Mitochondrial and ribosomal DNA analysis for identification of sibling species of the mosquito, Anopheles quadrimaculatu...

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Title:
Mitochondrial and ribosomal DNA analysis for identification of sibling species of the mosquito, Anopheles quadrimaculatus (Say)
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vi, 84 leaves : ill., photos. ; 28 cm.
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Mitchell, Sharon Elizabeth, 1951-
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Thesis (Ph. D.)--University of Florida, 1990.
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Includes bibliographical references (leaves 79-82).
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by Sharon Elizabeth Mitchell.
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Typescript.
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Vita.

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Full Text









MITOCHONDRIAL AND RIBOSOMAL DNA ANALYSIS FOR
IDENTIFICATION OF SIBLING SPECIES OF THE MOSQUITO,
Anopheles quadrimaculatus (Say)















By

SHARON ELIZABETH MITCHELL


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
UNIVERSITY OF FLORIDA LIBRARIES
































THIS WORK IS DEDICATED TO THE AUTHOR'S MOTHER,
ELIZABETH RUTH NEILL













ACKNOWLEDGEMENTS

The author wishes to express her sincere appreciation to Drs. J. A. Seawright

and A. F. Cockburn for their critical appraisal of the dissertation, and for their guidance

and friendship throughout the course of this study. Special thanks are extended to Dr.

S. K. Narang for his aid in interpretation of results obtained from protein

electrophoresis.

The author extends her gratitude to the graduate committee members Drs. J. E.

Maruniak, H. G. Hall and C. D. Chase for their encouragement and critical review of

the work presented. Very special thanks are extended to M. Q. Benedict for his help

with the computer analyses and continued friendship throughout the years.

The author wishes to thank the staff of the U. S. Department of Agriculture,

Insects Affecting Man and Animals Research Laboratory, Gainesville. She especially

thanks Dr. G. A. Mount for his support of this study. Finally, the author extends

special thanks to Ms. P. A. Martin for assistance in the preparation of this manuscript.













TABLE OF CONTENTS


PAGE
ACKNOWLEDGEMENTS ......................................... iii

ABSTRACT ...................................... ............. v

CHAPTER I. INTRODUCTION .................................. 1

CHAPTER II. CLONING AND ORGANIZATION OF THE MITOCHONDRIAL
GENOME OF Anopheles quadrimaculatus (Say) ............ 4

Introduction ............................. ......... 4
Materials and Methods ................................ 5
Results ................................... .. ..... 8
Discussion ....................................... 31

CHAPTER III. MITOCHONDRIAL AND RIBOSOMAL DNA VARIATION
AMONG MEMBERS OF THE Anopheles quadrimaculatus
SPECIES COMPLEX ................................ 36

Introduction ...................................... 36
Materials and Methods ............................... 38
Results ........................................ 43
Discussion .......................... ............ 69

CHAPTER IV. CONCLUDING REMARKS .......................... 77

LITERATURE CITED .......................................... 79

BIOGRAPHICAL SKETCH ....................................... 84













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


MITOCHONDRIAL AND RIBOSOMAL DNA ANALYSIS FOR
IDENTIFICATION OF SIBLING SPECIES OF THE MOSQUITO,
Anopheles quadrimaculatus (Say)


By

SHARON ELIZABETH MITCHELL

August 1990


Chairman: J. A. Seawright
Cochairman: A. F. Cockburn
Major Department: Entomology and Nematology


Work was done to investigate the feasibility of using DNA restriction patterns

for the identification of individual mosquitoes of the Anopheles quadrimaculatus species

complex. The entire 15 kilobase (kb) mitochondrial genome of A. quadrimaculatus,

species A was cloned in bacteriophage, then subcloned in plasmid vectors and

characterized. The cloned DNA was physically mapped with restriction endonucleases,

and several genes were mapped by sequencing the ends of A. quadrimaculatus subclones

and by hybridization with previously characterized Aedes albopictus mitochondrial DNA

(mtDNA) clones. These portions of the genetic map were identical in gene order to

those of Drosophila yakuba.








The cloned mtDNA and a previously isolated ribosomal DNA (rDNA) clone

were used as hybridization probes for analysis of individual mosquitoes from seven

populations containing more than one sibling species of A. quadrimaculatus.

Hybridization of both the mtDNA and rDNA clones to total genomic DNA digested

with several different restriction endonucleases produced species-specific restriction

patterns and identified low-frequency intraspecific variation.

The mtDNA of the four sibling species of the A. quadrimaculatus complex were

physically mapped with the same restriction enzymes employed in the population

analysis. Homologies among the mtDNA restriction patterns confirmed the genetic

relationships among the sibling species determined by isozyme analysis, chromosome

comparisons and hybridization studies.













CHAPTER I

INTRODUCTION


Cryptic species occur commonly in the genus Anopheles (Diptera: Culicidae).

Cryptic species are defined as genetically distinct, reproductively isolated but

morphologically indistinguishable taxa within what was previously considered to be a

single species (Narang and Seawright, 1990). The first species complex in anopheline

mosquitoes was recognized about fifty years ago for Anopheles maculipennis, an

important vector of human malaria in Europe (Kitzmiller, 1953). The discovery of this

species complex resulted from the observation that even though A. maculipennis was

widely distributed, the incidence of malaria was not. Further investigations indicated

that there were ecological differences among different geographic strains of A.

maculipennis and later these strains were found to correspond to eight sibling species

based on egg morphology, hybridization studies and chromosomal comparisons

(Kitzmiller et al., 1967). Since this initial discovery, species complexes have been

described for numerous anophelines including A. gambiae, A. culicifacies, A. balabacensis,

A. punctulatus, A. annulipes, A. coustani, A. sinensis, A. maculatus, A. subpictus, A.

marshallii and A. quadrimaculatus (see Narang and Seawright, 1990 for a complete

review). Some, but not all, members of these species complexes are important malaria

vectors in Africa, India and Asia.







2

Although it is not common knowledge, especially in North America, the fact

remains that malaria accounts for more morbidity and mortality, world-wide, than any

other transmissible disease. The World Health Organization estimates that at any given

time, about 100 million people show clinical symptoms of the disease and about 1

million die from malaria each year (Marshall, 1990).

From the epidemiological standpoint, it is important that only a few of the

sibling forms in the typical anopheline species complex may be involved in the

transmission of malaria parasites to humans. Therefore, correct identification of cryptic

species is necessary so that control measures can be taken in areas where high densities

of disease vectors occur and avoided if the nonvector, sibling species predominate.

Anopheles quadrimaculatus (Say), the common malaria mosquito, is widely

distributed throughout the eastern and central parts of the United States and ranges

north to southeastern Canada. When human malaria was prevalent in this country, A.

quadrimaculatus was the primary vector of this disease in the southern and eastern-

seaboard states. Presently, this mosquito is an important vector of canine heartworm,

Dirofilaria immitis, throughout its range (Lewandowski et al., 1980).

Recently, it has been found that the taxon A. quadrimaculatus is a species

complex containing at least four morphologically identical, sympatric sibling forms,

species A, B, C, and D (Lanzaro et al., 1988; Kaiser et al., 1988a,b; Narang et al.,

1989a,b). The genetic evidence for species designation of all four sibling types is

conclusive and includes hybridization crosses, polytene chromosome analysis and

diagnostic allozymes.







3

In addition to the traditional genetic techniques (i.e. cross-hybridization,

chromosome analysis and isozyme electrophoresis), DNA restriction analysis has become

a powerful method for studying the genetic structure of natural populations. The work

reported herein applies the techniques of molecular biology to the practical problem of

correct identification of morphologically indistinguishable species and to the more

theoretical aspects of evolutionary genetics (i.e. genetic diversity and phylogenetic

relationships among sibling species).

The objectives of this study were to (1) develop DNA probes for both

cytoplasmic (mitochondrial) and nuclear ribosomall) genomes of A. quadrimaculatus to

be used as hybridization probes for the analysis of individual mosquitoes; (2) investigate

the feasibility of using restriction site polymorphisms for both cytoplasmic and nuclear

DNAs to identify individuals of the A. quadrimaculatus complex to species, and; (3) use

the mitochondrial DNA restriction site data to estimate genetic distances and determine

phylogenetic relationships among the four sibling species.

This dissertation contains four chapters, two of which are currently being

submitted in modified form to scientific journals. Chapters II and III provide a short

introduction, a review of the relevant literature, a presentation of the results of the

work done and a discussion of the significance of the findings. A general summary and

concluding remarks are contained in Chapter IV.













CHAPTER II

CLONING AND ORGANIZATION OF THE MITOCHONDRIAL GENOME OF
Anopheles quadrimaculatus, SPECIES A


Introduction


Mitochondrial DNA (mtDNA) restriction analysis has become a powerful method

for studying natural populations (Brown, 1985). In order to analyze the mtDNA of

individual insects, it is useful to have purified or cloned mtDNA to use as a radioactive

probe (Cockburn and Seawright, 1988). Isolation of mtDNA from mosquitoes in

amounts sufficient for use as a probe is problematic because of the difficulty in

obtaining large quantities of live insects and low yields. The complete mitochondrial

genome has been cloned and sequenced from at least one insect, Drosophila yakuba

(Clary and Wolstenholme, 1985). Although Dubin and coworkers have cloned portions

ofAedes albopictus mtDNA (HsuChen et al., 1984; Dubin et al., 1986), cloning of the

complete mitochondrial genome for mosquitoes has not been reported.

To facilitate restriction analysis of mtDNA polymorphisms in the Anopheles

quadrimaculatus (Say) species complex, the entire mitochondrial genome from A.

quadrimaculatus, species A was initially cloned as three Eco RI fragments in

bacteriophage. A restriction map was prepared by digestion of the clones and confirmed

by comparison to the digestion pattern of native mtDNA. Subsequently, mtDNA inserts

from the bacteriophage clones were subcloned in plasmid vectors. Sequence data







5

obtained from 13 plasmid subclones allowed alignment of the physical map with the

genetic map derived from Drosophila. The clones and physical map will allow

comparison of mtDNA from the four species of the A. quadrimaculatus complex and

determination of the relationship of their maternal lineages.


