiCl, and
SSPE was substituted for SSC in the hybridization steps.
Genius probes were prepared by the random primed

20

incorporation of digoxygenin-tagged nucleotides, and
detected by immunoassay.

In the preparation of a Genius probe, linearized,
purified, heat-denatured probe DNA was mixed with the
supplied hexanucleotide mixture, dNTP labeling mixture, and
Klenow enzyme according to the instructions provided with
the kit, and incubated for at least 60 min at 37 °C. The
reaction was stopped by addition of ImM EDTA. The
unincorporated tagged nucleotide was removed by ethanol
precipitation, the probe DNA was resuspended, and was stored
at -20°C until used in a hybridization reaction.

The Genius prehybridization and hybridization buffer
was composed of 5X SSPE, 5% of the supplied blocking
reagent, 50% formamide, 0.1% sodium N-lauroylsarcosine, and
0.02% SDS. The temperature used for prehybridization and
hybridization, 42 °C, was that recommended for buffer with
50% formamide. Filters were hybridized overnight, then
washed twice for 5 min each wash at room temperature in 2X
SSPE, 0.1% SDS. Next the filters were washed twice for 15
min each wash at 68 °C in 0 . IX SSPE, 0.1% SDS. Detection was
performed immediately following these washing steps.

All steps in the Genius detection protocol were
performed at room temperature. The Genius detection was
begun by washing filters for 1 min in Genius buffer 1 (150mM

21

NaCl, lOOmM Tris, pH 7.5), then for 30 itiin in buffer 1 in
which had been dissolved 0.5% of the blocking agent. A brief
(1 min) rinse of the filters in buffer 1 was followed by
incubation for 30 min in a solution of antibody-conjugate,
prepared as a 1:5000 dilution of the supplied antibody-
conjugate in buffer 1. Unbound antibody-conjugate was
removed with 2 washes, each for 15 min, in buffer 1. Next
the filters were incubated for 2 min in buffer 3 (lOOmM
NaCl, 50mM MgClj, lOOmM Tris, pH 9.5), and finally in the
color solution (buffer 3 plus chromogenic substrates) under
reduced illumination until signals were properly developed.
The color reaction was stopped with ImM EDTA.

MOSQUITO SPECIES-SPECIFIC DNA PROBES
Isolation Methods and the Relevance of Genome Organization

Mosquito species-specific DNA probes were isolated by
the method of Cockburn (1990) . The method involves a search
for repetitive DNA clones from a library using differential
screening. The clones each contain a small piece of genomic
DNA. Two genomic DNA probes are used to screen clones for
the presence of a species-specific DNA insert. One
(homologous) probe is genomic DNA from the same species used
to prepare the library. The other (heterologous) probe is
genomic DNA from a different species. Only clones containing
a DNA sequence repeated many times in the genomic DNA probe
hybridize at detectable levels. To isolate a clone from the
C. niqripalpus libraries, DNA from the closely related
species C. salinarius or C. quinquefasciatus was used as the
heterologous probe. To isolate a clone from the A.
nuneztovari library, DNA from A. oswaldoi was used as the
heterologous probe.

The cloning strategy, including the choice of vector,
used in the preparation of DNA libraries to be screened for
probes determines the size (or range of sizes) of inserts
from the organism's DNA that end up in the clones. The
average size of the inserts in the library can affect the

22

23
outcome of the screening by differential hybridization, due
to peculiarities of genome organization.

The organization of the genomes of anopheline and
culicine mosquitoes is known to differ (Cockburn & Mitchell,
1989) . Both anopheline and culicine genomes contain regions
of repetitive DNA, but there are longer stretches of
interveniong nonrepetitive DNA between the repeats in
anopheline genomes, as compared to culicine genomes.

Species-specific probes can be isolated for Anopheles
species rather easily by differential hybridization, using
phage vectors that typically contain 10-15-kbp inserts
(Cockburn & Mitchell, 1989). The separation of repetitive
DNA in the Anopheles genomes allows large inserts to retain
species specificity when the insert contains only a single
species-specific repeat. The different interspersion pattern
of culicine genomes, however, causes large inserts to be
more likely to show cross-hybridization to heterologous DNA
used in differential screening, due to the presence of
nonspecific repetitive DNA scattered throughout the genome.

One way to enhance the possibility of isolating a
species-specific repetitive DNA probe from Culex DNA is to
use a vector which favors small inserts. This decreases the
chance that a clone carrying species-specific repetitive DNA
also contains a portion of nonspecific repetitive DNA. That

24

was the rationale for using the LainbdaGEM-4 and plasmid
vectors with double-digested genomic DNA in the attempts to
isolate a Cul ex-specific probe. The double-digested genomic
DNA used for preparation of libraries was mostly in the 100
base pair size range, and the LambdaGEM-4 vector excluded
inserts greater than 4,1 kbp. Cloning of small inserts thus
favored the isolation of a species-specific DNA probe from
Culex. using the differential hybridization method.

Isolation of Probe pCxl
In an attempt to isolate a C. nigripalpus-specif ic
probe by differential hybridization of genomic DNA from C.
nigripalpus and C. salinarius to recombinant LambdaGEM-4,
about 5000 recombinant phage containing C. nigripalpus DNA
were screened. From this primary screen, 10 plaques were
picked which gave some degree of differential signals. In no
case was the degree of hybridization to C. salinarius
genomic DNA negligible. However, two of the clones which
gave the best differential signals were chosen for further
characterization, because it was thought that they might
contain species-specific DNA along with nonspecific
sequences. The recombinant pGEM-4 plasmid was recovered from
the two clones (the plasmid is part of the phage vector
LambdaGEM-4), and the insert in both clones was found to be
about 1 kbp, but slightly different in size.

25
The DNA from the two recombinant pGEM-4 clones was cut
separately with 10 different restriction enzymes, each of
which cut the insert DNA into several fragments. These
digests were run on gels, and blotted to hybridization
filters to obtain equivalent targets that were hybridized
separately to C. niqripalpus and C. salinarius genomic DNAs.
The results of autoradiographic detection revealed that none
of the fragments hybridized differentially to the degree
necessary to distinguish the two species. Accordingly, work
with these clones was terminated.

Probe pCxl was isolated from a plasmid library of C.
nicfripalpus Hind III/ecoR I fragments which was screened with
C. guinquefasciatus and C. niqripalpus genomic DNAs. The
insert in pCxl was about 10 kbp. Squash blots with
radiolabeled pCxl provided detection of C. niqripalpus and
C. salinarius. compared to negligible signals to C.
guinquefasciatus and all other mosquito species used in this
study.

About 5000 colonies containing C. niqripalpus insert
DNA, and about 1000 colonies with C. salinarius insert DNA,
were screened for species specific sequences by differential
hybridization to C. niqripalpus and C. salinarius genomic
DNAs. None were found to display specificity sufficient for
a species-specific probe.

26

Isolation of Anopheles nuneztovari-specif ic Probes

A partial sau3A I library of A. nuneztovari fragments
(about 15 kbp insert size) in phage EMBL 3A was obtained
from A. F. Cockburn.

In an initial screen for A. nuneztovari-specif ic
probes, nine plaques were isolated which gave good
differential signals in hybridization to A. nuneztovari and
A. oswaldoi genomic DNAs. These phage will be evaluated to
determine if they can distinguish these and other species of
the A. nuneztovari complex.

Mapping and Sequencing of Anopheles quadrimaculatus- and
Anopheles freeborni-specif ic Probes

Physical mapping was performed with the clone pAfl-Sl,
using single and double digests. This resulted in the
localization of unique ecoR I, Hind III, sst I, and Kpn I
sites located at about 250, 500, 600, and 1400 base pairs,
respectively, from the xba I site in the vector. This
analysis also revealed the presence of four Pst I sites, and
the absence of sites for acc I, BamH I, sai I, and xba I, in
the insert. Many (more than 10) sau3A I sites were detected
in the insert, with several clustered within 200 base pairs
of the Pst I site in the vector. Deletion subclones were

27
constructed using the four unique restriction sites found in
the insert.

Analysis of the nucleotide sequence data obtained from
probes pBrpl-Sl (Figure 1), pAfl-Sl (Figure 3), and the Crp
plasmids (Figure 2) did not reveal any repeat structures
which might be important in conferring species specificity.
Of the three Crp plasmids, only pCrpl-S2 and pCrpl-S3 were
found to retain the specificity of the phage Crp probe, in
tests with quick blots (see below) .

Comparisons of all the sequence data obtained in this
study to the data contained in the EMBL and Genbank
databases revealed no significant findings (no contiguous
regions of mosquito DNA longer than about 2 0 nucleotides
were similar to sequences stored in the database) , with the
following exceptions. A small portion of sequence at one end
of the pAfl-Sl clone was found to show considerable homology
to several ribosomal sequences from plant and animal
sources, suggesting that the elimination of this small part
of the probe insert could result in increased specificity.
Comparisons of the sequence from pCrpl-S3 to cytochrome P-
4 50s from several sources may not be significant, as the
extent of similarity was not great; however, this finding
will be pursued further.

