Use of enzyme polymorphism and hybridization crosses to identify sibling species of the mosquito, Anopheles quadrimacula...

MISSING IMAGE

Material Information

Title:
Use of enzyme polymorphism and hybridization crosses to identify sibling species of the mosquito, Anopheles quadrimaculatus (Say)
Physical Description:
vi, 92 leaves : ill. ; 28 cm.
Language:
English
Creator:
Lanzaro, Gregory Charles, 1950-
Publication Date:

Subjects

Subjects / Keywords:
Anopheles quadrimaculatus   ( lcsh )
Mosquitoes -- Genetics   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1986.
Bibliography:
Includes bibliographical references (leaves 87-91).
Statement of Responsibility:
by Gregory Charles Lanzaro.
General Note:
Typescript.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 000897827
notis - AEK6512
oclc - 15527986
System ID:
AA00003808:00001


This item is only available as the following downloads:


Full Text












USE OF ENZYME POLYMORPHISM AND HYBRIDIAZTION CROSSES TO IDENTIFY
SIBLING SPECIES OF THE MOSQUITO, Anopheles quadrimaculatus (Say)


By


GREGORY CHARLES LANZARO


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


1986
































THIS WORK IS DEDICATED TO THE MEMORY OF THE AUTHOR'S

FATHER, FRANK















ACKNOWLEDGEMENTS

The author wishes to express his sincere appreciation to Dr.

J. A. Seawright for his guidance and continued friendship

throughout the course of the work resulting in this paper.

Special thanks are extended to Dr. S. K. Narang for his

instruction in electrophoretic techniques and interpretation of

results. The author extends his gratitude to the graduate

committee members Drs. D. W. Hall and S. C. Schank for their

encouragement and critical review of the work presented.

Very special thanks are extended to S. E. Mitchell and P. E.

Kaiser for their support and friendship. Thanks are extended to

B. K. Birky, L. A. Dickinson and M. Q. Benedict for helping in

many ways with this effort. Finally, special thanks are extended

to Ms. R. C. Brewington for assistance in the preparation of this

manuscript.
















TABLE OF CONTENTS

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

ABSTRACT...............................................

CHAPTER I. ISOZYME PHENOTYPES AND INHERITANCE PATTERNS
OF ENZYME VARIANTS IN Anopheles cuadrimaculatus
(Say) .. ............ ...................... 1
Introduction............................. 1
Material and Methods......................2
Results ................................ 14
Discussion...............................27
CHAPTER II. EXPERIMENTAL HYBRIDIZATION OF GEOGRAPHIC
STRAINS OF Anopheles quadrimaculatus
(Say)...................................... 28
Introduction............................. 28
Materials and Methods....................30
Results....................................35
Discussion............................... 52
CHAPTER III. ENZYME POLYMORPHISM AND GENETIC STRUCTURE
OF POPULATIONS OF Anopheles quadrimaculatus
Species A and B...........................58
Introduction..............................58
Materials and Methods....................59
Results...................................61
Discussion................................77
CONCLUSIONS........................................ ........85
BIBLIOGRAPHY ....................................87
BIOGRAPHICAL SKETCH.................................92









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




USE OF ENZYME POLYMORPHISM AND HYBRIDIZATION CROSSES TO IDENTIFY
SIBLING SPECIES OF THE MOSQUITO Anopheles quadrimaculatus (Say)



By

GREGORY CHARLES LANZARO

December 1986


Chairman: J. A. Seawright
Major Department: Entomology and Nematology



Work was conducted on the population genetics of the

mosquito, Anopheles quadrimaculatus (Say). The research

consisted of three parts: 1) electrophoretic techniques and

enzyme phenotypes; 2) hybridization experiment; and 3) population

genetics. Techniques were developed to visualize twenty-seven

enzyme loci. The phenotypes of these are described and the

inheritance patterns of nine of the polymorphic loci presented.

Hybridization experiments were conducted to determine the mating

compatibilities of nine geographic populations. Hybrid sterility

in males produced from some of these crosses revealed the

existence of two sympatic sibling species of A.quadrimaculatus at

three of nine sites. Analysis of isozyme frequencies of twenty

loci, also confirmed the existence of the sibling species.