Materials and Methods


Adult Anopheles quadrimaculatus, sp. A (ORLANDO strain) were obtained from

the USDA, Insects Affecting Man and Animals Research Laboratory, Gainesville,

Florida. Total mosquito DNA was prepared as in Cockburn and Seawright (1988).

Briefly, 2 grams of frozen mosquitoes were powdered in a mortar and pestle on dry ice

and suspended in 100 ml of cold homogenization buffer (0.1 M NaCI, 0.2 M sucrose,

0.01 M ethylenediaminetetraacetic acid (EDTA), 0.03 M Tris HCI, pH 8.0). After

homogenizing for a few strokes in a Wheaton dounce tissue grinder, the homogenate

was transferred to a centrifuge bottle. Twenty-five ml of lysis buffer (0.25 M EDTA,

2.5% sodium dodecyl sulfate (SDS), 0.5 M Tris HCI, pH 9.2) and 1 mg proteinase K

were added and the mixture was incubated at 550 C for 1 hr. Seventeen ml of 8 M

KCH3COOH was added, the bottle was placed on ice for 1 hr and then centrifuged at

17,000 g for 20 min. Three-hundred ml of ethanol was added to the supernatant and

the solution was incubated at 0 C for 1 hr. Nucleic acids were pelleted at 17,000 g in

a preparative centrifuge for 10 min and further purified by CsCI gradient centrifugation

(Maniatis et al., 1987).

Mitochondria were prepared by differential centrifugation. About 2 g of live

mosquitoes were thoroughly homogenized (Wheaton dounce homogenizer) in 100 ml








6

homogenization buffer on ice and centrifuged at 1,600 g for 5 min. The supernatant

was decanted and nuclei pelleted by centrifugation at 1,600 g for 10 min. Mitochondria

were pelleted by centrifuging the supernatant at 17,000 g for 30 min. The mitochondrial

pellet was resuspended in 25 ml homogenization buffer, centrifuged at 1,600 g for 10

min, the supernatant decanted, and centrifuged at 40,000 g for 10 min. The

mitochondrial pellet was suspended in 8 ml of homogenization buffer and lysed by the

addition of 0.5 ml 20% sodium lauryl sarcosinate. Cesium chloride (CsCI) (8.5 g) and

0.4 ml 10 mg/ml ethidium bromide were added, the solution was transferred to an

ultracentrifuge tube and centrifuged in a Beckman L8-M ultracentrifuge with a type 70.1

Ti fixed rotor at 35,000 RPM for 40 hr. The lower mtDNA band was visualized by UV

irradiation and removed. Ethidium bromide was extracted and purified mtDNA

precipitated using standard techniques (Maniatis et al., 1987).

Purified Eco RI digested mtDNA was cloned into the Eco RI site of

bacteriophage X gtlO (Stratagene) using standard procedures (Berger and Kimmel, 1987).

DNA was packaged into bacteriophage according to the manufacturer's instructions using

Gigapack Gold packaging mix (Stratagene). Agarose gel electrophoresis, nitrocellulose

plaque lifts, Southern transfer and nick translation of DNA were standard techniques

(Berger and Kimmel, 1987). Hybridizations were as in Cockburn and Seawright (1988).

Isolation of bacteriophage DNA, subcloning of fragments, transformation, plasmid

growth, isolation of plasmid DNA, and restriction digestion were standard techniques

(Berger and Kimmel, 1987). Plasmids were grown in Escherichia coli strain DH5a

(BRL). Mitochondrial DNA inserts from bacteriophage clones were purified from







7

agarose gels using the GENECLEANTM system according to the manufacturer's

instructions (BIO 101).

Two non-overlapping Aedes albopictus mtDNA clones, k8 and k14, were obtained

from Dr. Donald T. Dubin (University of Medicine and Dentistry of New Jersey).

Plasmid subclones were sequenced by the dideoxy chain-termination method

using the Sequenase version 2.0 kit from United States Biochemical Corporation. The

ends of all subclones were sequenced at least twice except when sequence data had

already been obtained from another clone which shared a common end. Sequencing was

done in one direction only. Therefore, sequence data was obtained for both mtDNA

strands only for the middle sections of relatively small subclones (i.e. clones p3X2 and

p3RX2).

Alignment of A. quadrimaculatus and D. yakuba mitochondrial sequences was

done with the aid of the Genetics Computer Group Sequence Analysis Software

Package (Devereaux et al., 1984). The D. yakuba sequence (Genbank) was searched

and regions of sequence similarity were identified using the WORDSEARCH program

(Wilbur and Lipman, 1983). Final alignment and calculation of percent similarity

between the two sequences was accomplished with the BESTFIT sequence comparison

program (Smith and Waterman, 1981). The MAP program was used to translate both

mosquito and Drosophila sequence data for protein comparisons. The Drosophila

mtDNA code was used to predict protein sequences. The validity of sequence

alignments in the A+T-rich region was evaluated by randomizing this section of DNA

from D. yakuba using the SHUFFLE program. Twenty randomized sequences were

generated and each of these was realigned with the mosquito sequence.








8

Results


Cloning

The complete mitochondrial genome of animals is frequently difficult to clone in

bacteria. For example, an A. quadrimaculatus total genomic DNA library was previously

screened for mtDNA clones. The library was made by cloning total DNA that had been

partially digested with Sau 3A into the bacteriophage X vector EMBL 3A. Although the

mtDNA represents about 1% of the total DNA, contains several Sau 3A restriction sites,

and would be near the optimal size to insert into this vector, no mtDNA inserts were

found in about 50,000 plaques screened. About 500 mitochondrial clones should have

been found if there was no bias against them.

Other workers have noted that cloning of mammalian mtDNA that is unstable in

bacteria can be successful if the DNA is cloned as relatively small fragments (W.

Hauswirth, personal communication). Previous experiments involving hybridization of

Eco RI digested total genomic DNA from Anopheles quadrimaculatus sp. A to purified

mtDNA revealed that the mtDNA of this species contains three Eco RI fragments (6.3,

5.6 and 3.4 kb). Therefore, an Eco RI library of purified mtDNA was constructed in A

gtlO. This library was screened with two non-overlapping Aedes albopictus mtDNA

clones, k8 and k14. Two clones were isolated, one (AqAml) homologous to k14 and

one (AqAm2) homologous to k8. The Eco RI inserts in these were subcloned into the

plasmid vector pIBI24. The mitochondrial library was rescreened with total mtDNA

from A. quadrimaculatus and counter-screened with a mixture of the plasmid subclones

(pAYCml and pAYCm2). A clone (AqAm3) was isolated that hybridized to mtDNA

but not the other two clones. This clone was subcloned into pIBI24 to form pAYCm3.







9

The three lambda clones each contained one of the three Eco RI fragments and

apparently represented the entire mitochondrial genome.

Analysis of the inserts from the three bacteriophage clones indicated that these

Eco RI fragments were identical in size to the native mtDNA fragments (data not

shown). Inserts from two of the three plasmid subclones, however, were not identical to

the parent clones (Figure 2-1). One subclone (pAYCml) contained a 5.1 kb insert

(about 1.2 kb had been deleted from one end). The deletion from the end of pAYCml

was probably due to cleavage at an internal Eco RI site, since an Eco RI site was

regenerated at this end. Another clone (pAYCm3) had a 6.3 kb insert (an extra 0.7 kb

was added to the interior of the clone). The 3.4 kb insert of the third subclone

(pAYCm2) was unaltered.

Inserts of AqAml and AqAm3 were cleaved into smaller fragments with several

different enzymes and, again, subcloned into plasmids. Because of poor growth and low

DNA yields of the pIBI-derived plasmids, pUC 19 was used as the plasmid vector. As a

result of this approach, several subclones were isolated from each bacteriophage (see

Figure 2-2), comprising the complete insert of each. The mtDNA insert from pAYCm2

was transferred into pUC 19, as well, to form p2R1.

The initial attempts to subclone the right end of AqAm3 failed. Several hundred

lac7 recombinant subclones from three separate digests (Eco RI/Stu I, Eco RIISst I and

Eco RI/Xba I) were isolated by X-gal selection. The identity of the subcloned fragment

was determined by digestion with the appropriate restriction endonuclease and gel

electrophoresis. The left end of the mtDNA and various vector fragments were

recovered many times from each digest, but the mtDNA sequences immediately adjacent

to the righthand Eco RI site were never obtained. Fortuitously, one subclone having a















































Figure 1-1. Eco R1 digests of Agt 10 and pIBI-24 mtDNA clones from Anopheles
quadrimaculatus.

Lane (A) AqAml; (B) pAYCml; (C) AqAm2; (D) pAYCm2; (E) AqAm3; (F)
pAYCm3; (G) A Hind III fragments. Note that the 32.7 kb and 10.6 kb fragments in
lanes A, C and E are X vector arms. Also the 2.9 kb fragment in lanes B, D and F are
the pIBI-24 vector.


A CDE



4 23.


#ag 9








11

partially nonrecombinant phenotype (lac*) was selected for analysis. This clone was

from an Eco RI/Xba I digest and contained the rightmost Eco RI/Xba I mtDNA

fragment (0.6 kb) plus the noncontiguous 2.3 kb Xba I mtDNA fragment. This subclone

was digested with Xba I and religated to make subclone p3RX2 (Figure 2-2), which was

strongly lac'. Further screening of lac* subclones yielded a subclone containing the 1.9

kb Eco RI/Stu I mtDNA fragment (p3RU2). Subclones containing the 4.2 kb Eco

RI/Sst I fragment (also lac') were rearranged. All of these lac* subclones had the same

Eco RI end (internal to the large ribosomal RNA coding sequence) ligated to the

plasmid fl-galactosidase a-peptide, so presumably the mitochondrial sequences upstream

from that site are responsible for the restoration of the lac' phenotype.


Restriction Analysis


Because of the initial difficulty encountered in obtaining unrearranged plasmid

clones, restriction sites were mapped in the three bacteriophage clones. Vector DNA

and DNA from the clones were cleaved with the appropriate restriction endonuclease(s),

loaded in adjacent wells and electrophoresed in 0.8% agarose gels. Single and double

digests were done for a total of 12 restriction enzymes. Ambiguities were clarified by

repeating the appropriate digests with purified mtDNA inserts excised from agarose gels.