28

The fact that no repeat sequences are reported here for
the pBrpl-Sl, pCrpl-Sl, pCrpl-S2, pCrpl-S3, and pAfl-Sl
probes does not indicate that the sequences conferring
species specificity to the probes were not found. Such
sequences may be present in the data, but the small amount
of sequence data obtained from these clones is just a start
in the effort to characterize them at the molecular level.
The sequences important for species specificity in these
clones may not be small repeats (as is the case for the Arp2
probe) , and the repetitive sequences providing specificity
may not become apparent even with the entire sequences in
hand, especially if only one repeat is contained in a given
clone. If this happens, subcloning and additional
specificity testing could narrow down the region conferring
specificity, and the species-specific subclones could be
used as tools to probe the genome directly.

Enough nucleotide sequence data were obtained from the
Nsi I subclones of pKA.2 to allow recognition of conserved
internal repeats (Figures 4 and 11) . This allowed the
specification of synthetic oligonucleotides. As the insert
in pKA2 was known to be rearranged with respect to that in
phage Arp2 , sequence data were also obtained from subclones
prepared directly from phage Arp2 (Figures 5 and 11) . The
latter data were thought to reflect more accurately the

29
actual sequence in the A. quadrimaculatus species A genome.
In the sequence data obtained from the phage Arp2 subclones,
it was found that in most every 200 base pair stretch of
contiguous sequence there were from one to five copies of a
given sequence motif, and two or three of the different
motifs, represented.

The differences between the sequence data obtained from
phage Arp2 and that from the pKA2 subclones suggest that the
sequence obtained from the phage Arp2 does not correspond to
the same regions of the mosquito genome as the sequence
obtained from the pKA2 subclones. However, the striking
conservation of consensus sequences in the data from both
sources (Figure 11) suggests that the pKA2 insert accurately
preserves at least some of the sequences found in the A.
quadrimaculatus species A genome. It also suggests a
mechanism that explains the shortening of the phage Arp2
insert in the subcloning step that generated pA2 (and thus
pKA2) from phage Arp2 : the multiple conserved consensus
sequences in the Arp2 insert provided a suitable substrate
for an internal recombination event in the bacterial host
that resulted in a large deletion. This type of event would
leave the majority of sequences within the pA2 insert intact
with respect to the corresponding regions in the phage Arp2
insert.

30
Forward Primer. 181 Nucleotides.

1 GACGTCCAGC TGCCGCTTCC TTCGTCTGcC GGCGTCGGAG TGACTTGTTG

51 GACGACGACG TCGGGCCGTT GCgcTCcCGC CAGCCGACGC TCACGCTGGT

101 GTACCGCATG AAGTTCCGCC ACGCGTTGGG CGTGGACTTC GCCATggccA

151 GGGTCTgcTT GTTCGACTAA TAGgCCAACC T

Reverse Primer. 275 Nucleotides.

1 ATCTCAGCTG ACTGCATAGT TTAGACGATT AACGTTGACT CGACCAAACA

51 ACGTCATGCA AACCAGCAAC TTTTGGTTGc CGTCGAATTT CCACCTCACA

101 TGGCAAAAGA GTGGACAGTC CTCGTTGTGT CGCTACGGTC AGCTACAATG

151 GCGCTcCCGT TAGAAGCCGA CCGCCGCCCA CATTCGTTCT TCTTAAAGAT

2 01 CGTCTTTATT AAAaGAACAC GCCGGTCCGT GGCGGTCAAA CCTAATGTGT
2 51 ACTGCCACTA TTTtCCTGGC CAGAA

Figure 1. Nucleotide Sequence from the A. quadrimaculatus Species
B-specific Probe, pBrpl-Sl. Lower case nucleotides indicate
uncertainty in the data at those positions.

31
pCrpl-Sl. Forward Primer. 264 Nucleotides.

1 AGCCAGCTGG ACGTCCAGTT GCCTAGTTCT CTTGCTTCTT GTCGTGTGAT
51 AGCCGNGCGA TTCGGTAATC GGCGCGTTGC CTACGGCANC GTGCTACCGT
101 GCCCGTTTGT CACCTAGGCA GCACATGCAG TCTTACAGTA GCACCAAaCG
151 GCTTACCAAA TGACGGGCTA GAGGCTATAC CTTGCGATAA CAGACTCTAA
2 01 CGATGATACG ATGGCGTTGC CAgGATgcAg GAAGCTCTtA aTGACAGTCA
2 51 CCAAGACACA CACG

pCrpl-Sl. Reverse Primer. 201 Nucleotides.

1 CTCAGCTGGT AAGCTGCTTA AAGATGnGGC GTAGCCGGGT GCCTGTCGGG

51 GTCTGCCTGT TGGGCGTCAT ACTGCATGTT GTTTCACGTT ACATCTTTTT

101 GTGTTGTGAT AAACTTCAAC AACCCTTGTC TTAgTTGGCn AcGGataTTt

151 CCATTAAGTG ACGTGAGTTt CATGTTGTTT tCCCGTATAT tGgAaTTGTa

201 A

pCrpl-S2. Forward Primer. 168 Nucleotides.

1 GGCGGCGGGT GTAAGCAAGA AGAATTTCTA GCAGAAATAA TTTtCTtGcT
51 GcCgGCCAGG CACCGCcCAG TTTGGATTAC ACATGACGGT GATAaAAAGG
101 ACCGGtCTGC CGGTCGCCGg TACAATgGcC ATCGCGTCTG ATACTTGGCG
151 CGTATAATCG nACTCGGA

Figure 2. Nucleotide Sequence from the A. quadrimaculatus Species
C-specific Probes pCrpl-Sl, pCrpl-S2, and pCrpl-S3. Lower case
nucleotides indicate uncertainty in the data at those positions,
and N (or n) indicates the occurrence of a nucleotide of unknown
identity.

32

pCrplS-S2. Reverse Primer. 90 Nucleotides.

1 TATCGGTTGG ACACGAGCAG AGCAAGGTGC GTGGATCGAC GGgCGGCTGG
51 TGAGGCTGTG CCGAGCTGCG CGAAAAGCTT CGGTATCACg

pCrpl-S3. Reverse Primer. 124 Nucleotides.

1 GAGCGTACGG CTGAACGACA TTTTCTACTG AGATATGACC AAACTTGTTT
51 GAATCCTTTC TTTGCTTTGC GTAGCTTCTG AGCTACGCTC CCAAACAATT
101 GCTCACTGCT AATGAaAGAA AAaG

Figure 2--continued.

33
Forward Primer. 177 Nucleotides.

1 GATCCGGGGT AGTCCACTAT AACACAAACA AACAACCAAA GGTCAGGAAT
51 GAGTAAATGG AGGTGCGTTG GGCTAGCTTG CCAACCGAAA CATAAGGAAT
101 GAGTACATGG AGTTGAGTTT GGTTTCCAAT CTACTATAAG GAAGCAAAAA
151 ACTTTACCTT AAATGAATTC TGCGTCA

Reverse Primer. 282 Nucleotides.

1 GTGTTGGATT GCTAGGAGGC GCTTgCgACC CCCAAATaCC ACGTTCGTAA

51 TGGATCGgAT GTcCGTACnC TGCGGATCGA CAAGTGCACC GCgGCCTtGC

101 ACgCcCGGGG GnCCACCGAC nggGCTGAAT gTCGCCCCGG TCTATTGAGT

151 TCAACGGGTT TGTTCCCCTA GGCAGTTTAC GTACTCTTTG ACTCTCTATT

2 01 CAGAGTGCTT TnAACTTtCC TCACGGTACT TGTTCGCTAT CGGCTCATGG

251 TGGTATTAGC TTAGAaGGAG TTCTCcACTT AG

Reverse primer. 160 Nucleotides.

1 GCAAAAAACT TTACCTAAAT GAATTCTGCG TCATATCATG GGTGTTCTAG
51 TCAAGTGGCC AAGATAACCA AGAGGTGCAG CAAATTACAA ATGAGAAGTT
101 GAGTATGCCT TCTCATATgA TAACCCTCTA ACAAAGTCAA TGACGCAAAT
151 CAACATTGGA

Figure 3. Nucleotide Sequence from the Anopheles f reeborni-
specific Probe pAfl-Sl. Lower case nucleotides indicate
uncertainty in the data at those positions, and N (or n)
indicates the occurrence of a nucleotide of unknown identity,

34
pKA2-Nl. Forward Primer. 85 Nucleotides.

1 TGCATACACC AATAGATGCA ATNAGTTTNg AGTATGTTCT ATGATAGGTT

51 TGTTAACAGA TGCCTAGATA TGGCATGTAT TCATA

pKA2-Nl. Reverse Primer. 294 Nucleotides.

^ GCATATAGCT_GGTGCTAGTT_TTTANANAGT GGNAGAACAT GGGAAATCTG

51 TGAAGCAAAC CAAGTCACAG GACAGACTCC GAAACTGATG GCATCTATTG

101 GGCTACGCAT GGAAAACCCG CTTTTTGCAT_ATAGCTGGTG_CTAGTTTTGG

151 ATATATNNTT GGGAATACGN CTGTTTGCGT_ATAGCTGGTG_CTAGTTTGGA

201 ACTGTGACAC AATTCAATCT GTTAGCAATC ATAGGACATA-THCAACTATG

251 GCATGATCGG TGTACGATGA ACgCTATTGC_TAGCTGGTGT_CTAG

Figure 4. Nucleotide Sequence from Nsil Subclones of Plasmid
pKA2 . Lower case nucleotides indicate uncertainty in the data at
those positions, and N (or n) indicates the occurrence of a
nucleotide of unknown identity. Internal repeats (conserved or
consensus sequences) used to specify the production of synthetic
oligonucleotides (Figure 11) are indicated as follows:
SEQUENCE _1; SEOUENCK^Z; SE0UENCE_3; and SEQUENCE 4.