Genotypic frequencies of heterozygotes for alleles at two enzyme

loci, Idh-1 and Idh-2, were significantly deficient for









heterozygotes at the same three localities identified in the

hybridization experiment. Heterozygote deficiency was also

observed at a fourth site not included in the hybridization

experiment. The IDH loci were identified as being diagnostic for

the two species and were used as a tool for assembling gene

frequency data into discrete populations of each. An analysis of

gene frequencies resulted in calculations of genetic distance

between the two species, tentatively designated A.

quadrimaculatus species A and B. The values obtained for genetic

distance were consistent with values previously published for

sibling species in the genus Anopheles.















CHAPTER I
ISOZYME PHENOTYPES AND INHERITANCE PATTERNS OF ENZYME
VARIANTS IN Anopheles quadrimaculatus (Say)


Introduction

The southern house mosquito, Anopheles quadrimaculatus

(Say) is one of the five species comprising the Nearctic

Anopheles maculipennis complex. Work on the genetics of

these species is limited. Salivary gland chromosomes have

been described and polytene maps have been prepared for all

the species (Kitzmiller, et al., 1967). Of the five species

in the group, A. quadrimaculatus has been most studied

genetically. The inheritance of DDT and dieldrin resistance

have been described (Davidson, 1963; French and Kitzmiller,

1964) in this species.

In addition the inheritance of a number of morphological

mutants have been described. These include stripe (French

and Kitzmiller, 1963), red-stripe (Mitchell and Seawright,

1984b), black body (Seawright and Anthony, 1972) and brown

body (Mitchell and Seawright, 1984a).

The karyotype of this species is comprised of two

metacentric autosomes (chromosomes 2 and 3) and a pair of

heteromorphic sex chromosomes (Kitzmiller and French, 1961).









Recently the mutants brown-body and stripe were assigned to

chromosomes 2 and 3, respectively (Mitchell and Seawright,

1984a).

In the present study electrophoretic techniques were

developed for the visualization of twenty enzyme systems

representing twenty-seven enzyme gene loci. The inheritance

patterns for nine of the polymorphic loci are presented.

This study provides the groundwork for mapping studies using

enzyme genes and a tool for the analysis of the population

genetics of this species.



Materials and Methods

Gels were made using three parts Connaught starch

(Connaught Laboratories Limited, Willowdale, Ontario, Canada)

and one part Electrostarch (Electrostarch Company, Otto-

Hiller, Madison, Wisconsin). A 12.5% (w/v) solution of the

starch mixture in the appropriate gel buffer was heated over

a gas flame in a 1000 ml Erlenmeyer filtration flask. The

mixture was continuously swirled by hand during the entire

cooking process, which generally took 4-5 minutes. When the

solution came to a vigorous boil the heating process was

terminated. The solution was immediately degassed by

attaching the sidearm of the flask to a vacuum line. A

vacuum was drawn over the solution until all small air

bubbles were removed. The gel solution was then poured into

a mold. The gel molds were constructed of 1/4 inch

plexiglass with the top horizontal surface measuring 20 cm x






3

12.6 cm x 1 cm. This surface held that portion of the gel

through which the samples migrated during electrophoresis.

Each of the long sides of the horizontal was connected to a

leg running perpendicularly, so that the entire surface

formed an inverted U shape. Each of the perpendicular side

walls was open along the bottom. The openings were closed

with two inch masking tape when the gel was poured. A volume

of 400 ml of buffer yielded a gel 1 cm thick. After pouring,

the gel was cooled for about two hours at room temperature,

then covered with saran wrap. The cast gels were further

cooled, for at least two hours prior to loading, in a

refrigerator at 50C.

Samples were prepared for electrophoresis by first

making a crude homogenate of individual adult mosquitoes. A

block of 3/4 inch plexiglass containing sixty-four 1/4 inch

deep wells was used to hold samples for homogenization. Each

well was filled with thirty microliters of deionized water,

and the block was then wrapped in saran wrap and cooled in a

refrigerator for at least one hour. The block was placed in

a container of crushed ice, and an individual adult mosquito

was placed in each of thirty wells. The specimens were

homogenized by means of stainless steel rods, which were

attached to a brass plate in four rows of four rods per row.