Clones were oriented and the resulting composite map was verified by comparison to

restriction digests of total cellular DNA blotted to nitrocellulose and probed with

mtDNA. For example, pAYCml contained two Eco RIAva I fragments (5.2 and 1.1

kb). Clone pAYCm2 also contained two such fragments (2.2 and 1.2kb). To comprise

the 3.3 kb Ava I fragment observed in native mtDNA (see Figure 2-3, third lane), the








12

1.1 kb Eco RI/Ava I of pAYCml and the 2.2 kb fragment of pAYCm2 must be adjacent

to one another.

This restriction mapping indicated that the three mtDNA clones had the same

profile as total cellular mtDNA (Figure 2-2). The integrity of all plasmid subclones

subsequently isolated was evaluated with respect to the restriction map obtained from

the bacteriophage clones and by DNA sequencing.


Mapping of Aedes albopictus mtDNA clones


Animal mitochondrial DNAs studied so far code for 2 ribosomal RNAs (rRNAs),

22 tRNAs, an A+T-rich region which has no corresponding transcripts) and contains

the origin of mtDNA replication and 13 hydrophobic proteins (Garesse, 1988). The

protein coding regions code for subunits of enzyme complexes associated with the inner

mitochondrial membrane and include cytochrome b (Cytb), two subunits of the ATPase

(6 and 8), three subunits of the cytochrome c oxidase (CO I, II and III) and seven open

reading frames which have recently been identified as subunits of the NADH reductase

complex (ND1, 2, 3, 4, 4L, 5 and 6) (Chomyn et al., 1985).

Dubin and coworkers have previously cloned and partially sequenced three small

parts of the A. albopictus mtDNA. These consisted of 1) 2.8 kb containing the 3' end

of the CO III gene, tRNAG", the ND 3 gene, a cluster of tRNAs (tRNAA,9 tRNA^,

tRNAA', tRNAS, tRNA'G and tRNAPh') and the 3' end of the ND 5 gene (clone k8)

(Dubin et al., 1986); 2) the Hind III D fragment, 1.9 kb containing the 3' end of the

large subunit rRNA gene (lrRNA), tRNA', and the 5' end of the ND 1 gene (clone.kl4)















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15

(HsuChen et al., 1984); and 3) the Hind III E fragment, 0.5 kb containing the 5' end of

the large ribosomal RNA (IrRNA) gene and tRNAm (HsuChen et al. 1984).

Two of these cloned DNA sequences (k8 and k14) were hybridized to total A.

quadrimaculatus DNA cleaved with various enzymes to determine the regions of

homology. Plasmid subclone k8 hybridized to fragments contained in AqAm2, including

the 3.4 kb Eco RI fragment, the 6.3 kb and 2.2 kb Hind III fragments, the 3.3 kb Ava I

fragment, the 10.2 kb Cla I fragment, the 13.0 kb Bgl II fragment, and the 13.1 kb and

2.2 kb Sca I fragments (Figure 2-3). Therefore, the k8 homologous region extends

from just left of the Eco RI site between the 3.4 kb and 6.3 Eco RI fragments to the

Ava I site between the 3.3 kb and 2.5 kb Ava I fragments. These results are consistent

with the restriction map generated from the cloned A. quadrimaculatus mtDNA and with

the isolation of AqAm2 using a k8 probe.

Clone k14 hybridized to the 6.3 kb Eco RI fragment, the 1.85 kb Hind III

fragment, the 9.2 kb Ava I fragment, the 10.2 kb and 5.1 kb Cla I fragments, the 2.3 kb

and 13.0 kb Bgl II fragments, and the 13.1 kb Sca I fragment (Figure 2-3). The k14

homologous region appears to be identical to the 1.85 kb Hind III fragment, which is

found in all four species of the A. quadrimaculatus complex. In fact, the restriction

patterns of mtDNAs from the two mosquito genera are very similar in the region of the

IrRNA gene. The three Hind III sites that create the D and E fragments appear to be

conserved as well as the Eco RI site in the Hind III D fragment.














k14


R H A


BS


R HA


C BS


S* *0


-w


0.6 -








Figure 2-3. Total genomic DNA from Anopheles quadrimaculatus digested with various
restriction enzymes and probed separately with the k8 and k14 mtDNA clones from
Aedes albopictus.
Note: Abbreviations for the restriction enzymes are the same as in Figure 2-2.
Positions of the A Hind III marker fragments are noted in the far left column.


k8


23.1 -
9.4 -
.5 -
4.4 -

2.3-
2.0 -


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17

Sequencing of the ends of subcloned A. quadrimaculatus mtDNA


Sequence information was obtained from the ends of 13 plasmid subclones as

well as an internal region of AqAml (the right end of pAYCml) (Table 2-1; Figures 2-4

and 2-5). The sequencing results and the cross-hybridization with the A. albopictus

clones allowed the orientation of the physical restriction map with the D. yakuba genetic

map derived from the complete sequence information (Clary and Wolstenholme, 1985).

Seven out of 13 protein coding sequences, both rRNA coding sequences, 6 out

of 22 tRNA coding sequences, and the A+T rich region were localized and occupied

the same positions as in Drosophila (Figure 2-2). When these results are combined with

the information derived from the A. albopictus clones, it appears that the gene order in

Drosophila and mosquitoes is identical.

In general, there was considerable homology at the DNA level between the

mosquito and Drosophila sequences in coding regions (Table 2-1). Similarity at the

DNA level ranged from 86.8 % for the 5' end of the CO I gene to 71.4 % for the 3'

end of the ND 5 gene. Significant homology is also seen in the predicted amino acid

sequences of the protein coding regions. It should be noted that percent similarity on

the protein level (Table 2-1) was calculated on an absolute basis, without correcting for

conservative substitutions. Therefore, this similarity figure substantially underestimates

the structural similarity of these proteins. The major difference observed in protein

coding sequences of the two organisms is that the A. quadrimaculatus ND 1 gene

apparently terminates 11 amino acids upstream of the D. yakuba ND 1 gene (Figure 2-

41). It is not known if this discrepancy represents a real difference in the protein

product, a cloning artifact, or the use of alternative stop codons in the mosquito.




























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28

The stop codon (TAA) is generated by an insertion (AAT) in the mosquito sequence at

positions 50-48 (Figure 2-41). Most Drosophila mitochondrial protein genes end in a

"T' or a "TA" and the complete stop codon is generated by polyadenylation (Clary and

Wolstenholm, 1985). Sequence for the end of the CO II gene (Figure 2-4f) was

obtained for A. quadrimaculatus. In this case, the stop codon is not completely encoded

in the mtDNA and is apparently created by polyadenylation. Therefore, it would seem

likely that TAA is recognized as a stop codon in the mosquito and the ND 1 gene is, in

fact, truncated.

An ATG, ATT or ATA codon occurs at the beginning of all of the D. yakuba

mitochondrial protein genes except the CO I gene where the tetranucleotide ATAA

appears to serve as the translation initiation codon (Clary and Wolstenholme, 1983).

Limited sequence was obtained from the 5' end of the ATPase 8 gene of A.

quadrimaculatus (Figure 2-4f). In this case, either an ATC triplet (positions 180-182)

functions as the initiation codon or translation begins seven bases downstream at ATA.

In Drosophila melanogaster, the ATC codon appears internally and is not used to initiate

translation (Garesse, 1988).

The tRNA genes are extremely well conserved between A. quadrimaculatus and

Drosophila (Table 2-1; Figures 2-4a, 2-4f and 2-4n). In general, the mosquito tRNAs

are slightly longer than those in Drosophila and have fewer bases separating the genes

(Figure 2-4a).

The A+T-rich region has little or no detectable homology to the Drosophila

sequence, aside from that due to their highly skewed base composition (Table 2-1,

Figure 2-5). When the D. yakuba A+T rich region was randomly shuffled and tested


























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31

for homology to the A. quadrimaculatus A+T rich region, the homology was as good as

that of the unshuffled sequence. The similarity statistics calculated for the best

alignment of the unshuffled Drosophila sequence and clones p3RU1 and p3X1 were

71.3% and 69.7%, respectively. The mean similarities for alignments of 20 randomized

Drosophila sequences were 72.8 2.3% for p3RU1 and 73.0 3.7% for p3X1.

The mtDNAs of D. yakuba and A. quadrimaculatus differ in size by about 0.7

kb (D. yakuba mtDNA is 16 kb and A. quadrimaculatus is 15.3 kb). Since all genes

were mapped to the same locations in both species, the length difference of the

mitochondrial genomes is attributed to variation in length of the A+T rich origin of

replication.


Discussion


Animal mtDNAs are small molecules and should be easy to clone in bacteria.

However, many groups have found it difficult or impossible to clone an entire

mitochondrial genome in one piece (W. Hauswirth, personal communication). Many

DNAs that have been found to be "unclonable" contain segments capable of forming 2

and 3 structures such as cruciforms (due to the presence of inverted repeats) (Erlich,

1989) or Z-DNA (within stretches of alternating purine-pyrimidine residues) (Santella et

al., 1981). Sequencing has shown that the mtDNA of A. quadrimaculatus, like

Drosophila, is extremely A+T-rich (about 20% G+C). Long adjacent stretches of A's

and T's and AT repeats are frequently encountered particulary in the tRNA and rRNA

genes and in the region of the replication origin). It has been demonstrated that

cloning the mitochondrial genome of mosquitoes in bacteriophage can be achieved by








32

cleaving the genome into several pieces. This success may be due to: (1) the

interruption of segments that have a propensity to form non-standard structure; (2) the

fortuitous destruction, by the cloning strategy, of a single inserted gene that may be

lethal to the bacterial host; (3) differences in host/vector systems, or; (4) the separation

of several genes that interact to make the mtDNA unstable or lethal to bacteria. The

three Eco RI sites used in the cloning interrupt the IrRNA gene, the CO II gene, and

the ND 5 gene (Figure 2-2).

In order to subclone the bacteriophage inserts into plasmids, it was necessary to

cut the mtDNA into still smaller fragments. Apparently there are even greater

restraints on DNA inserted into plasmids than on DNA inserted into bacteriophage.