Note the overlaps of some of the repeats at their ends.

35
pKA2-N2. Data from Forward & Reverse Primers. 267
Nucleotides.
1 rr,PTn'I*rTGg_ATATAACTAG_TGCTAGATTT GGATATATGG_CACAAATGTC

51 AAATCTGTTA GCAAATCAAT CATAGGACAT ACTTCAAACT CATGGCATCT

101 ATTGGTGTAC GCATGGTAAT CCGCTGTTTGCATATAGCTGGTGCTAGTTT

151 GAGATATATG GCACAAATGT GATCAATTGT CATATCTAGG CATCTGTTAG

201 CAAACCAATC ATAGGACATA CTCCAAACTC ATTGCATCTA TTGGTGTATG

251 CAGGTCGACT CTAGAGG

pKA2-N3. Data from Forward & Reverse Primers. 113

Nucleotides.
1 CGCTGTTTGC ATATAGCTGG_TGCTAGTTTG AGATATATGG_CAAAAATGTC

51 AAATCTGTTA GCAAAGCAAT GATAGGACAT ACTCCAAACT CATTGCATCT

101 ATTGGTGTAT GCA

Figure 4--continued.

36
pArp2-Nl. Reverse Primer. 135 Nucleotides.

1 CAAGCTTGCN TNCCTGCATA CACCAATANA TGCAATGAGT ITOq^qTATG

51 TCCTATGATT GGTTJGCTAA CAGATTTGAA_ATTTGTGTCA_CAGTTCCAAA

101 ACCAGCACCA GCCATATGCA AACAGCGTAT TCCCA

pArp2-N3. Reverse Primer. 262 Nucleotides.

1 TAGCTOGTOC_TAGrrTTTTA_TATATGGCAA_ACATGTCAAA_TCTGTTAACA

51 AACCAATCAC AGGACATACT CCAAACTCAT GGCATCTATT GGTCTACGcC

101 ATGAAAACCg CcGcTTTTTG CATATAGCTG GTGCTAGTTT TGGATATATG

151 CTTGGGAATN nNTGTTTGCG TATANTGGTG_CTAGTTTNNN AaCTGTGACA

2 01 CAAATTTCAA AtctGattaG CAaATCAATC ATAGGACAT.A^_CI£AaACTAT

2 51 GGCATGTATC GG

pArp2-N5. Reverse Primer. 94 Nucleotides.

1 CTATGATTGA TTTGCTAAAA_GATTTGACAT_TTGTGcCCAT ATATCCAAAA

51 CTAGCNCCGG_CTATAACCAa_ACAGCGTATT TCCATGCAGG TCGA

Figure 5. Nucleotide Sequence from Subclones of Phage Arp2 . Lower
case nucleotides indicate uncertainty in the data at those
positions, and N (or n) indicates the occurrence of a nucleotide
of unknown identity. Internal repeats are identified (see Legend
for Figure 4) .

QUICK BLOTS
Experiments Leading to the Quick: Blot Protocol

In attempts to use nonradioactive detection systems
with mosquito species-specific probes, it was found that an
improved method for preparing targets from a series of
individual mosquitoes was needed. Table 1 provides a summary
of the experiments that led to the development of this
method.

Experiment 1 (Table 1) demonstrated the effectiveness
of SA-AP detection, using mosquito genomic DNA as both the
target and the probe, with dot blots. It revealed that 10 ng
of target DNA could be detected with homologous probe, even
when background was very high.

Experiments 2 and 3 (Table 1) suggested that the high
background seen in experiment 1 could be reduced by
substituting nitrocellulose filters for nylon without
sacrificing sensitivity. Using SA-AP detection with
nitrocellulose filters, a sample of 10 pg of target DNA was
detected on dot blots. Experiment 4 was performed in order
to determine if results differed when NFDM was substituted
for BSA (bovine serum albumin) in the SA-AP detection
protocol. Either ingredient could be used without effect on
the sensitivity of detection or the level of background.

37

38

Thus, NFDM was used in place of BSA in all subsequent SA-AP
detections.

In experiment 5 (Table 1) and in other experiments
(Tables 1 and 3) , the recombinant plasmid pKA2 was used as a
probe. Using nick translation and autoradiography for
labeling and detection, respectively, it was found that
radiolabeled pKA2 provided specific detection of A.
quadrimaculatus species A, without showing significant
detection of A. quadrimaculatus species B, C, and D, A.
crucians, A. albimanus. A. aegypti. A. taeniorhynchus. C.
quinauef asciatus . and C. perturbans.

Experiments 5 and 6 (Table 1) showed that an unmodified
SA-AP detection protocol could not be used for species
identification of mosquitoes using the squash blot protocol
(Cockburn, 1990) with species-specific probes, due to
nonspecific detection. The problem was thought to be caused
either by residual streptavidin-binding substance (perhaps
biotin) or alkaline phosphatase activity in the target areas
on the filters. Results of experiment 7 (Table 1) suggested
that the former was the cause, since no signals were formed
upon equivalent detection when both the SA-AP enzyme complex
and biotinylated probe were omitted. Thus, improvements to
the SA-AP detection system were needed, which would allow
specific detection of mosquitoes using species-specific

39

probes. It was thought that the streptavidin-binding
substance might be removed or neutralized by certain
treatments of the filters following sample application but
prior to the prehybridization step. These treatments are
hereafter referred to as post-application treatments or
post-application washes, as they were applied to blots after
the binding of sample (target) DNA to the filters.

Experiments 8 and 9 (Table 1) suggested that an
unmodified ECL detection protocol would not be useful for
mosquito identification using species-specific probes in
either the dot blot or squash blot systems, due to a
relatively high level of nonspecific detection. Experiment
10 showed that the ECL system was functioning in the
detection of non-mosquito control DNAs in the dot blot
system. Hence it was obvious that alterations would have to
be made before the ECL detection system could be used with
species-specific mosquito DNA probes. Experiment 11 (Table
1) was designed to find out whether the ECL detection
protocol would produce signals on nitrocellulose squash
blots of mosquitoes in the absence of hybridized probes.
Indeed it did, suggesting that residual peroxidase activity
in the target areas may have contributed to nonspecific
detection. It was thought that certain post-application wash

40
conditions might be found to inactivate this activity on
filters before prehybridization steps were performed.

In order to discover post-application treatments of the
filters which would eliminate the barriers to
species-specific nonradioactive detection of mosquito squash
blots using DNA probes, various washes of the filters were
tested. In experiments 12a and 12b (Table 1) , squash blots
were washed separately for 45 min at room temperature in the
following solutions : 10% SDS, 8M urea, 0.5 M HCl , 10% meat
tenderizer in IX SSPE, 10% LA FRANCE (whitener/brightener
powder containing protease. The Dial Corporation) , 8M urea
plus 10% SDS, or 8M urea followed by 10% SDS. In these
experiments, the washing step was followed by detection
steps, and the prehybridization and hybridization steps were
omitted.

When the ECL detection protocol was applied to these
unprobed squash blots (experiment 12a, Table 1) , all the
washes were found to be useful in greatly reducing
background levels. However, various levels of nonspecific
detection were observed. The lowest signals occured on nylon
filters which received the urea and the urea-then-SDS
treatments, and on nitrocellulose filters which received the
LA FRANCE or urea-then-SDS treatments (see notes on Table 1
for more experimental details) .

41
Another set of squash blots treated as described above
was subjected to the SA-AP detection protocol (experiment
12b, Table 1) . High background levels were observed on all
of the nylon filters, whereas all nitrocellulose filters
showed very low background. The nylon and nitrocellulose
filters which received the LA FRANCE treatments showed
negligible signals, apart from background, whereas all other
filters had a moderate level of nonspecific signals.
Experiments 12a and 12b (Table 1) revealed that
specific treatments of squash blots were effective for
reducing background and/or nonspecific detection. The
results suggested that nitrocellulose squash blots washed
with LA FRANCE could be used with DNA probes to provide
specific detection of mosquitoes with either the SA-AP or
the ECL systems.

First Attempts at Making Quick Blots
Although experiments 12a and 12b (Table 1) demonstrated
the potential usefulness of post-application treatments for
improving the specificity of nonradioactive detection with
squash blots, it was obvious that further improvements could
be made in signal-to-noise ratio, as well as in
standardization of sample application. It was thought that
an adaptation of the 96-well dot blot manifold might allow
the use of batch-processing techniques and ensure uniform

42
sample application. Further improvement in detection might
also be achieved with this apparatus by using a selective
barrier for excluding cuticle and large pieces of tissue
from binding to the filters. A dot blot manifold was used
essentially as in a standard dot blot protocol, but with the
important modification of placing a filter paper above the
blotting filter instead of below it.