These were positioned on the brass plate so that the wells in

the plexiglass block served as a template into which the

sixteen rods fit. By rocking the plate rapidly from side to

side sixteen samples could be homogenized simultaneously.









The homogenates were each absorbed onto 9 x 3 mm wicks

cut from Whatmann 3 MM filter paper. Thirty samples and

three bromphenol blue dye markers were inserted into an

incision in the gel at a position 2.5 cm from the cathodal

end.

Prior to loading the gels the electrode buffer chambers

were filled with the appropriate electrode buffer and placed

in the refrigerator. The chambers were rectangular boxes

measuring 23 x 7 x 4.5 cm constructed of 1/4 inch plexiglass.

The chamber was partitioned by a divider into two subchambers

one 3.5 cm wide the other 2 cm wide. The smaller subchamber

contained the electrode (20 gauge platinum wire) which was

connected to a banana plug set in one end of the chamber.

The large subchamber provided a place for the leg of the gel

mold to be set. A set of two chambers, anode and cathode

completed the apparatus. Each chamber held 250 ml of

electrode buffer.

The loaded gel was readied for the electrophoretic run

by first removing the masking tape from the openings in the

legs of the gel mold and then setting each leg in an

electrode chamber. This arrangement allowed current to pass

through a continuous, U shaped gel so that no sponge or paper

was used to connect the gel to the electrode buffer.

Although this required using more starch, it provided a

superior connection, since sponge or paper connectors can

become dislodged or dry out.









The entire apparatus was placed in a refrigerator at 50C

to keep the gel cool. In addition, the top surface of the

gel was covered with saran wrap and a plastic box containing

crushed ice was placed on top, for additional cooling.

Current, 125-250V, was applied to the gel by using an ISCO

regulated high voltage supply unit Model 493.

Three buffer systems were required for electrophoresis

of the enzymes included in this study. A description of the

buffers follows:

1. CA-8 Tris-Citrate (Steiner and Joslyn, 1979)

gel buffer: .074 M Tris hydroxymethyll)
aminomethane (Tris)
.009 M citric acid
pH 8.45
dilution: none
electrode buffer: 1.37 M Tris
.314 M citric acid

dilution: Cathode; 1:3 dH20
anode; 1:4 dH20
2. Ayala-C (Ayala, et al., 1972)

gel buffer: .009 M Tris
.003 M citric acid
pH 7.0

electrode buffer: .135 M Tris
.040 M citric acid
pH 7.0

dilution: none





3. TC-5.5 (Selander, et al., 1971)

gel buffer: .064 M Tris
.026 M citric acid
pH 5.5

dilution: 1:2 dH20









3. Continued.

electrode buffer: .223 M Tris
.093 M citric acid
pH 5.2

dilution: 3:1 dH20


The buffer system used for each specific enzyme is

listed in Table 1. The electrophoretic run was terminated

when the bromphenol blue dye markers had migrated to the end

of the gel (8.5 cm). The 1 cm thick gel was removed from the

gel mold by making an incision through the leading edge, just

in front of the dye marker. The gel was then cut into five,

1.5 mm thick slices by placing the gel on a plexiglass guide

and using a .012 inch diameter guitar string mounted in a

hack saw frame. Each slice was then stained for a particular

enzyme.

Twenty enzyme systems, representing the products of

twenty-seven loci were assayed. The names and Enzyme

Commission numbers (E.C. No.) for each enzyme, as provided by

the Commission on Biochemical Nomenclature (1972), are listed

in Table 1. The abbreviation listed will be used throughout

this report to indicate the enzyme system (all upper case

letters) or genetic locus (only first letter capitalized).



The staining methods described below are those from

Steiner and Joslyn (1979), unless otherwise noted. The

quantities listed were for 50 ml of staining solution, the

volume required to stain a 1.5 mm gel slice. The following









Table 1. Names, Enzyme Commission numbers, locus
designations and buffer system for the
enzymes assayed in this study.