This is probably because bacteriophage are less dependent on the proper working of the

cell, since after replicating they lyse the cell and infect another. Plasmids, which are not

capable of infection, will be sensitive to any sequence that disrupts the long term

functioning of the cell.

Identification of the plasmid subclones containing part of the IrRNA gene was

difficult because they restored the lac* phenotype to recombinant plasmids. The

multiple cloning site of plasmid vector pUC 19 is located within the E. coli B-

galactosidase ct-peptide gene. The multiple cloning site in pUC 19 interrupts the f-

galactosidase gene 18 base pairs downstream from the translation start site of the ta-

peptide. Therefore, insertion of DNA at the multiple cloning site usually results in

interruption of the a-peptide, producing lac- colonies, which can be identified on media

containing the appropriate chromogenic substrate. All subclones having the aberrant







33

phenotype contained the 0.6 kb Eco RI/ Xba I fragment which consisted of a tRNA and

the 5' end of the IrRNA gene (see Figure 2-4n).

Restoration of gene activity occasionally occurs due to the insertion of a

fragment containing a completely open reading frame but sequencing has shown that

such an open reading frame does not exist in subclone p3RX2. Alternatively,

restoration of /-galactosidase activity would be possible if sequences exist within this

stretch of mtDNA that act as a strong promoter in bacteria, contain a transcriptional

start site, and contain an in-frame translation start site.

Bacterial promoters possess two highly conserved sequences. A sextamer

(TTGACA) occurs about 35 bases upstream of the transcription start site. This

sequence is recognized but is not bound by RNA polymerase. The Pribnow box

(TATAAT) is located 5-9 bases upstream from the transcription startpoint and because

of its high A+T content is probably involved with initial melting of DNA into single

strands. The transcription start site is not highly conserved (Lewin, 1985).

There are several sequences in p3RX2 that are similar to the conserved -35

sextamer of bacterial promoters. Two of these sequences occur within three bases of

each other (TTGATA at positions 551-556 and TTGTTA at positions 560-565) (Figure

2-4n). One of the reading frames opens at position 633. The stretch of DNA (about

70 bases) between the "-35-like" sequences and this open reading frame is extremely

A+T-rich. The distance between the conserved sextamer and the transcription initiation

site, however, is about half this length in bacterial promoters (35 bases). A translation

start codon (ATG) is present in this reading frame at positions 650-652. The DNA

sequence obtained for subclone p3RX2, unfortunately, did not include the Eco RI







34

cloning site. Therefore, it is not known if the insert sequence is in frame with the

remainder of the a-peptide sequence contained in the plasmid vector and, as a result,

restoration of J-galactosidase function in this clone remains a mystery.

Even though the A+T-rich region has been successfully cloned in Drosophila

yakuba, several groups have failed to clone the A+T-rich origin of replication and

flanking sequences of D. melanogaster mtDNA either in bacteriophage or in plasmids

(Mason and Bishop, 1980; Garesse, 1988). The 5.6 kb Eco RI fragment containing this

region of A. quadrimaculatus mtDNA was readily cloned in bacteriophage. However,

when initial attempts were made to subclone this fragment into plasmids, a rearranged

subclone was obtained (pAYCm3). This subclone had a 0.7 kb insertion located within

the 2.7 kb Hind III fragment which contains the A+T-rich region. Subsequently, several

additional subclones of this fragment were isolated and all had deletions within this same

region. Stable, unrearranged plasmid subclones (p3X1 and p3RU2) were obtained by

subcloning DNA containing the A+T-rich region as small fragments. Unrearranged

subclones that contained the 4.2 kb Eco RI/ Stu I fragment from AqAm3, however,

were not recovered. Apparently only plasmids having about 1.5 kb or less DNA

flanking the A+T-rich region were stable in bacteria.

The genetic organization of the mtDNA of A. quadrimaculatus is similar to that

of Drosophila. All of the protein coding sequences that have been localized were in the

same positions in both species. Considerable homology is seen between protein coding

regions, though no homology was seen between the A+T-rich origins of replication. Six

tRNAs were identified (tRNALy and tRNA7 from subclone p2R1, tRNA', tRNAG' and

tRNAf' from p3RU1 and tRNAV from clone p3RX2). These tRNA genes were located







35

in the same positions as in Drosophila. In Aedes albopictus two of the six contiguous

tRNA genes located between ND 5 and ND 3 are inverted with respect to the

Drosophila map (Dubin et al., 1986). It is not known if this reshuffling of tRNAs is also

present in A. quadrimaculatus, since sequence information has not yet been obtained

from this region.

The cloning of the complete mtDNA genome from a mosquito should greatly

improve the accuracy of mtDNA restriction analysis. This powerful tool is now being

widely used to study the population genetics of wild mosquitoes, and with the use of

mosquito mtDNA as a probe, can be used to study the mtDNA of individual mosquitoes

(Cockburn and Seawright, 1988). The A. quadrimaculatus species complex consists of at

least four sibling species: species A, species B (Lanzaro et al., 1988; Kaiser et al.,

1988a), species C (Kaiser et al., 1988b; Narang et al., 1989a), and species D (Narang et

al. 1989b). The restriction profiles of the mtDNA of the four species of this complex

have been determined, and this information will be used to estimate the degree of

diversity between different populations and to find diagnostic restriction sites for

identifying individual insects.













CHAPTER III

MITOCHONDRIAL AND RIBOSOMAL DNA VARIATION AMONG MEMBERS
OF THE Anopheles quadrimaculatus SPECIES COMPLEX


Introduction


The use of restriction endonucleases to measure mitochondrial DNA (mtDNA)

sequence relatedness in natural populations has, over the past few years, become an

important tool for population biologists. Genetic variation in natural populations has

traditionally been studied by electrophoretic analysis of the protein products of nuclear

genes. Identification of enzyme variants (isozymes) by electrophoresis has proven to be

a valuable and versatile technique but this method is limited by certain fundamental

characteristics of the nuclear genome. Since the nuclear genome is highly recombinant

and gene flow between populations (via migration or dispersal) can lead to genetic

homogenization, the reconstruction of historical relationships within a species becomes

difficult and subtle population structuring can be missed (Avise et al, 1979; Saunders et

al., 1986). Animal mitochondrial DNA, on the other hand, is maternally inherited,

haploid, and nonrecombinant (Dawid, 1972; Hutchinson et al., 1974; Lansman et al.,

1983; Gyllensten et al., 1985). Therefore, the mitochondrial genotype (as determined by

restriction site maps) can be used as a marker to trace maternal lineages and the genetic

relationships among individuals can be established. Also, mtDNA analysis may be a

more sensitive way to detect population structure than the traditional electrophoretic








37

analysis of nuclear enzyme-coding loci since mtDNA is less susceptible to interpopulation

genetic homogenization due to gene flow and more susceptible to population bottlenecks

because of its maternal mode of transmission and haploid copy number (Birky et al.

1983; Wilson et al., 1985).

Until recently, the mosquito, Anopheles quadrimaculatus (Say), which is widely

distributed over the eastern United States, was considered to be a single species.

Results of hybridization, cytogenetic and electrophoretic studies, however, have lead to

the discovery of at least four sympatric sibling forms, species A, B (Lanzaro 1986;

Lanzaro et al., 1988; Kaiser et al., 1988a), C (Kaiser et al., 1988b, Narang et al., 1989a),

and D (Narang et al., 1989b). Over the past three years, extensive electrophoretic data

have been collected for 147 natural populations of A. quadrimaculatus. The populations

studied were located primarily in the southeastern United States. Diagnostic allozyme

loci were described for each sibling species and electrophoretic keys have been published

(Narang et al., 1989a,b).

This study reports the results of the analysis of mtDNA restriction site

polymorphisms in seven mixed populations (i.e. more than one sibling form present) of

A. quadrimaculatus. The primary objective of this study was to investigate the feasibility

of using mtDNA and ribosomal DNA (rDNA) restriction patterns for the identification

of individuals and species of mosquitoes. Since the rDNA profiles in conjunction with

isozyme analysis provided markers for the nuclear genomes of individual mosquitoes,

interspecific hybridizations and introgression of mtDNA between species could

potentially be detected. In addition, the mtDNA restriction site data were used to

evaluate the genetic relationships among the four sibling species of the A.

quadrimaculatus complex.







38

Materials and Methods


Collection sites


Adult A. quadrimaculatus from seven natural populations (four from Florida,

one from Alabama and two from Mississippi) (Table 3-1), were collected by vacuum

aspiration from tree holes or artificial resting stations. The taxonomic key of Darsie and

Ward (1981) was used to identify the adult mosquitoes. All confirmed specimens were

frozen in liquid nitrogen for transport back to the laboratory where they were stored

at -80 C until analyzed.


Isozyme analysis


Frozen mosquitoes were placed on ice and decapitated. Horizontal starch gel

electrophoresis was performed on protein homogenates obtained from the head of each

mosquito. Samples from 50 individuals were analyzed for each of the seven populations

surveyed. Electrophoresis was done according to Steiner and Joslyn (1979) with minor

modifications as described by Narang et al. (1989a). Gels were stained for enzymes that

were known to be diagnostic for the four sibling species of the A. quadrimaculatus

complex (Lanzaro, 1986; Narang et al., 1989a,b). A single locus or combinations of loci

have been considered to be diagnostic if the probability of correct assignment of a

mosquito to a species was >99% (Ayala and Powell, 1972). For most populations, two

loci each of isocitrate dehydrogenase (Idh-1 and Idh-2), hydroxy acid dehydrogenase

(Had-1 and Had-3) and glutamate oxaloacetate transaminase (Got-1 and Got-2) were

scored as well as one locus for phosphoglucose isomerase (Pgi-1) and malic enzyme

(Me-1) (Table 3-1).


