Experiments 13a and 13b (Table 1) represent the first
attempts at implementing the QB protocol based on some of
the ideas described above. In experiment 13a, a thick filter
paper pad was used as a blocker. In experiment 13b, a thin
tissue was used. The filters were probed with radiolabeled
pKA2 , and detection was by autoradiography. Detection of DNA
was superior when the thin tissue was used as the blocker.
The thicker paper apparently blocked the DNA from reaching
the blotting filter. After this experiment all other QBs
were produced by using the thin tissue.

43

Table 1. Experiments Performed to Develop the Quick Blot
Protocol .

EXPT FILTER/SAMPLE PREHYB/HYB PROBE DETECTION RESULTS

NY dot blot of
of AqA DNA

NC dot blot of
biotinylated

#14 7 DNA

NC dot blot of
#147 DNA
(unlabeled)

same as for
expt 2

NC squash blots
with Ac, AqA,
Cq, and Cs

same as for
expt 5

NC squash blots
with AqA, Cs,
and Cq

Hy-ECL dot blot
(with AqA, Cn,
Cq, and Cs)

NC squash blots
with AqA, Cs,
and At

std

std

std

std

std

std

NONE

ECL

ECL

AqA

SA-AP

10 ng det

-BIOTIN

unequivocal;
very high bg

NONE

SA-AP

100 pg det
unequivocal ;
10 pg barely
distinguish-
able; very
low bg

#147

SA-AP

same as for

-BIOTIN

expt 2

NONE

SA-AP

same as for

(BSA)

expt 2

pKA2

SA-AP

nonspecific

-BIOTIN

det of all
samples; very
low bg

NONE

SA-AP

same as for
expt 5

NONE

SA-AP
(without
SA-AP
reagent)

no det

pKA2

ECL

nonspecific
det ; very
low bg

pKA2

-ECL

ECL nonspecific
det (signals
for all three
species) ;
very low bg

44

Table 1 — continued,

EXPT FILTER/SAMPLE PREHYB/HYB PROBE DETECTION RESULTS

10

11

NY dot blot and ECL
Hy-ECL dot blot
(both with lambda
DNA dilution series)

lambda
-ECL

NC squash blot
with AqA, Cs,
and At

NONE

NONE

ECL 10 pg det

unequivocal
with NC and NY
filters;
1 pg det
(barely) on NY

ECL Nonspecific
det; low bg

12a NC and NY

squash blots
with AqA, Cs,
and At

(VARIOUS POST-
APP WASHES:
SDS, urea,
HCl, mt, LF,
urea-SDS,
urea-then-SDS)

ECL

NONE

ECL Nonspecific

det with very
low bg, on all
filters,
but very faint
signals only
on NY filters
which received
the urea
and the
urea-then-SDS
treatments,
and lowest
signals among
the NC filters
which were
treated with
urea-then-SDS
or LF

45

Table 1 — continued,

EXPT FILTER/SAMPLE PREHYB/HYB PROBE DETECTION RESULTS

12b same as for
expt 12a

std

NONE

SA-AP All NY filters
showed high
bg, and all NC
filters showed
very low bg.
Nonspecific
signals seen
on all targets
on all filters
EXCEPT that
virtually no
signals seen
on both NC and
NY filters
which received
the LF washes

13a NY QB but std

with filter
paper blocker;
AqA

13b NY QB (with std
tissue paper
blocker) ; AqA

pKA2
-32P

pKA2
-32P

AR

AR

only 3 6 (out
of 94) spots
show a signal

all (of 94)

spots

show a signal

Notes for Table 1. EXPT = experiment (number). PREHYB/HYB = type
of prehybridization and hybridization conditions used. AqA = A.
quadrimaculatus species A (its DNA, when used in probe column) ;
Ac = A. crucians; Cq = C. quinquef asciatus ; Cs = C. salinarius;
Cn = C. niqripalpus. At = A. taeniorhynchus . bg = background
level of signal development (i.e., signal intensity where target
DNA was not applied to the filter) . #147 DNA = HspVO deletion
subclone from A. albimanus in pUC19, from Mark Benedict. pKA2 =
plasmid pK19 with part of the mosquito DNA insert from phage Arp2
(Cockburn, 199 0) . lambda = phage lambda DNA.

46

Notes for Table 1 — continued. Std = standard prehybridization (1%
NFDM, 0.2% SDS, 55 "C for at least one-half hour) and
hybridization (30% formamide, 5X SSPE, 1% NFDM, 0.2% SDS, plus
probe) conditions. SA-AP (BSA) = the GENE-TECT detection
protocol, Clontech Laboratories, Inc., with SSPE substituted for
SSC (saline sodium citrate) . SA-AP = the GENE-TECT detection
protocol, but with NFDM substituted for BSA, and SSPE used
instead of SSC. NC = BA-85 nitrocellulose filter, Schleicher &
Schuell. QB = Quick Blot. NY = ZetaProbe nylon filter, Bio-Rad.
Hy-ECL = HyBond ECL filter (nitrocellulose; Amersham) . NONE = no
treatment, or no probe. BIOTIN = biotinylated probe prepared by
nick translation. ECL = ECL hybridization buffer (for
prehybridization and hybridization steps), as supplied plus 0 . 5M
NaCl, or ECL probe prepared according to protocol provided by
manufacturer (Amersham) . pg = picogram(s) . ng = nanogram(s) . det
= detection, no det = no signals or only very faint (barely
observable) signals. Dot blots = standard dot blot procedure
using dilution series of purified DNA applied to the filters,
with separate spots containing different amounts of the same DNA.
Squash blots prepared as described by Cockburn (1990) . POST-APP =
post-application treatments, or washes of filters after target
DNA samples were applied (all for 45 min at room temperature) :
SDS = 10% SDS; urea = 8M urea; HCl = 0 . 5M HCl ; mt = 10% meat
tenderizer (Tone's Meat Tenderizer, Tone Bros., Inc.); LF = 10%
LA FRANCE (whitener/brightener powder containing protease, Dial
Corporation) ; urea-SDS = 8M urea, 10% SDS; urea-then-SDS = first
wash with 8M urea, second wash with 10% SDS. Nitrocellulose
filters were subjected to standard vacuum-baking procedures
following application of samples, and nylon filters were
subjected to standard UV-fixation (ultraviolet light treatment)
following application of samples (except where otherwise noted) .
However, results of equivalent experiments in which these
fixation steps were omitted suggested that non-fixed filters of
both types yield signals equal in intensity to fixed filters.

47
Steps in the Quick Blot Protocol

This section describes the steps in the QB protocol in
detail. A list of materials and apparatus used in the
preparation of QBs is given in Table 2, and a picture of the
apparatus is shown in Figure 6.

Mosquitoes (larvae, pupae, or adults) were placed
individually into the wells of a 96-well microtitration
plate. Denaturing buffer (Table 2) was then added. For
standard sized wells of 10 mm deep and 13 mm in diameter, a
maximum of 200 /Ltl per well of denaturing buffer was used.

The DNA was then released from the tissues by grinding
with the steel pegs of the Replaclone for about three min in
the orientation that allowed all of the Replaclone pegs to
be inserted into wells of the plate. The progress of this
grinding step was checked visually at 30 s intervals by
inspecting the coloration of the sample buffer and noting
whether any large tissue fragments were attached to the
proximal part of the steel pegs. A given mosquito species or
life stage produced a characteristic (usually slightly
brownish or yellowish) coloration of the buffer when
grinding was sufficient. In about 5% of adult mosquito
samples, tissue fragments required being pushed back into
the buffer by using a pin or fine forceps.

48
The plate was incubated for 3 0 min at room temperature,
and then neutralization buffer (Table 2) was added and mixed
thoroughly in the sample wells using the Replaclone (or
micro-pipette) . The volume of neutralization buffer added
per well was one-fourth of the volume of denaturing buffer
added previously. Use of a multi-channel pipette for all
transfers of solutions significantly decreased the time and
effort required to complete the protocol, but was not
required.

A blotting filter (nitrocellulose or nylon) was cut to
size and wet in water. The base of the dot blot manifold was
positioned for convenient access to the vacuum source, and
then the middle block of the manifold was set in position
over the base. Next, the wetted filter was placed over the
top of the middle block of the manifold so that it was more
or less centered over the sample application areas. The two
corners of the filter were trimmed where the metal pegs
arose from the manifold, so that the entire filter was flat.
A tissue was used to keep larger pieces of cuticle and
other debris off the blotting filter. The tissue was wetted,
then placed over the surface of the filter by starting from
one edge or corner. In this way, large bubbles did not
become trapped between the tissue and the filter.

49
The top portion of the manifold was clamped tightly
over the tissue, filter, and lower portions of the
apparatus. Vacuum was applied (usually with a trap and valve
mechanism so that a low level of suction was applied) . Then
samples from the microtiter plate were applied to the dot
blot manifold wells, preserving the relative orientation of
sample locations between the microtiter plate wells and the
manifold wells. Once the samples had been aspirated through
the membrane, wash buffer (Table 2; about 350 ^1 per well)
was added to the wells to wash portions of the samples
remaining on the walls of the manifold wells onto the
filter. When all the buffer had been washed through the
filter, the vacuum was removed from the manifold, the
manifold was disassembled, and the filter removed.