Enzyme name E.C. No. Abbrev. Locus Buffer
System


Acid phosphatase

Aconitase

Adenylate kinase

Catalase

Esterase


Glutamate oxaloacetate
transaminase

alpha-Glycerophosphate
dehydrogenase

Hexokinase


Hydroxyacid
dehydrogenase

Isocitrate dehydrogenase


Lactate dehydrogenase

Malic dehydrogenase

Malic enzyme

Mannose phosphate
isomerase

Peptidase

Phosphoglucomutase


6-Phosphogluconate
dehydrogenase


3.1.3.2 ACPH Acph

4.2.1.3 ACON Acon

2.7.4.3 ADK Adk

1.11.1.6 CAT Cat

3.1.1.1 EST Est-1
Est-2
Est-3
Est-4

2.6.1.1 GOT Got-1


CA-5.5

CA-7.0

CA-7.0

CA-8.0

CA-7.0
CA-7.0
CA-7.0
CA-7.0

CA-8.0


1.1.1.8 GPDH Got-2 CA-8.0


2.7.1.1 HK


1.1.1.30


1.1.1.42


1.1.1.27

1.1.1.37

1.1.1.40

5.3.1.8


3.4.1.1

2.7.5.1

1.1.1.43


HAD


IDH


LDH

MDH

ME

MPI


PEP

PGM


Hk-1
Hk-2

Had


Idh-1
Idh-2

Ldh

Mdh

Me

Mpi-1
Mpi-2

Pep

Pgm


CA-7.0
CA-7.0

CA-8.0


CA-8.0
CA-8.0

CA-8.0

CA-8.0

CA-8.0

CA-7.0
CA-7.0

CA-8.0

CA-8.0


6-PGD 6-Pgd CA-5.5










Table 1 continued.


Enzyme name


E.C. No. Abbrev. Locus Buffer
System


Phosphoglucose isomerase 5.3.1.9 PGI

Sorbitol dehydrogenase 1.1.1.14 SODH

Xanthine dehydrogenase 1.2.1.37 XDH


Pgi

Sodh

Xdh


CA-8.0

CA-8.0

CA-8.0









abbreviations are used: MTT ([3-(4,5 Dimethylthiazol-2-yl) -

2,5-diphenlytetrazolium bromide]), NAD (nicotinamide adenine

dinucleotide), NADP (Nicotinamide adenine dinucleotide

phosphate), and PMS (phenazine methosulfate).

All reagents were purchased from Sigma Chemical Co., St.

Louis, Missouri.

1. ACPH acid phosphatase: sodium alpha naphthyl acid

phosphate, 50 mg; polyvinylpyrolidine, 100 mg; 0.1

M manganese chloride; 0.5 ml; sodium chloride, 500

mg; 0.05 M acetate buffer pH 5.0, 50 ml. After

incubating at 370C for 30 minutes 50 mg of Fast

Blue RR was added.

2. ACON aconitase (Shaw and Prasad, 1970): cis-

aconitic acid, 60 mg; 0.1 M magnesium chloride,

0.5 ml, NADP, 10 mg; isocitrate dehydrogenase, 20

units; MTT. 10 mg; 0.2 M Tris-HCl pH 8.0, 50 ml

After incubation at 370C for 30 minutes 5 mg of PMS

were added.

3. ADK adenylate kinase: glucose, 200 mg; adenosine

diphosphate, 40 mg; 0.1 M magnesium chloride, 5 ml;

NADP, 10 mg; glucose-6-phosphate dehydrogenase, 30

units; hexokinase, 60 units ; MTT, 10 mg; PMS, 5

mg; 0.2 M Tris-HCl pH 8.0, 50 ml.

4. CAT catalase (Shaw and Prasad, 1970): 35% hydrogen

peroxide, 0.1 ml; dH20, to 100 ml. Following

incubation at room temperature for 15 minutes the









solution was drained and the gel rinsed with water.

Solutions of 2% potassium ferricyanide, 25 ml and

2% ferric chloride, 25 ml were added and the

mixture agitated until white bands appeared on the

gel.

5. EST esterase: alpha-naphthyl acetate, 40 mg; beta-

naphthyl acetate, 20 mg; 0.2 M phosphate buffer pH

6.4, 50 ml. After incubating for 30 minutes 50 mg

of Fast Blue RR was added.

6. GOT glutamate oxaloacetate transaminase: L-aspartic

acid, 400 mg; alpha-ketoglutaric acid, 185 mg;

pyridoxal-5-phosphate, 10 mg, 0.2 M Tris-HCl pH

8.5. After incubating for 30 minutes at 37*C, 50 mg

of Fast Blue RR was added.