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DNA Analysis


Total genomic DNA was extracted from the remainder of each decapitated

mosquito by the procedure of Cockburn and Seawright (1988). The individual DNA

preparations were stored as ethanol precipitates at -200 C. Nucleic acid precipitates

were recovered by centrifugation for 5 min. in a microfuge. The pellets were washed

twice with 70% ethanol, air dried and redissolved in 25 gl TE (10 mM tris HCI, 1 mM

EDTA, pH 8.0) containing preboiled RNase A at a concentration of 1 /Ig/ml. After

incubation at room temperature for 30 min, restriction enzyme digestions were

performed according to protocols provided by the manufacturer (Bethesda Research

Laboratories). The total genomic DNA preparations from individual mosquitoes were

divided into five portions. Each of four aliquots was cleaved separately with a different

restriction enzyme ( Ava I, Hind III, Pst I or Pvu II). Restriction enzyme digestions

were done at 370 C for 2 hr with a ten-fold excess of enzyme. The remaining one-fifth

of the mosquito DNA preparation was retained in case of failure of one of the cleavage

reactions or for further analysis. The digested DNA samples were electrophoresed in

0.8% agarose gels and Southern blots were prepared according to established procedure

(Berger and Kimmel, 1987).

Southern blots were first hybridized to a mixture of 32P-radiolabeled mtDNA

clones (plRN1, plRN2, p2R1, p3RU1 and p3RU2). These plasmid clones, described in

Chapter II, comprise the entire mitochondrial genome of A. quadrimaculatus species A.

Nick translations were done with purchased reagent kits and according to the

manufacturer's instructions (Bethesda Research Laboratories). Nitrocellulose filters were







41

blocked and hybridizations were performed as in Cockburn and Seawright (1988).

Autoradiography was a standard technique (Berger and Kimmel, 1987).

Nitrocellulose filters were stripped of bound mtDNA probe by brief immersion

in boiling water followed by several washes in warm water. The filters were blocked, as

before, and rehybridized to an rDNA clone, QB1, from A. quadrimaculatus, species B.

The QB1 clone was isolated from a total genomic DNA library in bacteriophage A

vector EMBL3A (Cockburn and Mitchell, 1989). This mosquito clone was homologous

to pBC2, a pBR322 clone that contained the entire 8.4 kb rDNA repeat unit from the

fungus gnat, Sciara coprophila (Renkawitz and Gerbi, 1979). Since the QB1 clone has

not been extensively characterized, whether or not it includes the entire rDNA cistron

of A. quadrimaculatus species B is unknown.


Restriction Site Mapping of the Mitochondrial Genomes


Restriction sites for Ava I, Hind III, Pst I and Pvu II were mapped for the

mtDNA of one isofemale line of each of the four sibling species. The cleavage-site

maps were derived for the most common restriction patterns of each species and the

positions of rare sites were deduced from these maps.

Total genomic DNA was isolated as described above and complete single and

double digests were done for all combinations of the four restriction enzymes listed

above. The cleavage reactions were electrophoresed in 0.8 % agarose gels and

Southern blots were made and hybridized to the mtDNA plasmid clones as previously

described.











Cleavage-site mapping was based on the analysis of fragment sizes estimated

from the electrophoretic patterns obtained for the single and double digests. The Hind

III fragments of phage X DNA were used as size standards and a Lotus program

(FRAGMENT) was used to calculate fragment sizes based on the distance migrated.

The FRAGMENT program was obtained from M. Q. Benedict (USDA-ARS, Insects

Affecting Man and Animals Research Laboratory).


Estimation of Genetic Distance


Sequence divergence, expressed as the frequency of nucleotide substitutions (6),

was estimated for the four sibling species of A. quadrimaculatus from the number of

sites mapped and the number of sites shared (S) in the genomes according to formulae

(8) and (10) of Nei and Li (1979). Sample variances of S and 6 were estimated by

formulae (19) and (20) of Nei and Tajima (1981). Pairwise comparisons were made for

all haplotypes. Interspecific distance calculations for each enzyme class were combined

after weighting by haplotypic frequency and the proportion of the total number of

observed restriction sites contributed by each group of enzymes across all species.

Genetic distances were used to construct an unrooted phylogenetic tree based on the

neighbor-joining method of Saitou and Nei (1987). Tree construction was accomplished

with the aid of a FORTRAN program that was kindly furnished by Dr. Masatoshi Nei

(University of Texas).







43

Results


Species-Specific Restriction Site Polymorphisms

Mitochondrial DNA analysis

Complete isozyme and mtDNA restriction data were obtained for 288 individual

mosquitoes out of a total of 350 analyzed. Comparable sample sizes were obtained for

all populations except Pickwick Reservoir (PIC). Suitable isozyme and restriction data

were obtained from only 40% (20/50) of the individuals analyzed from Pickwick and the

poor quality of these results was attributed to sample degradation.

The mitochondrial DNA restriction patterns for each enzyme were designated

by Arabic numbers (e.g. Ava-1, Ava-2 etc.). These designations indicated the order in

which the various polymorphic restriction patterns were noted on autoradiograms. For

all species combined, 7 different patterns were observed for Ava I, 10 for Hind III, 6 for

Pst I and 4 for Pvu II. The sizes of the fragments observed or inferred in these

alternative restriction patterns are shown in Table 3-2 and pattern distribution by species

and population is presented in Table 3-3. Restriction site maps for each sibling species

are included in Figure 3-1. In these maps, variable restriction sites for each enzyme

were denoted by lower case letters (eg. Ava-a, Ava-b, etc.).

All four of the restriction enzymes used in this study produced one major

electrophoretic pattern for each species with minor variants occurring at frequencies that

ranged from 1-20% (Table 3-3). For Ava I, a three-banded pattern (Ava-2) was most

common in species A and C while a two-banded variant (Ava-1) was predominant in

both species B and D (Figures 3-2a and 3-3a). Minor variants were shared in about 1%

of species A and B (Ava-3), and species A and C (Ava-5). One species C individual










Table 3-2. Fragment sizes observed in alternative mtDNA restriction patterns
of Anopheles quadrimaculatus sibling species.




Enzyme Variant Fragment sizes (kb)

Ava I 1 12.1; 3.3
2 9.4; 3.3; 2.7
3 9.4; 3.3; (1.35)a; (1.35)
4 9.4; 3.1; 2.7; 0.3
5 9.4; 6.0
6 12.7; 2.7
7 15.4

Hind III 1 6.3; 2.7; 2.0; 1.9; 1.7; 0.5; 0.3
2 6.3; 2.7; 2.3; 1.9; 1.7; 0.5
3 6.3; 2.7; 3.6; 1.9; 1.7; 0.5; 0.3
4 4.4; 2.7; 2.0; 1.9; 1.7; 0.5; 0.3
5 3.4; (2.7); (2.7); 2.3; 1.9; 1.7; 0.5
6 3.9; 3.4; (2.7); (2.7); 1.9; 0.5
7 5.6; (2.7); (2.7); 1.9; 1.7; 0.5
8 6.3; 3.9; 2.7; 1.9; 0.5
9 (2.7); (2.7); (2.7); 2.2; 1.9; 1.7; 0.9; 0.5
10 (2.7); (2.7); (1.9); (1.9); (1.7); (1.7); 0.5

Pst I 1 7.65; 4.35; 3.3
2 15.4
3 (7.65); (7.65)
4 10.95; 4.35
5 10.3; 5.0
6 6.95; 4.35; 3.3; 0.7

Pvu II 1 11.8; 3.5
2 6.2; 5.6; 3.5
3 9.7; 5.6
4 15.4


"Inferred fragments are enclosed in parentheses.









45

Table 3-3. Number of individual mosquitoes observed for alternative restriction patterns for
four enzymes in natural populations of Anopheles quadrimaculatus sibling species.



SPECIES BY POPULATION

Enzyme
and LOC DLK WHR NOX
Variant A B A B A B A B D


Ava I
1...
2...
3...
4...
5...
6...
7...


Hind III
1...
2...
3...
4...
5...
6...
7...
8...
9...
10...

Pst I
1...
2...
3...

4...
5...
6...

PvuII
1...
2...
3...
4...


35 1






1


33

2


2 23
4
16 1


20
6
4 1


7 37 26 12 27 21 9 36












Table 3-3. ---- continued.


SPECIES BY POPULATION


PIC BBS LCK TOTAL
A B D A C A B C A B C D


Ava I
1...
2... 2
3...
4...
5...
6...
7...

Hind III
1...
2... 2


1 20


1
1 44 6


1 1
1


28 80
1


106 1
72


72 20
1
2


39
1 1 1
5
19 2


1 7 2


26 85 65
63 2 1
2 14 7
4 22 2
1


79 109


Pst I
1...
2... 2
3...
4...
5...




1... 2
2...
3...
4...

















Uh








U -~



.o,



I -,

E U


o~
-t U
Y








o o ~

U Ua









a' II .m







-.t



U2 -:

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E
U .-

o 0U
.t ZiL














I -

I -










I


> __-
I





0 --
o nI --




S-



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I


*0- ---A


*1


*-(- -- a


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oI -








SI -








a- --
I
0 m















--






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-0



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--4



- <
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49

from the Bear Bay Swamp population had the Ava-1 pattern which was prevalent in

species B and D (Table 3-3).

Approximately 95% of species C and D and one species A individual from the

Lake Octahatchee population had the Hind-5 mtDNA pattern (Figures 3-2b and 3-3b).

The majority (97.6%) of species A mosquitoes, however, possessed a pattern that was

unique to that species (Hind-2). All of the species B variants observed were unique to

that species because of the presence of the diagnostic Hind-e site which cleaves the 2.3

kb Hind III fragment common to the other 3 species (Figure 3-1).

Slight variation in the mobility of the Hind III fragment containing the A+T-

rich replication origin was noted in a few of the species A and species B mosquitoes

analyzed. The size of this DNA fragment was usually 2.7 kb in all four sibling species.

The species B individual in Figure 3-2b (third lane), however, had a 2.9 kb Hind III

fragment. The length variation observed for this fragment was typically on the order of

a few hundred base pairs. Because of the weak hybridization to the A+T-rich region

and the small differences in mobility between these size variants, all individuals were not

classified with regard to this length polymorphism. This variation was also not included

when the maps in Figure 3-1 were developed.

It should be noted that the 0.5 kb Hind III fragment common to all of the A.

quadrimaculatus siblings and the 0.3 kb Hind III fragment present in species B were

observed on autoradiograms only if larger quantities of total DNA were loaded (at least

0.5 pg, which is about one-half of one individual mosquito DNA preparation) and

exposure times were lengthened (Figure 3-2c).

