By splitting the sample solutions into several
aliquots, a given set of samples was used to produce
multiple equivalent filters. Duplicate filters were prepared
in the dot blot manifold, samples being applied from the
same microtiter well plate, until the total sample volume
had been used. In this way, many (up to 10) equivalent
filters were produced with a single set of samples.
Nitrocellulose filters containing samples were
vacuum-baked. Then the filters were used as hybridization
targets with DNA probes. Nylon filters were either air-dried
or UV-fixed and then air-dried.

50

Table 2 . Materials and Apparatus for the Quick Blot
Protocol .

Plastic Microtitration Plate, 96-well (flat-bottom wells;
many sources)

96-Place Microsample Filtration Manifold (Dot blot manifold;
Schleicher & Schuell)

Micro-Pipette (Multi-channel preferred; many sources)

Pipette Tips (many sources)

Replaclone (96-prong model; L.A.O. Enterprises)

Filters for Nucleic Acid Blotting (nitrocellulose, such as
BA-85 from Schleicher & Schuell; or nylon, such as
Zeta-Probe from Bio-Rad) , cut to 12 x 8 cm size

Laboratory Tissues (such as Kimwipes from Kimberly-Clark, or

Stirling Light Duty Wipes from Stirling Converting
Company, Inc.)

Vacuum Source (sink aspirator or pump)

Buffers

Denaturing Buffer: 0.5M NaOH, 1.5M NaCl

Neutralization Buffer: 3N sodium acetate, 2N acetic acid

Wash Buffer: 2X SSPE

51

Figure 6. Apparatus used to prepare quick blots. The 96
steel pegs of the Replaclone (left) fit into the wells of
the microtiter plate (lower right) when grinding the
mosquitoes. The dot blot manifold is shown with blotting
filter in place, overlaid with a tissue to prevent bits of
cuticle and cell debris from adhering to the blotting
filter. Before samples are applied to the blotting filter,
the top portion of the manifold is clamped into place, and
the vacuum source is attached. The optional multichannel
pipette speeds transfer of solutions.

52

Experiments to Optimize Use of Quick Blots
with Mosquito Species-specific Probes

Table 3 summarizes the results of experiments performed
to evaluate and refine the QB protocol for use in
identification of mosquito species by DNA hybridization
using species-specific probes. These experiments were
required for the optimization of results when using various
detection systems for species-specific DNA probes with
mosquito QBs.

Experiments 14 and 15 (Table 3) were performed to
evaluate the effectiveness of various post-application
treatments of QBs probed with either one or two probes. In
experiment 14, various post-application washes of the
filters were tested to maintain conditions that would reduce
the level of nonspecific detection. In experiment 14, the
probe was omitted from one set of filters which were treated
with the same washes, and it was found that washes that
contained a whitener/brightener with protease (LA FRANCE)
were effective at improving the specificity of detection
(filters C, D, G, and H in Figure 7) . Even though specific
signal strengths were decreased somewhat by the use of LA
FRANCE, the overall effects were desirable due to a dramatic
reduction in nonspecific detection (compare filters E and F
with filters G and H in Figure 7) .

53
In experiment 15 (Table 3) , standard ECL
prehybridization was used, and ECL detection was performed
before SA-AP detection. The pCxl-ECL probe, when used alone
or in combination with a biotinylated probe, gave at least a
medium level of specific detection and a low level of
nonspecific detection with any of the post-application
treatments. When SA-AP detection of biotinylated pKA2 was
performed after ECL detection, very strong specific
detection was achieved. This occurred after post-application
washes, with both the urea-SDS-LF and the LF-then-urea-SDS
treatments (see notes to Tables 1 and 3 for more details on
post-application washes) . These results suggested that some
aspect of the ECL prehybridization and/or detection was
enhancing the results of SA-AP detection, since SA-AP
detection of biotinylated pKA2 using standard
prehybridization resulted in nonspecific detection

(experiment 5, Table 1) and/or unacceptably high background

(experiment 15, Table 3) .

Regarding experiments 14 and 15, there was considerable
variation in intensity between spots from different
mosquitoes. Since this was seen with spots which received
the same amount of homogenate (starting with a single
mosquito for each homogenate) , the variation was probably
due to one or a combination of the following: a variable

54

amount of DNA was released from each mosquito which was
ground by this method, or a variable amount of target
repetitive DNA sequences in the genomes of individual
mosquitoes. Another result of these experiments which was
seen consistently in the QB results was the concentration of
signal in a small spot in the center of the circular area
where samples were applied. The latter effect was probably
due to tangential flow toward the center of the wells in the
dot blot apparatus during preparation of QBs. Also,
identical filters were given the post-application treatments
described, but using a wash temperature of 45 °C. The results
were virtually identical to those obtained for similar
filters which received the post-application treatments at
room temperature.

Experiments 16a and 16b (Table 3) were performed to
confirm the utility of a combination of biotinylated and ECL
probes to QBs used in a single hybridization experiment,
when using stepwise LA FRANCE and urea-SDS post-application
treatments and ECL prehybridization. The two probes were
labeled reciprocally in the experiments, in hopes of
distinguishing effects from the detection system from
effects resulting only from properties of the particular
probes. Although some of the latter effects were manifested,
the results prove the utility of the conditions used for the

55
specific detection of these hybridized probes in a
sequential application of detection protocols.

In experiments 16c through 16f (Table 3) , the same
post-application and prehybridization treatments were used
as in experiments 16a and 16b, but the filters were dot
blots instead of QBs, so that a rough quantitation of the
sensitivity of specific detection could be obtained.
Detection levels were in the range of 1-10 ng for ECL,
SA-AP, and autoradiographic detection.

Experiment 17 (Table 3) was performed to assess
quantitatively the levels of detection possible when ECL-
labeled and biotinylated probes were used in a single
hybridization step. A standard dot blot strip was used for
this experiment. It is not known why the sensitivity of
detection of the ECL-labeled probe was lower than that found
when only a single probe was used in the hybridization step
(experiment 10, Table 1) . However, the results suggested
that detection in the 10 ng (or higher) range should be
sufficient for properly scoring results of various detection
systems using mosquito QBs. Also, this experiment showed
that when using a biotinylated probe in ECL prehybridization
and hybridization conditions, detection levels were lowered
considerably as compared to the levels obtained when
standard prehybridization and hybridization conditions

56

(i.e., those used with radiolabeled probes) were used
(experiments 2 through 4, Table 1).

Experiment 18 (Table 3) revealed the level of detection
attainable with radiolabeled probe hybridized to homologous
target DNA in a dot blot. Detection levels in the ng range
were achieved consistently in several experiments using
these conditions, and detection in the pg range has been
observed on occasion.

Figure 8 reveals the effects of using different filter
types and DNA binding conditions on the results of SA-AP
detection (experiment 19, Table 3). UV fixation of nylon
filters did not in itself affect the detection levels
(Figure 8 B and C) when QBs were subjected to SA-AP
detection. A degradation of specificity resulted when
alkaline binding was used in preparing a QB with a nylon
filter (Figure 8 A) , as compared to that obtained when the
other conditions were used.

Experiments 20a and 20b (Table 3) were designed to
further test whether UV fixation of nylon QBs would improve
the detection of hybridized species-specific DNA.
Radiolabeled probe was detected by autoradiography, and the
QB which did not receive the UV fixation yielded signals
equivalent to those produced from the UV-fixed filter.

57
Three different nonradioactive detection systems were
used separately with QBs (experiment 21, Table 3; Figure 9).
Whereas reliable specific detection was obtained with the
ECL and SA-AP systems, the Genius system detection was quite
variable. Many of the experiments in Tables 1 and 3 were
performed with the Genius system, which was used as
suggested by the supplier, except for substituting SSPE for
SSC, and changing the formamide concentration in the
hybridization buffer to 50% for hybridization at 42 °C.
Figure 9 B is typical of the results with the Genius system,
as a patchy distribution of high background often interfered
with interpretation.

Experiment 22 (Table 3), shown in Figure 10, confirmed
some of the results of experiments 15, 16, and 17. This
proves that probes with different specificities can be used
in a single hybridization step and detected differentially
with a sequential application of nonradioactive detection
methods.

58

no probe pka2-bi0tin probe

"aq aa cn at aq aa cn at

Am i I «

• •••••

* F

• • : e • ■

fO

« H

9

A,E: no wash

B,F: urea-SDS

C,G: LP (LA FRANCE)

D,H: urea-SDS-LF

Figure 7. Effects of various post-application treatments of quick
blots on the specificity of SA-AP detection. Quick blots were
prepared using nitrocellulose filters, with each spot receiving
one-tenth of the solution in which a single mosquito was
macerated. Six (two rows of three) spots per filter contained DNA
from different individuals of a single mosquito species, as
follows: AQ = A. quadrimaculatus species A; AA = A. albimanus ; CN
= C. niqripalpus; AT = A. taeniorhynchus . Certain filters
received treatments between target DNA application and
prehybridization. These treatments were called post-application
washes (or treatments) , and were performed at room temperature
for 45 min. Filters A and E received no post-application wash.
Filters B and F received a post-application wash of urea-SDS (8M
urea, 10% SDS) . Filters C and G received a post-application wash
of 10% LA FRANCE (Dial Corporation) . Filters D and H received a
post-application wash of urea-SDS-LF (8M urea, 10% SDS, 10% LA
FRANCE) . Filters were prehybridized and hybridized (with or
without biotinylated probe pKA2 , as indicated) according to the
GENE-TECT protocol (Clontech Laboratories, using BRL reagents,
except that NFDM was substituted for BSA and SSPE was substituted
for SSC) .