7. a-GPDH alpha-glycerophosphate dehydrogenase: alpha-

glycerophosphate, 50 mg; NAD 20 mg; MTT, 10 mg;

0.2 M Tris-HCl pH 8.0, 50 ml. After incubating at

37*C for 30 minutes, 5 mg of PMS were added.

8. HAD hydroxyacid dehydrogenase: D-gluconic acid, 100

mg; 0.1 M magnesium chloride. 0.5 ml; sodium

chloride, 100 mg; 0.2 M Tris-HCl pH 8.0, 50 ml;

After incubating at 37*C for 30 minutes, 5 mg of

PMS were added.

9. HK hexokinase: glucose, 50 mg; adenosine

triphosphate, 40 mg; 0.1 M magnesium chloride, 1 ml;

NADP, 10 mg; glucose-6-phosphate dehydrogenase, 20

units; MTT, 10 mg; 0.1 M Tris-HCl pH 7.5, 50 ml. After









incubating at 37" for 15 minutes, 5 mg of PMS were

added.

10. IDH isocitrate dehydrogenase: sodium isocitrate,

50 mg; 0.1 M magnesium chloride, 2 ml; NADP, 10 mg;

MTT, 10 mg; 0.1 m Tris-HCl pH 7.5, 50 ml. After

incubating for 15 minutes at 37"C, 5 mg of PMS were

added.

11. LDH lactate dehydrogenase: lithium lactate, 300

mg; NAD, 20 mg; MTT, 10 mg; 0.2 M Tris-HC1 50 ml.

After incubating for 60 minutes at 37"C, 5 mg of

PMS were added.

12. MDH malic dehydrogenase: 2.0 M DL-malate pH 7.0, 3

ml; NAD, 20 mg, MTT, 10 mg; 0.2 M Tris-HCl pH 8.0,

50 ml. After incubating at 37"C for 30 minutes, 5

mg of PMS were added.

13. ME malic enzyme: 2.0 M DL-malate pH 7.0, 2 ml; 0.1

M magnesium chloride, 2.5 ml; NADP, 10 mg; MTT, 10

mg; 0.1 M Tris-HCl pH 7.0. 50 ml. After

incubating at 37"C for 30 minutes, 5 mg of PMS were

added.

14. MPI mannose phosphate isomerase: (Harris and

Hopkins, 1976): mannose-6-phosphate, 20 mg; 0.1 M

magnesium chloride, 1 ml; NADP, 10 mg; glucose-6-

phosphate dehydrogenase, 20 units; phosphoglucose

isomerase, 20 units; MTT, 10 mg; 0.2 M Tris-HCl pH

8.0, 50 ml. After incubating at 37"C for 30

minutes 5 mg of PMS were added.









15. PEP peptidase: L-leucyl-tyrosine, 20 mg;

peroxidase, 25 mg; amino acid oxidase, 30 mg; 0.1 M

Tris-HCl pH 7.5, 50 ml. After incubating at 37C

for 30 minutes, 20 mg of 0-dianosidine-HC1 was

added.

16. PGM phosphoglucomutase: sodium glucose-1-

phosphate, 35 mg; glucose-1, 6-diphosphate, 0.45

mg, 0.1 M magnesium chloride, 4 ml; NADP, 10 mg;

glucose-6-phosphate dehydrogenase, 20 units; MTT,

10 mg; 0.1 M Tris-HCl pH 7.5, 50 ml. After

incubating at 37'C for 15 minutes, 5 mg of PMS were

added.

17. 6-PGD 6-phosphogluconate dehydrogenase: 6-

phosphogluconate, 50 mg; 0.1 M magnesium chloride.

0.5 ml; NADP, 10 mg; MTT, 10 mg 0.1 M Tris-HC1 pH

7.5, 50 ml. After incubating at 37C for 15

minutes, 5 mg of PMS were added.

18. PGI phosphoqlucose isomerase: fructose-6-phosphate,

10 mg; 0.1 M magnesium chloride, 4 ml; NADP, 10 mg;

glucose-6-phosphate dehydrogenase, 10 mg; MTT, 10

mg; PMS, 5 mg, 0.1 M Tris-HCl pH 7.5, 50 ml.