Figure 3-2. Variable mitochondrial DNA restriction patterns for Anopheles
quadrimaculatus sibling species.

(a) Ava I mtDNA restriction patterns; (b) Hind III mtDNA restriction patterns; (c)
Overexposure of autoradiogram shown in panel (b). Upper arrow marks the position of
the 0.5 kb Hind III fragment common to all four sibling species. Lower arrow denotes
the 0.3 kb Hind III fragment present in species B; (d) Pst I mtDNA restriction patterns;
(e) Pvu II mtDNA restriction patterns; (f) Cla I mtDNA restriction patterns.

Note: S=Hind III fragments of bacteriophage X. Lanes containing sibling species were
loaded in the same order from left to right for all panels. A=species A from the
ORLANDO laboratory strain; A=species A from Lake Panasofkee, Florida; B=species
B from Wheeler Reservoir; C=species C from Bear Bay Swamp; D=species D from
Pickwick Reservoir. Pattern identification numbers are listed beneath each species
designation.









Aw I Hind I
SA A C D A A C D S
2 2 1 2 1 2 2 1 9 5


23.1
9.4
6.5
4.4


kIb


Pt I Pvu II
S A ABC D A A I C D
S 1 1 4 1 1 1 3


Cla I
S A A C D


23.1
9A.
4.4 'VI,

2.3
2.0





0.6-


i)


23.1
6.5
* 4A

23
2.0





-0;6


0.6-


23.1
9.4
6.51
4.4 -


0.6-

a


'14


























Figure 3-3. Representative mitochondrial DNA restriction patterns for field-collected
samples of Anopheles quadrimaculatus sibling species.


(a) Ava I restriction patterns; The arrow on the bottom panel indicates that the aberrant
migration of the DNA fragments in this lane is due to distortion of the gel during the
Southern blotting procedure and not to differences in molecular weight. (b) Hind III
restriction patterns; (c) Pst I restriction patterns; (d) Pvu II restriction patterns.

Note: Letters above the lanes indicate the various species identifications made on the
basis of diagnostic isozymes and representative restriction patterns. S=Hind III
fragments of bacteriophage A. Pattern identification numbers are listed below each
species designation.













CC CCCC CC CA CCSCCCCC
aI;p ia a 22 23 2 52


I:2


w *
CCCC AASIA IBAA IAA
ll2 2L &1 I


V

1..


CCCCCC CCC ACC S
A3 1 ( 1 4 1I 2

Sf4 ;4-


CCCC CCSC C ACC
5s Ija a 7 25 ,


CCCCCC
SB5 7 5


1Wi


CCCC AAS A &A
111s 221s 2 11


RD a


A AA
2332


-e9


cccc C SCCCACCS ccCccc
3 3 333 3 3 34 3 1 3 33 3 3 33


"I


89AA
I I u







54

The Pst I cleavage patterns were not useful for species identification. The

majority of species B (78%) and species C (85.5%) had the Pst-1 pattern (Figures 3-2d

and 3-3c). The most common variant in species A was Pst-2 which was also present

infrequently in the other sibling forms. Species D had the Pst-4 restriction pattern

which, again, was represented in all the other siblings. It is of interest that this pattern

(Pst-4) occurred in 43% of the species B mosquitoes collected in Florida but was absent

in species B from the Alabama (WHR) and Mississippi (NOX and PIC) populations.

Most of species A (96%) and all the species B mosquitoes examined possessed

the same Pvu H restriction pattern (Pvu-1) (Figures 3-2e and 3-3d). Species D had a

unique pattern (Pvu-2). Ninety-seven percent of all species C also had a distinctive

pattern (Pvu-3). There were a few species C individuals, however, that shared a minor

pattern (Pvu-4) with species A from the Dead Lake population.

None of these four restriction enzymes, when used alone, was capable of

discriminating all four sibling species on the basis of their mtDNA cleavage patterns. If

data generated by any pair of the three enzymes Ava I, Hind III or Pvu II were

combined, however, all siblings could be distinguished. For example, Hind III yielded

distinctive patterns for species A and B but not for C and D. The latter 2 species could

be distinguished by their unique Pvu II patterns, however. Pst I had little diagnostic

value.

The four restriction enzymes used in this study were selected on the basis of

preliminary data which indicated that they produced variable patterns among the four

sibling species for both the mitochondrial and ribosomal genomes. Even though none of

the restriction enzymes used in this population survey produced diagnostic mtDNA







55

patterns for all of the sibling species, other enzymes such as Cla I (Figure 3-2f) had this

capability.


Ribosomal DNA analysis


Most of the restriction enzyme digestions that were hybridized to the rDNA

probe produced smears of high molecular weight bands along with discrete, smaller

DNAs. The indistinct nature of these bands was attributed to length variation between

individual copies of the rDNA (probably in the non-transcribed spacer region) and not

to poor gel resolution or Southern transfer, since mtDNA bands as large or larger than

these were quite sharp.

Species specific rDNA patterns were obtained with Ava I, Hind III and Pvu II

(Figures 3-4 and 3-5). For Ava I, species A did not have a 2.4 kb fragment that was

present in species B (Figure 3-4c). Both species C and D possessed a slightly faster

migrating fragment (about 2.3 kb) but species D lacked a 0.7 kb fragment common to

the other siblings (Figure 3-4a-c). Species C had a diagnostic 4.8 kb fragment (Figure

3-4b).

The Hind III digests also yielded diagnostic rDNA patterns (Figure 3-4d-f).

Diagnostic fragments of 1.2 kb in species A, 0.9 kb in species B (Figure 3-4f) and 1.0 kb

in species D were obtained (Figure 3-4d). The species C pattern was three-banded with

smaller fragments of 1.5 and 1.6 kb (Figure 3-4e). Species A and B also shared a 0.2

kb repeat which was absent in both species C and D (Figure 3-4d and e).















C4






C,4



L 2o


boo
a OO


ba: E CL
o u
0-4-PC


















M 0 Z


U 09
Eu E


bo 0u m-Cu
C,,z 0
~;) .-.






- -
~
i~
~s
~P
-a ~ Jr!
3 L nv
A:ap I



C u


e E '0U2




E -pc E.


o ~ P
i; 1~
of ~ cc
Cum -
gUu
*,U 0l.9.
cA

*a '* -
Uo~ .o~
L40C --CuE









0


0
a
a
a
* Cd


aI
a
*

a

a
aD


3, (I
'U.',


-. (.

@13


em
S.
em


B.
mmC
emI


S0


0N
a
a
a
a
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a
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td


'U
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2 a
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I- T
43
C.)

4-1


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0
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60

The Pst I rDNA digestion patterns were not particularly illuminating. All four

species shared a 3.8 kb fragment (Figure 3-5 a-c).

Restriction patterns for Pvu I were specific for species A and C but did not

distinguish between species B and D. The latter 2 species contained multiple fragments

in the 20-10 kb size range (Figure 3-5d). The species A pattern consisted of a fairly

discrete 6.5 kb band (Figure 3-5f) and species C had three fragments (15, 5.6 and 4.2

kb) (Figure 3-5e).


Comparison of Isozyme and DNA Analyses


Species identifications made on the basis of electrophoretic analysis of isozymes

and DNA restriction fragment patterns agreed for 99% of the mosquitoes evaluated.

Three individuals out of a total of 288 were misidentified by the isozyme procedure.

Two mosquitoes, one from Wheeler Reservoir and one from Noxubee Wild

Life Refuge, were identified as species A based on isozymes but these individuals had

the mtDNA genotype of species B. Since only the heads of individual mosquitoes were

available for protein analysis, a minimal number of diagnostic enzyme loci were

examined. Species A and B can be differentiated by using a combination of 2 enzyme

loci (Idh-1 and Idh-2) (Lanzaro, 1986). Initially, these were the only two loci employed

for identification in populations believed to consist entirely of species A and B. The

two mosquitoes in question were heterozygous at the Idh-1 locus (Idh-ll'/Idh-1) and

homozygous for Idh-2"7. Species A is polymorphic at the Idh-1 locus. In this species,

the Idh-11 allele is the most frequent electromorph (allele frequency = 0.82) but







61

Idh-1s occurs in about 16% of species A examined from natural populations. Species B,

on the other hand, was believed to be fixed for Idh-1s (Lanzaro et al., 1990). The

Idh-2'~ allele for these mosquitoes was diagnostic for species B (allele frequency = 1.0)

but, again, species A is polymorphic at this locus and homozygotes for Idh-217 have been

encountered (Lanzaro et al., 1990).

Identification of these specimens as species A was primarily indicated by the

presence of the Idh-110 electromorph. Since the mitochondrial genotype of both

mosquitoes was typical of species B, the possibility of interspecific hybridization was

considered. Results from the rDNA analysis, however, clearly indicated that these

mosquitoes were species B. The rDNA patterns for these individuals are shown in

Figure 3-4 panels (b) and (e) (eleventh lane) and Figure 3-4 panel (f) (first lane). It is

apparent, therefore, that species B also exhibits a low degree of polymorphism at the

Idh-1 locus.

Another individual, also from Noxubee, was identified as species A on the basis

of isozyme analysis but had the mtDNA and rDNA restriction patterns typical of species

D. Species D, like species A, is polymorphic at Idh-1 and Idh-2 so these loci are

incapable of distinguishing these two species (Narang et al., 1989b). Since the Noxubee

population was believed to contain only species A and B, diagnostic loci for the other

sibling species (such as Had-3 and Pgi-1 for species C and ME-1 and Got-2 for species

D) were not evaluated.







62

Analysis of Mitochondrial DNA Haplotypes


The simplest measure of polymorphism for a particular segment of DNA

nucleonn) is the number of haplotypes (k) observed in the sample (Nei, 1987). This

number is dependent on sample size so comparable sample sizes must be used when

evaluating the extent of polymorphism present in different populations or species. For

the sibling species of the A. quadrimaculatus complex, a total of 33 haplotypes were

observed for all species combined. Nine haplotypes were found in species A and eight

in species B, so these species exhibited about the same amount of polymorphism in their

mitochondrial genomes (Table 3-4). Species C was the most polymorphic with a total of

13 haplotypes observed from 2 populations. It was interesting that in the species C

populations studied (BBS and LCK), the majority of rare haplotypes were not shared

between these populations although they were located only 15 miles apart (Table 3-4).