59

AQ

AA

CN

AT

A

•
•

•
• i

• • •

1 • • •

«

• •
9 %

1

B

,• • •

•

4i

_

1

C

• • •
1

1

D

• •

9

1

1

;

(

1

■1

A: NYLON: alkaline binding

B: NYLON: no UV

C: NYLON: UV- fixed

D: NITROCELLULOSE: baked

Figure 8. Effects of filter type and DNA-binding conditions on
SA-AP detection of probe pKA2 hybridized to quick blots. All four
filters were subjected to the urea-SDS-LF post-application
treatment before being subjected to prehybridization and
hybridization with biotinylated pKA2 probe (see legend to Figure
7 for details of post-application treatment, prehybridization and
hybridization conditions, and abbreviations). The method used for
fixing the DNA to the filters during sample application was
varied. Filter A was prepared according to the normal QB
protocol, except that the samples in the denaturing buffer were
applied to the blotting filter without being mixed with
neutralization buffer (Table 2) . Nylon filters B and C were
prepared according to the normal QB protocol, except that no
vacuum-baking step was performed, and filter C was treated with
UV light after sample application. Nitrocellulose filter D was
prepared according to the unmodified QB protocol.

60

AQ

AA

CN

AT

SA-AP

GENIUS

ECL

Figure 9. Different nonradioactive systems used for detection of
probe pKA2 hybridized to quick blots. All three nitrocellulose
filters received the urea-SDS-LF post-application treatment
before being hybridized with pKA2 probe. See legend to Figure 7
for details on the post-application treatment and abbreviations) .
Probe DNA was labeled according to the different methods
appropriate for detection by the SA-AP, Genius, and ECL
protocols. Prehybridization and hybridization conditions were as
follows: for the SA-AP and ECL filters, conditions were as for
the filters on which SA-AP and ECL detection was performed in the
experiments described in Table 3; for the Genius filter,
conditions were as suggested by the manufacturer (Boehringer
Mannheim Biochemicals) , except for substituting SSPE for SSC, and
changing the formamide concentration in the hybridization buffer
to 50% for hybridization at 42 "C.

61

AQ

AA

CN

AT

PKA2-ECL

pCxl-BIOTIN

Figure 10. Sequential use of nonradioactive detection systems
following a single hybridization of two probes to a quick blot,
quick blot was prepared in the same way as filters D and H of
Figure 7 (standard quick blot protocol, with urea-SDS-LF post-
application treatment; see Figure 7 legend for experimental
details, and for abbreviations) . Two probes, biotinylated pCxl
and ECL-labeled pKA2 , were hybridized to this filter, using the
ECL prehybridization and hybridization conditions (ECL
hybridization solution supplied in the kit plus 0.5M NaCl) . The
bound pKA2 probe was detected first, using X-ray film according
to the ECL protocol, then the pCxl probe was detected using the
SA-AP detection protocol.

62

Table 3. Experiments Performed to Evaluate and Optimize the Quick
Blot Protocol.

EXPT FILTER/SAMPLE PREHYB/HYB PROBE DETECTION RESULTS

14 NC QB with AqA,
Aa, Cn, and At,
WITH VARIOUS
POST-APP
TREATMENTS :

none

std NONE

SA-AP light to med
nonspecific
det at all
spots; no bg

urea-SDS

std NONE

LF

urea-SDS-LF

std NONE

std NONE

SA-AP variable
(light to
strong)
nonspecific
det at all
spots ; no bg

SA-AP no signals;
no bg

SA-AP no signals;
no bg

none

std pKA2

-BIOTIN

SA-AP very strong

specific det;
light to med
nonspecific
det; no bg

urea-SDS

std pKA2

-BIOTIN

SA-AP very strong

specific det;
light to med
nonspecific
det; no bg

63

Table 3 — continued.

EXPT FILTER/ SAMPLE PREHYB/HYB PROBE DETECTION RESULTS

14 (continued)

LF

urea-SDS-LF

std pKA2 SA-AP light to med
-BIOTIN specific det;

no nonspecific
det; no bg

std pKA2 SA-AP light to med
-BIOTIN specific det;

no nonspecific
det; no bg

15

NC QB; Aa, At,
AqA, Cq, Cs
WITH VARIOUS
POST-APP
TREATMENTS :

LF

urea-SDS

urea-SDS-LF

ECL pCxl-ECL ECL med level of

specific det,
but light
nonspecific
det of all
other spots ;
med bg

ECL pCxI-ECL ECL med level of

specific det,
but light
nonspecific
det of all
other spots;
med bg

ECL pCxI-ECL ECL med level of

specific det,
but light
nonspecific
det of all
other spots;
med bg

Table 3 — continued,

64

EXPT FILTER/SAMPLE PREHYB/HYB PROBE DETECTION RESULTS

15 (continued)
LF-then-
urea-SDS

LF

urea-SDS

ECL pCxl-ECL ECL ECL:

med level of
specific det,
but light
nonspecific
det of all
other spots;
med bg

std pKA2 SA-AP med

-BIOTIN nonspecific

det on all
spots ;

high uneven bg
(perhaps some
specific det
but high bg
makes

interpretation
difficult)

std pKA2 SA-AP med

-BIOTIN nonspecific

det on all
spots ;

high uneven bg
(perhaps some
specific det
but high bg
makes

interpretation
difficult)

65

Table 3 — continued.

EXPT FILTER/SAMPLE PREHYB/HYB PROBE DETECTION RESULTS

15 (continued)

urea-SDS-LF

std pKA2 SA-AP med

-BIOTIN nonspecific

det on all
spots ;

high uneven bg
(perhaps some
specific det
but high bg
makes

interpretation
difficult)

LF-then-
urea-SDS

std pKA2 SA-AP some

-BIOTIN nonspecific

signals but
high bg
made

interpretation
difficult

urea-SDS-LF

ECL pKA2 ECL ECL: good

-BIOTIN then specific det;
and SA-AP but also
pCxl-ECL very light

nonspecific
det; very low
bg

SA-AP: very
strong

specific det;
no bg
except

blotches due
to filter
overlap

66

Table 3--continued.

EXPT FILTER/SAMPLE PREHYB/HYB PROBE DETECTION RESULTS

15 (continued)
LF-then-
urea-SDS

16a NC QB with

Aa, At, AqA,
Cq and Cs .
WITH POST-APP
TREATMENT :
LF-then-
urea-SDS

ECL pKA2 ECL ECL: good

-BIOTIN then specific det;
and SA-AP but also
pCxl-ECL very light

nonspecific
det; very low
bg

SA-AP: very
strong

specific det;
no bg, except
for blotches
due to filter
overlap

ECL pCxI-ECL ECL ECL: specific
and then (Cs) signals

pKA2 SA-AP somewhat
-BIOTIN higher than

others but
nonspecific
signals med;
med bg
SA-AP:
specific
signals med,
and all other
spots show
very faint
signals ;
low bg

(UNEQUIVOCAL
DET.)

67

Table 3 — continued,

EXPT FILTER/SAMPLE PREHYB/HYB PROBE DETECTION RESULTS

16b

16c

NC QB with
Aa , At , AqA ,
Cq and Cs.
WITH POST-APP
TREATMENT :
LF-then-
urea-SDS

ECL

NC dot blots

with L-HI and

pKA2

WITH POST-APP

TREATMENT :

LF-then-

urea-SDS

pCxl

ECL

-BIOTIN

then

and

SA-AP

PKA2-ECL

ECL

L-HI-ECL

ECL

and

then

pK19

SA-AP

-BIOTIN

ECL: strong
specific (AqA)
SA-AP det;
nonspecific
signals and bg
undetectable
except at very
long exposures
SA-AP: signals
to Cs strong,
signals to Cq
weak; no other
signals (no
nonspecific
signals) ;
no bg

ECL: specific
(L-HI) det at
10 ng with 20
sec exp, to 1
ng with 10 min
exp
SA-AP:
specific
det at 10 ng
(and barely
seen at 1 ng) ;
no nonspecific
det; very low
bg

68

Table 3 — continued.

EXPT FILTER/SAMPLE PREHYB/HYB PROBE DETECTION RESULTS

16d

16e

NC dot blots

with L-HI and

pKA2

WITH POST-APP

TREATMENT :

LF-then-

urea-SDS

ECL

NC dot blots

with L-HI and

pKA2

WITH POST-APP

TREATMENT:

LF-then-

urea-SDS

ECL

L-HI

ECL

ECL: specific

-BIOTIN

then

(pK19) det at

and

SA-AP

10 ng with 20

pK19-ECL

sec exp, to 1
ng with 10 min
exp
SA-AP:
specific
det at 10 ng
no nonspecific
det; very low
bg

L-HI

AR

3 day RT exp:

-32-P

specific det
faint but
unequivocal
at 1 ng

16f NC dot blots ECL
with L-HI and
pKA2

WITH POST-APP
TREATMENT :
LF-then-
urea-SDS

17 NC dot blots ECL
with L-HI and
pKA2
(NO POST-APP! ! )

pK19

AR

3 day RT exp:

-32-P

specific det
faint but
unequivocal
at 1 ng

PK19-ECL

ECL

ECL: specific

and

then

det at 10 ng

L-HI

SA-AP

(5 min exp) ,

-BIOTIN

at 1 ng at

longer

(40 min) exp

but with

increased bg

SA-AP:

specific

(but light)

det at 10 ng;

light bg

69

Table 3 — continued.