After incubating at 37"C for 15 minutes, 5 mg of

PMS were added.

19. SODH sorbitol dehydrogenase (Shaw and Prasad,

1970): sorbitol, 250 mg; NAD 20 mg; MTT, 10 mg;

0.2 M Tris-HCl pH 8.0, 50 ml. After incubating at

37*C for 45 minutes, 5 mg of PMS were added.











20. XDH xanthine dehydrogenase: hypoxanthine, 100 mg;

NAD, 20 mg; MTT, 10 mg; 0.2 M Tris-HCl pH 8.0, 50

ml. After incubating at 37"C for 30 minutes, 5 mg

of PMS were added.

The name and number for enzyme loci and alleles were

assigned as follows: The first letter of the locus name was

capitalized. The loci were numbered in order, with the locus

having the highest mobility as number one. A biochemical

marker strain, called Q2, was developed by sub-culturing the

ORLANDO strain of A. quadrimaculatus. With the exception of

Got-2 and Mpi-2, the Q2 strain was fixed for a single allele

at all the enzyme loci included in this study. Numbers were

assigned to each allele based on its mobility relative to

that of the allozyme found in the Q2 strain. Except for Idh-

2 and Mpi-1, the Q2 allozyme (designated as 100) represented

the allozyme most common in field populations. In the case

of Got-2 and Mpi-2 the allele with the highest frequency in

the Q2 strain was designated as 100.

Crosses to determine inheritance patterns of enzyme

phenotypes were achieved using the induced copulation

technique, as described by Baker et al., (1962). The Q2

strain was crossed to F1 individuals reared from a field

population located at Ginnie Springs, Florida. Both the

parents and F1 progeny were electrophoresed and stained for

the various enzymes.









Results

Enzyme Phenotypes

Techniques for visualizing enzyme phenotypes needed to

be developed before more detailed genetic studies could be

undertaken. A variety of buffer systems were available for

enzyme separation (Steiner and Joslyn, 1979; Selander, et

al., 1971; Ayala et al., 1972). The systems used were

obtained empirically, depending on banding quality and

consistent reproducibility. The banding phenotypes are

illustrated in Figures 1, 2, 3, and 4. All polymorphic loci

are illustrated in these figures either by gels containing

the most common allele and variants found in a field

population (Figures 1 and 2) or by gels representing the

results of genetic crosses (Figures 3 and 4). These

observations also provided information concerning enzyme

quaternary structure. Table 2 describes the number of bands

observed in putative heterozygotes representing individuals

from the field and/or heterozygotes resulting from genetic

crosses. Enzyme structure was inferred from the number of

bands present in heterozygotes (Harris and Hopkins, 1976).

Nine of the enzymes(Acon, Adk, Est-l, Est-2, Est-3, Hk-l, Hk-

2, Mpi-1, and Pgm) were identified as monomers which appeared

as one band in homozygotes and two bands in heterozygotes.

There were ten enzymes which appeared to be dimers (Got-l,

Got-2, a-Gpdh, Had, Idh-l, Idh-l, Mdh, Pgi, Sodh, and Xdh).

These were always presented as three-banded heterozygotes.

The three banded phenotype always presented as two homodimer

































(A 0
ZOH

0 ril -

(a (d td 4-
0) k 0
r- 0H- 0 MO
;jrC$ : 0 ) tp *
0H -rI 4 4 >1
(d > do N adH
j~i-H 14 0 0 00a)
-4' 10 4 r
4 9 10 () ~0 rd a) 1
rdHH I.r4- 0 --1 j En104
a) r-c a0) U4-Jrd ) d U
-rq rl 4 -4-r-I p r-
t cc -H 4-4Hrd 0>I)
wr.m 000oFO EAC 0p
CO X '.O 10-I

0)1 H rl O ~ 0OC C
a)H 41~ 0 1fH0 (0 ) dd44 > -40
.4IMd P H 0i~ 4 -) Z 4E
p p 44 -H 0 (0(0 1):jo
1 (-4n4 LH O rd(A rd004
(P4rd H 9H d H
a) (1) (0 -1 0 N 1 440r: ) >4 I
p 4j 0 0 0 e X 0 ( (d-P (
0 a) 0-40 a) CO .-. C$4 a)
r-i r-I >0 r. 4-) Q)A4 r- 0 04 Q)
WH a) r-4 44 -4 d (1Q) -1 '0 r-4 CO4-) d
Q) ,0 'dr.4 4f)-i 00a)