Three haplotypes were noted for species D. The small sample size obtained for this

species, however, invalidates any direct comparison to the other three species.

For each species, the mtDNA haplotypes were connected in a "most

parsimonious" network (Figure 3-6) which gives the minimum number of mutational

steps required to connect all the morphs. The network represents a possible

evolutionary pathway for the mtDNA of each species of the A. quadrimaculatus

complex. These networks, however, do not have directionality and, therefore, do not

reveal which one is the most ancestral haplotype. In similar studies, it has been

assumed that haplotypes composed exclusively of the major patterns of each enzyme and

having the most widespread distribution are the oldest extant haplotypes and are

ancestral to the others (Hale and Singh, 1987). For the A. quadrimaculatus species









Distribution of mtDNA haplotpes in natural populations of Anopheles


quadrimaculatus sibling species.

POPULATION

Haplotype' LOC DLK BBS LCK WHR PIC NOX TOTAL


2 8


21 1 32
2
1


Total


44 41 48 38 48 23 46


288


' Haplotypes are designated according to species (Al denotes the major haplotype for
species a; B1 for species B, etc.) and by the restriction patterns for Ava 1, Hind III, Pst
I and Pvu II, in that order (numbers in parenthesis separated by commas).


1 4
2


Al
A2
A3
A4
A5
A6
A7
A8
A9
B1
B2
B3
B4
B5
B6
B7
B8
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
Cll
C12
C13
D1
D2
D3


(2,2,2,1)
(2,2,3,1)
(2,2,4,1)
(3,2,2,1)
(2,8,2,1)
(6,2,2,1)
(2,2,5,1)
(2,2,2,4)
(2,5,2,1)
(1,,11,1)
(1,3,1,1)
(1,4,1,1)
(1,1,4,1)
(3,,1,11)
(1,3,4,1)
(1,1,2,1)
(7,1,11)
(2,5,1,3)
(2,5,3,3)
(2,5,6,3)
(4,5,1,3)
(2,5,1,4)
(2,6,1,3)
(1,5,4,3)
(2,5,4,3)
(2,7,1,4)
(5,5,1,3)
(2,7,1,3)
(6,5,2,3)
(2,9,1,3)
(1,5,4,2)
(1,10,4,2)
(1,5,2,2)


Table 3-4.

























W
.0












0

0.





0
U,












u0
a











.0
.0




Cu





YLu



U,x
0








ioE
UO



aCu


oo~




0.

Cl.






Lu
o w Cu u
~boUo

0- 0
LU











00
0 0
o czc










-0
o ~fa, 0d~i













cc 0 wi
0















Ut 00.
z c
B 0c



o o



u u
'4-
Z5 .n1 o..a C












65








1-



cul
oo o




:, W7v
N/~l
V

h0 N

0 NI .t -





9









0) 1
CO
mlli






5%r



L) vi 0
OS -?






lz 03
0 -























ot
0"0





N









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t n
co n



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r-e
5% 5%
U sI
U) U)
0)s 0







66

complex, the most common haplotypes have been designated as Al, B1, C1 and D1 for

each of the respective sibling species. It is evident from Figure 3-6 that the central

positions of these major haplotypes make it necessary to pass through them in order to

go from any one pattern to all the others within each species.

Examination of some minor restriction site variants shared between different

species, however, did not support the notion that the most common haplotype is always

indicative of the ancestral lineage of a species. For example, the Ava-3 pattern was

present in 1% of the species A and B individuals analyzed. This pattern was created by

the presence of one extra site (Ava-d) in species A (haplotype A4) and two sites (Ava-e

and Ava-d) in species B (haplotype B5) (Table 3-5). It is parsimonious to assume that a

restriction site variant does not arise independently more than once and that the

presence of a restriction site is more informative than the absence of a site (because the

presence of a site is determined by a sequence consisting of multiple bases but mutation

in any one of the bases of the recognition sequence results in the loss of the site).

Therefore, the Ava-3 pattern present in haplotypes A4 and B5 is most likely to be

ancestral for species A and B. Haplotypic frequency, therefore, may merely reflect

genotype fitness under current selective pressures or random drift and not the ancestral

character state of a species.



Genetic Distance Based on mtDNA Restriction Site Data


Distance calculations for pairwise comparisons for all haplotypes observed in

each of the four mosquito species are presented in Table 3-6. Since two classes of







67

Table 3-5. Presence/absence of varied restriction sites in mtDNA haplotypes of Anopheles
quadrimaculatus sibling species.

Enzyme
Ava I Hind III Pst I Pvu II
Haplotype" abcde abcdef abcd ab

Al (2,2,2,1) + +- + ---+ + -- +- +
A2 (2,2,3,1) +- +-+ ---+-+ +- +- +
A3 (2,2,4,1) +-+- + ---+-+ + +- +
A4 (3,2,2,1) + + + ---+-+ -- +- +
A5 (2,8,2,1) +-+-+ ---+-- --+- +
A6 (6,2,2,1) --+-+ --- +-+ --+- +
A7 (2,2,5,1) +-+-+ ---+-+ -+-+ +
A8 (2,2,2,4) +- +-+ ---+-+ -- +- --
A9 (2,5,2,1) +- +-+ +--+- + -+- +
B1 (1,1,1,1) +-+-- ---+++ + + +- +
B2 (1,3,1,1) +-+-- ---++- + + +- -+
B3 (1,4,1,1) +-+- --+ ++ + + +- -+
B4 (1,1,4,1) +-+-- ---+++ -++- -+
B5 (3,1,1,1) +-+++ +++ + + +- -+
B6 (1,3,4,1) +- + -- ---++- + +- +
B7 (1,1,2,1) +-+-- ---+++ --+- -+
B8 (7,1,1,1) +---- ---+++ +++- -+
C1 (2,5,1,3) +-+-+ +--+-+ +++- +-
C2 (2,5,3,3) +-+-+ +--+-+ +- +- +-
C3 (2,5,6,3) +- +- + + + + + -+ + + + +-
C4 (4,5,1,3) + + +-+ +--+-+ + + +- +-
C5 (2,5,1,4) +- +- + +--+-+ +++- --
C6 (2,6,1,3) +-+-+ +--+-- +++- +-
C7 (1,5,4,3) +- + -- +--+-+ -++- +-
C8 (2,5,4,3) +-+-+ +--+-+ -++- +-
C9 (2,7,1,4) +-+-+ +----+ + + + --
C10 (5,5,1,3) +--- + +--+-+ + + +- +-
C11 (2,7,1,3) +-+-+ +---+ + + + +-
C12 (6,5,2,3) --+-+ +--+-+ --+- +-
C13 (2,9,1,3) +-+-+ ++-+-+ +++- +-
D1 (1,5,4,2) +-+-- +--+-+ -++- ++
D2 (1,10,4,2) +- +-- +- + +-+ + +- ++
D3 (1,5,2,2) +-+-- +--+-+ --+- ++

Haplotypes are designated according to species (Al denotes the major haplotype for
species A; B1 for species B, etc.) and by the restriction patterns for Ava 1, Hind III, Pst I
and Pvu II, in that order (numbers in parenthesis separated by commas).

Note: A plus sign (+) denotes presence of the site; a minus sign (-) denotes absence of
the site.









00





1dde 4ddddddd ddd ddddddd


8 Ml;iiH@illlil S lsEsHESWlll j I

6dddddddddd6ddddddddadddd






6oo ddeddddd dddddddeddi c


ddcddddddd ca
gIga 4i
d' d i ae e wd d d"'ne -. d d 'no S J cc d C
a assSesssssll Jist i ss F











|d 6aaH88 d d
djdededd 'dd e d d i ise
ddddddddededd dedd%18
dd edddd eddd cs




ee dd44444 dddd ddd
- Yt38t60fl O c-c. edc, ci00t-,0. r- tfltflScn ici r., -












8 d d d C; dt d d 6S













S S eieiedeii degsieisisi ieed i g
dd~ ddedddd ddddddd ddd


















d | dc 66dr eicii i 11 II 5 8 d
ddd saddddd d 053dddd QSddddded a dddd
'deddde'dededdn d dc 000


~;"E~18~~~~ 8~~~S8~64Cu



'n'n-c'n'n~a dd 4)d



~~333'ais~33-
dcjsi~..e C
"~6OB ~~na~-~
Cua~~~g~~~~ 42
4-dd ddiia~ ddddd CU
ECu'

*0 Cu-ddddddd d

u~o as
ddd~a- Cudd
-Je~ s B
Cud YIQP~v~iU--,-
H O6~~~U3~ uooo~







69

restriction enzymes were used (Hind III, Pst I and Pvu II each recognize one unique six-

base sequence while Ava I recognizes four different six-base sequences), distances were

reported separately for each enzyme class.

Pooled distance calculations based on all mtDNA restriction site variants

observed within each species for both enzyme classes are reported in Table 3-7.

Distance values are presented for each separate enzyme class as well as combined

weighted distances for both classes. The pooled distance values were weighted by the

proportion of the total number of sites contributed by each enzyme class across all

species (1:5 weighting).

The genetic distances calculated from restriction site data indicated that A.

quadrimaculatus species A is more closely related to species B than to species C or D.

Species C and D also cluster and an unrooted tree based on these data is presented in

Figure 3-7.


Discussion


The data presented in this study confirm that restriction site polymorphisms

unique to the mitochondrial and nuclear (rDNA) genomes of members of the A.

quadrimaculatus complex can be used for identification of individual mosquitoes to

species. The DNA-based techniques were slightly more labor-intensive than isozyme

analysis but the molecular methods proved to be more reliable for correct species

assignment than protein electrophoresis, especially when a small number of diagnostic

isozymes was evaluated.













Table 3-7. Pooled interspecific genetic distances for all haplotypes and including both
restriction enzyme classes.