EXPT FILTER/SAMPLE PREHYB/HYB PROBE DETECTION RESULTS

18 NC dot blots
with L-HI and
pKA2

(NO POST-APP! !)

19 QB with AqA,
Aa, Cn, and At.
WITH POST-APP
TREATMENT :
urea-SDS-LF
WITH DIFFERENT
FILTERS AND/OR
BINDING CONDITIONS

std

std

L-HI
-32-P

pKA2

-BIOTIN

AR

SA-AP

specific
det at 1 ng;
light bg

NY: alkaline
binding

NY: no UV
binding

NY: UV binding

NC

nonspecific
det variable:
light to
strong
det at all
spots; low bg

med specific

det;

no nonspecific

det; low bg

med specific

det;

no nonspecific

det; low bg

light to med
specific det;
no nonspecific
det; low bg

2 0a NY QB; AqA

std

pKA2
-32P

AR

all spots
where sample
applied show
clear signal

70

Table 3 — continued.

EXPT FILTER/SAMPLE PREHYB/HYB PROBE DETECTION RESULTS

2 0b NY QB; AqA
UV-fixed

21 NC QB with AqA,
Aa, Cn, and At.
WITH POST-APP
TREATMENT :
urea-SDS-LF
WITH DIFFERENT
NONRADIOACTIVE
DETECTIONS:

std

pKA2
-32P

AR

same
expt

as for
20a

std

pKA2
-BIOTIN

Gen-2 pKA2

-GENIUS

SA-AP light to med
specific det;
faint to no
nonspecific
det ; med bg

Genius light specific
det; faint to
no nonspecific
det; heavy bg
in areas that
tend to
obscure
signals

ECL

2 2 NC QB with AqA.
Aa, Cn, and At.
WITH POST-APP
TREATMENT:
urea-SDS-LF

ECL

pKA2

ECL

med specific

-ECL

det;

no nonspecific

det; no bg

pKA2

ECL

ECL: strong

-ECL

then

specific det;

and

SA-AP

no nonspecific

pCxl

det; low bg

-BIOTIN

SA-AP: strong
specific det;
no nonspecific
det; low bg

71

Table 3 — continued.

EXPT FILTER/ SAMPLE PREHYB/HYB PROBE DETECTION RESULTS

2 3 NC QB with AqA,
AqB, AqC, AqD,
Aa, and Cn.
WITH POST-APP
TREATMENT :
urea-SDS-LF

oligo

Arp-1

through

Arp-4

oligo

probes

(32P)
(used
separately)

AR

Arp-1 and
Arp-4 probes:
strong

specific det;
no nonspecific
det; no bg.
Arp-2 probe:
strong
specific
det; light
nonspecific
det; high bg
Arp-3 probe:
faint specific
det;

no nonspecific
det; no bg

Notes for Table 3. see notes for Table 1, and the following. AqB
= A. quadrimaculatus species B. AqC = A. quadrimaculatus species
C. AqD = A. quadrimaculatus species D. AR = autoradiography; L-HI
= phage lambda DNA restricted with Hind III. Aa = A. albimanus.
Gen-2 = GENIUS prehybridization/hybridization mix modified for
use at 42 °C by including in the recommended mix 50% formamide,
and substituting SSPE for SSC. med = medium. Postapplication
treatments were performed as described in the notes on Table 1,
plus: urea-SDS-LF = 8M urea, 10% SDS, 10% LA FRANCE; LF-then-
urea-SDS = first wash with 10% LA FRANCE, then second wash with
8M urea, 10% SDS.

SYNTHETIC OLIGONUCLEOTIDE PROBES
Four oligonucleotides were synthesized based on
nucleotide sequence data obtained from Nsi I deletion
subclones derived from pKA2 , in an attempt to (1) obtain
greater specificity in identification of A. quadrimaculatus
species A, as compared to that obtained by using pKA2 as a
probe, and (2) demonstrate that synthetic oligonucleotides
can provide valuable tools for identification of cryptic
mosquito species. Sequence data obtained from the Nsi I
subclones of pKA2 (which were used to specify the
oligonucleotide sequences) were compared to sequence
obtained from subclones of phage Arp2 (Figure 11) , since the
latter was thought more likely to preserve the sequences
present in the mosquito genome.

Figure 11 shows the sequence of the synthetic
oligonucleotides compared to sequence data obtained from
subclones of phage Arp2 and plasmid pKA2 . Here, differences
between an oligonucleotide sequence and the sequence from a
given phage subclone do not necessarily reveal the result of
molecular rearrangement in the pKA2 subclones. Rather, they
probably reflect small differences between the many repeat
elements found within the mosquito genome. The large

72

73
differences among the four sequence motifs detected in the
pKA2 subclones (and represented in the oligonucleotide
sequences) contrast with the similarities among the
sequences of a specific motif, whether from pKA2 or Arp2
subclones.

In the limited sequence data obtained from the pKA2 and
pArp2 subclones, the motifs 1 and 3, and the motifs 2 and 4,
were found to be adjacent or overlapping in at least six
instances per combination. Motifs 1 and 2 were also found
nonoverlapping, in at least four and three instances,
respectively (Figures 4 and 5) .

The oligonucleotides were radiolabeled and used as
probes to quick blots prepared with A. quadrimaculatus
species A, B, C, and D, A. albimanus. and C. niaripalpus
(Table 3, experiment 23). The results of autoradiographic
detection of hybridized oligonucleotide probes is shown in
Figure 12. A long exposure of ten days (at -80 °C with
intensifying screens) revealed that the specificity of
oligonucleotide probes Arp-1 and Arp-4 (and probably also
Arp-2) was greater than that of the phage Arp2 probe
(Cockburn, 1990) . These results, when considered in light of
the sequence data obtained from the plasmid subclones,
suggested that the sequence elements present in A.
quadrimaculatus species A DNA which conferred species

74
specificity to the phage Arp2 probe are short, nonidentical
(but very similar) repeats, and that there exist three or
more distinct motifs which contribute to this specificity.
These sequence motifs are not tandem repeats, and are
present in some cases in inverted orientations with respect
to one another.

There are several possible advantages in using
oligonucleotides over cloned DNAs for the preparation of
hybridization probes. Usually a cloned insert will be a much
longer segment of DNA than the oligonucleotide, so there is
more chance for a degeneracy in specificity to be manifested
by some portion of the cloned DNA sequence. Thus, the
oligonucleotide may provide increased specificity if it
lacks nonspecific sequences found in the cloned DNA. Another
advantage of synthetic oligonucleotide probes is that since
their chemical structure is completely defined, new lots of
the probe may be produced at any facility set up for
synthesis of oligonucleotides. Incompletely characterized
DNA probes contained in plasmid vectors must be prepared
using suitable (bacterial) host strains and sufficient
amounts of recombinant plasmid. A third possible advantage
of oligonucleotides over recombinant plasmids for use as
probes is their purity. Probe DNAs propagated in recombinant
plasmids must be purified to remove bacterial nucleic acids.

75
proteins, and lipids. While these purification steps are
usually adequate for most applications, DNA modifying
enzymes (such as those used in the labeling of hybridization
probes) are often inhibited by trace contaminants. This is
not a problem with synthetic oligonucleotides, which are
typically free of contaminants.

Figure 11, Synthetic oligonucleotide sequences compared to
phage Arp2 and plasmid pKA2 subclone sequences. The
sequences of the four oligonucleotides used in this study
are each shown above sequence information obtained from
subclones of the insert from phage Arp2 and the subclones of
plasmid pKA2 . The sequences of the oligonucleotides were
determined from sequence data obtained from the pKA2
subclones, and are shown with their 5' ends to the left.
Since there was some doubt about whether the pKA2 sequence
accurately represented sequence from the phage (Arp2)
insert, subclones from the phage (including those listed in
Figure 11 as subclones 1, 3, and 5) were sequenced and found
to be similar to the sequences of the oligonucleotides.
Since the insert in phage Arp2 was isolated from genomic DNA
of A. quadrimaculatus species A (Cockburn, 1990) , this
figure reveals the close similarity of the sequences of the
oligonucleotides to the repeats in the mosquito genome,
oligo = oligonucleotide.