Weight" Genetic distance (6)
A:HPV Species A B C D


A 0.00485
(0.00080)b
B

C

D


A 0.00171
(0.00030)


A 0.00547
(0.00090)


0.02709
(0.00470)
0.00352
(0.00059)


0.04484
(0.00799)
0.00413
(0.00075)


0.02354
(0.00405)
0.00340
(0.00056)


0.03574
(0.00652)
0.03682
(0.00661)
0.00347
(0.00058)



0.00290
(0.00051)
0.04554
(0.00820)
0.00407
(0.00071)



0.04231
(0.00773)
0.03508
(0.00629)
0.00335
(0.00055)


0.02993
(0.00521)
0.02548
(0.00449)
0.02117
(0.00364)
0.00108
(0.00018)

0.04336
(0.00760)
0.00209
(0.00038)
0.04409
(0.00783)
0.00000
(0.00000)

0.02725
(0.00474)
0.03016
(0.00531)
0.01659
(0.00280)
0.00129
(0.00021)


" Weighting factor for Ava I (A) and for Hind III, Pst I and Pvu II combined (HPV).

b Variance associated with the estimation of 6 is given in parentheses.




















species A


species C


14.5


15.0


12.5 species D
species B









Figure 3-7. Unrooted phylogenetic tree for the four sibling species of the Anopheles
quadrimaculatus complex.

Note: This tree was based on pooled distance values derived from restriction site
analysis of the mitochondrial genomes of all sibling species. Branch lengths denote the
number of substitutions per kilobase pair.







72

The four siblings species of the A. quadrimaculatus complex are sympatric and in

some populations adults of all species can be collected from the same tree hole. There

has been no genetic evidence (from extensive cytological studies or from surveys

designed to detect hybrid males based on abnormal testes morphology) for interspecific

hybridization between species A and B in the wild (Lanzaro, 1986). One presumed A/C

hybrid, however, has been detected by DNA hybridization to cloned species-specific

repetitive sequences (Cockburn, 1990). If interspecific hybridization occurs between

species A and C, it does so at an extremely low level since the supposed hybrid

individual comprised only 0.3% of the mosquitoes analyzed (Cockburn, 1990).

The present study represents the first attempt at a systematic correlation of the

cytoplasmic and nuclear genomes of individual mosquitoes from mixed populations of A.

quadrimaculatus sibling species. The fact that DNA analysis found no evidence for

interspecific hybridization confirms previous observations and indicates that strong

prezygotic isolating mechanisms must be operating to preclude matings among the

various sibling forms in their natural environment.

Recently, it has been noted that heteroplasmy, in which two mtDNA variants

occur abundantly in an individual, occurs fairly frequently in both vertebrate and

invertebrate taxa (Hale and Singh, 1986; Harrison et al., 1985; Hauswirth and Laipis,

1982; Solignac et al, 1983). A few of the mtDNA restriction patterns generated by Ava

I, at first glance, indicated that some individuals (both species A and species C) might

be heteroplasmic for the Ava-1 and Ava-2 patterns (Figure 3-3a, most lanes of top

panel). Further analysis revealed, however, that these mixed patterns resulted from

recalcitrant digestion of the Ava-e site and not heteroplasmy.








73

Variation in the length of the A+T rich region of the mitochondrial genome is

also common in a variety of different animals (Brown, 1987). Since this region does not

code for a gene product, deletions and/or insertions are readily maintained without

apparent harm to the organism. Polymorphism in the length of the A+T-rich region, as

evidenced by slight differences in the mobility of the 2.7 kb Hind III fragment, was

noted for both species A and species B of the A. quadrimaculatus complex.

Several genetic studies have shown that species A and B are more related to each

other than either is to the other two sibling forms. This conclusion has been supported

by cytological, isozyme and hybridization studies (Kaiser et al., 1989; Narang et al., 1989a

and 1989b). Phenograms of genetic relationships based on electrophoretic data have

not yet included species D, but cytological analyses indicate that species C and D are

probably more closely related to each other than to species A or B (Kaiser et al., 1989;

Narang et al. 1989b). The genetic distances calculated from the mtDNA restriction site

data and the clustering of taxonomic units in the unrooted tree generated from these

data generally agreed with the interspecific relationships described above. However,

some of the genetic relationships estimated from restriction site data were contrary to

those derived from hybridization experiments. For example, hybridization studies have

shown that: (1) species C is more related to species B than to species A, and; (2)

species A is more related to species C than to species D (Kaiser et al., 1989; Narang et

al.,1989b). The genetic distances reported in this study suggest that species A is closer

to species D than to C, and that species A is more related to species C than species B

is. The distance data also suggest that species B is slightly closer to species D than to







74

species A. Unfortunately, hybridizations between species B and D have not yet been

performed.

The validity of the genetic distance values derived for members of the A.

quadrimaculatus complex was influenced by two factors. First of all, the interspecific

distances were very small. For example, the distances calculated for the mosquito

siblings ranged from about 2-4% as compared to the 5-10% nucleotide divergence

reported for 8 species of the D. melanogaster subgroup (Solignac et al, 1986). Secondly,

the variances associated with the estimation of 6 were fairly large, averaging about 16%

the value of 6. The variance of 6 is large when the average number of restriction sites

surveyed is small (Nei and Li, 1979). Therefore, the reliability of the estimate is

increased if many different restriction enzymes are used. Since the primary objective of

this study was to develop a simple and rapid means of identifying large numbers of

mosquito species, a limited number of restriction enzymes was employed. Nevertheless,

many of the interspecific relationships derived from these distance calculations were

biologically meaningful and were in agreement with relationships deduced from other

genetic methods.

There are several methods used for reconstructing phylogenetic trees from

molecular data. Distance matrix methods, such as the neighbor-joining method used in

this study, employ genetic distances computed for all pairs of species and a phylogenetic

tree is constructed by considering the relationships among the distance values.

Maximum parsimony methods, on the other hand, deduce the nucleotide sequences of

ancestral species from those of extant species and a tree is produced by minimizing the

number of evolutionary changes for the entire tree. Trees constructed by parsimony







75

methods rely on the existence of "informative" restriction or nucleotide sites (Nei, 1987).

A site is informative only if there are at least two kinds of sites, each represented at

least two times. For example, in the A. quadrimaculatus complex a restriction site is

informative only if it is shared between two species and is absent in the other two

siblings. There are a total of five informative restriction sites produced by the

restriction enzymes used in this study. Three of these sites (Pvu-a, Ava-d and Hind-a)

are shared by species A and B and species C and D, respectively. Conversely, the Ava-

e site is present in the majority of species A and C and the rare Hind-c site is shared by

species B and D. Three informative sites, therefore, cluster species A with B and C

with D while the other two sites indicate that A/C and B/D is the correct relationship.

In order for maximum parsimony methods to be used to discern the relationships among

members of the A. quadrimaculatus species complex, a more thorough study utilizing

many different restriction enzymes is needed.

The value of using mtDNA restriction site data in studying closely related species

has been highly touted because of the apparent rapid evolution of mtDNA in

vertebrates (Brown et al. 1979; Avise et al., 1979). It has been estimated that the

mtDNA is evolving about ten times more rapidly than the nuclear genome in mammals

(Brown, 1985). The nuclear genome of insects, however, seems to be evolving at a

much higher rate than vertebrate nuclear DNAs. For example, in a study of D.

melanogaster and D. simulans, it has been suggested that mtDNA and allozyme loci

evolve at similar rates (Shah and Langley, 1979). Comparisons of the reassociation

kinetics of mtDNA and single copy nuclear DNAs in D. melanogaster have confirmed







76

this observation (Powell et al., 1986). If further studies on insects substantiate these

findings, the implication is that mtDNA restriction analysis may not prove to be as

useful for studying closely related insect species as it has for vertebrates.













CHAPTER IV

CONCLUDING REMARKS


The major thrust of the work reported in this study was twofold. First, the

mitochondrial genome of Anopheles quadrimaculatus, species A was cloned and

characterized. Secondly, these mtDNA clones, along with a previously isolated rDNA

clone, were used as radioactive probes for identification of species-specific DNA

restriction patterns for individual mosquitoes from natural populations containing more

than one sibling species of A. quadrimaculatus.

The sequence content and organization of both the mitochondrial genome and

nuclear genes that code for ribosomal RNAs have been highly conserved over the

course of evolution. The DNA probes that were developed for A. quadrimaculatus,

therefore, can be used as a general tool for the investigation of genetic differences in

any mosquito or even more distantly-related species.

Anopheline mosquitoes belonging to the subgenus Nyssorhynchus are extremely

difficult to differentiate taxonomically. Some species of this subgenus are important

vectors of malaria in South and Central America. Preliminary results indicate that the

mtDNA probes developed as a result of this study can be used to differentiate many of

these species (e.g. A. nuneztovari, A. triannulatus, A. oswaldoi, A. marajoara and A.

albimanus). The mtDNA probes will be used for further study of the genetic







78

relationships among these important mosquitoes. Hopefully, knowledge gained by

careful study of the genetics, vector capacity and ecology of anopheline mosquitoes will

ultimately contribute to a reduction in the incidence of malaria in the third-world.













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BIOGRAPHICAL SKETCH


The author was born on September 15, 1951, in Fayetteville, Tennessee. Her

family moved to Tallahassee, Florida, in 1957 where she attended elementary and

secondary school and received a B.S. in biology from Florida State University in 1975.

In 1978 the author moved to Gainesville, Florida, to begin graduate studies at the

University of Florida in the Department of Entomology and Nematology. During this

time, she was employed by the USDA-ARS, Insects Affecting Man and Animals

Research Laboratory, Genetics Research Unit. The author completed her M.S. in

entomology in 1984 and continued employment with USDA. In 1987 she began

doctoral studies at the University of Florida, Department of Entomology and

Nematology. After completing the Ph.D., the author plans to accept a Research

Associate position with USDA in population genetics and molecular biology. The

author is a member of Gamma Sigma Delta (the honor society of agriculture) and the

American Genetic Association.








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.


ck A. Seawright, Chair
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, Cochair
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.


Ja s E. Maruniak
As ciate 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.


H. Glenn Hall
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.

6Au7 h. QkaiM
Christine D. Chase
Assistant Professor of
Horticultural Science









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.


August 1990
Dean, llege of Agric re


Dean, Graduate School









































UNIVERSITY OF FLORIDA
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