77

TTTGCATATAGCTGGTG-CTAG-TTT Oligonucleotide Arp-1

-. . . pArp2-N3

G -. .G.-. . . pArp2-Nl

-... pArp2-N3

GT C. .N.-. ...... . pArp2-N5

G. . . .-N -. . . pArp2-N3

-... PKA2-N1

-• . . PKA2-N1

G -....-... PKA2-N1

GC.ATTGC T. . . . pKA2-Nl

A. .A. . .- A. . . PKA2-N2

. . . PKA2-N2

. . . pKA2-N3

GCAAACCAA-TCATAGGACATACTC Oligonucleotide Arp-2

- pArp2-Nl

A -. . .C pArp2-N3

T. . .- pArp2-N3

T...- pArp2-N5

A T.- A PKA2-N1

G...C G PKA2-N1

TGTT.G...- PKA2-N1

T...- T pKA2-N2

- PKA2-N2

- pKA2-N3

TTTGAGATATATGG-CACAAATGTGATCAATT Oligo. Arp-3

. . .TG G C.AATC. . pArp2-N5

. . .TTT -. .A.C C.AATC. G pArp2-N3

. . . TG . . AC . G . . A- T . C . AATC . G pArp2 -Nl

G- - . . .A. PKA2-N2

- pKA2-N2

-. .A . . .A. PKA2-N3

CTCCAAACTCATTGCATCTATTGGTGTATGCAG Oligo. Arp-4

N pArp2-Nl

G C. .C. .CA pArp2-N3

. . .N N PKA2-N1

. .T G C. . .T PKA2-N2

PKA2-N2

PKA2-N3

78

B

Arp-1
Arp-2
Arp-3

Arp-4

Figure 12. Results of using radiolabeled oligonucleotide
probes with quick blots for species-specific detection.
Quick blots were prepared, with each filter containing two
spots from each of six species. Each spot received one-tenth
of the solution in which a single insect was macerated. A,
B, C, D = A. quadrimaculatus species A, B, C, and D,
respectively. E = A. albimanus. F = C. niaripalpus. The
figure shows the results of autoradiographic detection (ten
days at -80 °C with intensifying screens) .

CONCLUSIONS AND SUMMARY
Discussion of the Efforts to Isolate a Culex-specif ic Probe

The difficulty encountered in isolating a C.
niqripalpus-specif ic DNA probe indicates that the vast
majority of species-specific repetitive DNA in the genome of
C. niqripalpus, if this exists, is closely linked with
nonspecific DNA sequences. One study (Cockburn & Mitchell,
1989) indicated that the level of repetitive DNA
interspersion in C. quinquefasciatus DNA was higher than
that found in anopheline DNA, although lower than that found
for Aedes aegypti (Linnaeus) DNA. Even if some repetitive
DNA is clustered within the C. niqripalpus genome, these
clustered repeats may not be species-specific. Indeed, the
results of the attempts to isolate a C. niqripalpus-specif ic
probe indicate a paucity or lack of species-specific
sequences in the genome.

The fact that the pCxl probe could be isolated from a
C. niqripalpus library by screening with C. quinquefasciatus
DNA, and the observation that the insert in pCxl is large,
indicate that the conclusions above regarding C. niqripalpus
versus C. salinarius do not apply when comparing the genomes
of C. niqripalpus and C. quinquefasciatus . These results
support the close phyletic relationship of C. niqripalpus

79

80
and C. salinarius. with C. guinguefasciatus a more distant
relative. They also show that the techniques used are
capable of isolating differentially repeated sequences when
they exist.

Significance of Synthetic Oligonucleotide Probes and
Characterization of Other Mosquito Species-specific Probes

The ease with which potential species-specific
synthetic oligonucleotide probes were specified from the
sequence data obtained from the pKA2 subclones, and the
success in using the synthetic oligonucleotides as species-
specific probes, indicate that this approach to obtaining
probes from clones thought to contain numerous repeats due
to a paucity of restriction sites is a valuable one. The
improved specificity of the synthetic oligonucleotide probes
showed these can be valuable tools for mosquito species
identification.

It may not be possible to identify repeat sequences in
clones containing only one (or a portion of one) repeat.
This could be one reason why repeats were not identified in
the sequence data obtained for pAfl-Sl, pBrpl-Sl, pCrpl-S2,
and pCrpl-S3. Nevertheless, in some cases testing of the
hybridization specificity of synthetic oligonucleotides
specified by sequence data from such clones may provide
species-specific probes as well as the localization of

81
repetitive species-specific sequences. For example, since
pCrpl-S2 and pCrpl-S3 retain the specificity of Crpl, it is
likely that a species-specific repeat spans the genomic
region corresponding to the junction of these clones. Thus,
oligonucleotides could be synthesized based on the sequence
data obtained for these two clones, and tested for
hybridization specificity. In this case, restriction
analysis could be used to define which of the ends composed
this junction.

The physical map of pAfl-Sl, and the sequence data
obtained from pAfl-Sl and the various A. quadrimaculatus-
specific probes, provide a foundation for further
characterization of the sequences which confer species
specificity in these clones. The data are also valuable for
providing a beginning of a more in-depth study of repetitive
DNA of mosquitoes, which might include transposons or other
interesting mobile genetic elements. There is a possibility
that the pCrpl-S2, pCrpl-S3, or other clones may contain
such mobile elements. The discovery of mobile genetic
elements in mosquitoes could provide valuable tools for
genetic engineering of these organisms.

82

Significance of the Quick Blot Protocol and Nonradioactive

Detections
There are several methods available for preparing
targets for nucleic acid hybridization experiments, and the
decision of which method to use in a particular situation
should be based on a number of considerations. These include
the specific goals of the experiment and the advantages
afforded by use of a particular method of preparing the
target(s). In the simplest of cases, where a single probe is
to be used with a single type of target, a slot blot, dot
blot, or squash blot may be appropriate. It may be more
advantageous to use a quick blot, however, for an experiment
requiring a single sample of tissue to be probed separately
with many probes, or for an experiment requiring many
samples to be probed.

Most of the features of quick blots are available in
one or more of the other types of blots, but none of the
others provides the unique combination of traits of quick
blots. Also, the ability to prepare multiple sets of
equivalent targets with a given set of samples, with little
additional effort, is a feature that is shared by the quick
blot, dot blot, and slot blot protocols, but not possible
with squash blots. The QB protocol can be used to prepare
sets of nucleic acid samples in a form suitable for various

83

types of nucleic acid analysis. The nucleic acids could
potentially be derived from any of a wide range of tissues
from various animals, including insects and other
arthropods, soft tissue samples from various non-arthropod
animals, and plants. It is well suited for analysis of
nucleic acids extracted from entire insects in the 1-2 0 mg
size range, or body parts or isolated tissues from larger
individuals. We have used the QB protocol to analyze DNA
from individual mosquitoes.

The availability of nonradioactive detection systems
has allowed nucleic acid hybridizations to be carried out in
laboratories not equipped for handling radioactive reagents.
Many agencies or groups not able or willing to comply with
regulations or safety requirements relating to radioisotopes
thus have the opportunity to use DNA probes in their basic
or diagnostic research.

Nonradioactive detection systems have been used in many
ways with various types of blots (Mclnnes & Symons, 1989b) ,
but their potential usefulness with quick blots is
especially great in those situations where nonradioactive
detection must be used with multiple probes to a given set
of samples. This is due to the ease of preparation of
multiple equivalent blots with the quick blot protocol.
These multiple blots can serve as targets for probes of

84

different specificities. This study has shown the
feasibility of using quick blots to screen any number of
mosquitoes with as many as ten different probes. The probes
may be species-specific, allowing the detection of different
mosquito species, or some may be pathogen-specific, allowing
the detection of particular mosquito-transmitted diseases
among the samples. Simple treatments of blots were described
which effectively reduce nonspecific background. The
procedure presented in this study for the nonradioactive
detection of two probes hybridized simultaneously would
allow a species-specific probe and a pathogen-specific probe
to be used in the same hybridization step.

Nucleic acid hybridization probes will be used in
increasing ways in basic and applied research, as they allow
rapid, accurate, and often extremely sensitive detection of
nucleotide sequences. In particular, it is expected that
these techniques will become more important in the
development of animal and plant breeding programs, and in
the diagnosis and treatment of many types of diseases. The
advances in DNA probe techniques described here are part of
a trend to moving DNA probes beyond the laboratory and into
the field. The advances may eventually allow field
epidemiologists and others to possess field kits which can
identify a putative vector, show what it is infected with,
and show what it has fed on, in a few simple steps.

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Cockburn, A.F. 1990. A Simple and Rapid Technique for

Identification of Large numbers of Individual Mosquitoes
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San Diego, Academic Press, pp 572-581.

BIOGRAPHICAL SKETCH
I was born in Columbus, Ohio, on June 7, 19 59. I moved
to Florida when I was 11 years old, and received the B.S. in
Microbiology & Cell Science from University of Florida in
December, 1981. I received the M.S. in Microbiology from The
Florida State University in August, 1984.

88

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

/?

ick A. Seawright, Znair
Associate Professor of
Entomology and Nematology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

Andrew F. Cockburn
Assistant Professor of
Entomology and Nematology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

jEl

k

J. Howard Frank

Professor of

Entomology and Nematology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

>vJ^ C

David G. Yoiw^
Associate Scientist of
Entomology and Hematology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

t Hiebert '

Ernes
Professor of
Plant Pathology

This dissertation was submitted to the Graduate Faculty
of the College of Agriculture and to the Graduate School and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.

December, 1990

r.i

Dean, aollege of Agriculture

Dean, Graduate School

UNIVERSITY OF FLORIDA

3 1262 08553 8287