> C M H COW0 U)Q) rO r P4 p
4c) En HO-H4 9 4CO0)< 0 U a)
0 P 4-) 9 rl -4l a) -P P -H

,0)O4d CO4P Q)O 0) 04-4 0 0 0
Q~~~i idC2C 4J .iit H Hc
04 0 (n M 4- 4-) E r-q r-
rd 4J V)Hd0H Z -~ (0( > n n0 -
(1 a) Q)a) -14 0 0 A r-4 0 0 1 ( rd
0 P-z 0 >4 0 >, rd 0 0 to dj
Qi- 0) z 0 tp N 4 V 0) td m M4 0 .-
N rd ; 0 U>0 Q) P '0 >4 >1 04-HO
0 P J N P 94 0 N N >NN O
HW4 E ro 0 1) (d0 a0) 4 0 0 4 -HHir-q
H a) F4-) 044) r4
r: En 00O (1) En 4M00 r. "t
-r-i d 0 (a -HA ( d C .4 4 U H-H-


0
0



0
N
o

o


'. O
a) "A


0
U LO ~44
H 0)


r$ (a tpr.
-H NOCOmCm
*- 0 4 -HI -H
r.L a)
HO 0 4-ir--0


(0 ,40) H0
r. 0 -N, tpa)a
a) 44 -- >i 4) 4-
ZP0 0 Nc 0 0
0Eo H 0 md

60 NF 0) 0)


p OOH 0 > >
ro 0.4, 0) N N
> M 1x 41 0 0
,c >4X a)
0)N z a Q
d 0 Pd Z 4J 4
g0o Ln L 0))
a) 0 4-4 U)O 0~,


4 a0) NO 0 0
4i-) $40 OH

CEd O~ .H N
ard >-CO 4m
00C NO dOo 0
OH XH H
rc 04 r
z 0 Far. d X






























U


=e\


9lC





























o c
P r-i
(Do

I I I 4-I
4 0 4-) H 0 0
0 0 p Cn o o
En Tl3 U En >4 r4- o
SC i- N *N C r-
H (0a ) .d 0 a) I C )
P 0 P 4- M IC-
Sr 0) r d 0) 0

0 -4 .O o o 0)
S0 U > 0)0o a o) > P

-u g 4 C \ o WH -,0 (O
4 (d H 0) O-4 w 0 0 0z
0) O r --, P o H m e
U I CZ 0 I En 4- H -40 00 Q- r-l P

-n 0 r-i +1 or 0 o O E A I i a)

O 4 -r- C) *r-1 0 (d '0-H H *H 0) j x 0
r' (5 4J (0- 1 r .* 4 0 Z M 0J
) 4-l E( 4d E0 r0l 0) 10 -( r( cO0 >rH 0
m :j H 4 O W H-i (d uO W Q a) (10 (a


0i H P 0 > ( O i H' r-0 0p 0N r-l
0 .o M (a ( (0 ( QO (0 o r-O


F -a-) 0 4 1 O O U ) ) '- 0 0 O H *Hi
z; C (0 < wl z 0 tpM m w d0-w >





r +O1 H0 4 0 -H (d0-H c 00o P Nq
o0 4 r 0 O M *-MOLn N C 9 )
En > 1) (0 0 4O> >1 >i *0 0o-H r O i C 1 r- H 41




-4 *- H 0 0 N N 4 >- C- 0 r- N0 0
(u C'0 m44 r4 M 1 0 ad N u) 0c tTI b)




40 9,- C f 0-H & 0000 P 0) 0 O O >0
> 1 O M x r^O 0 4) 0 -pl 0 0 r- a) N


C1 M 0 0 > O\ OU4 r4 0 Oz O C-10 c o O
0 0 U) -H 4 CM (a (MN >N o
z O1rl 0 tp 4) \I U >1 Ul I H N Q)
(1) (O W1 ^1 CP4J (d W (00 N 0) (0 X\ Q) 4 C P

04 :1 0 P 0 rO 0 ) S O 0 k r a Or 0 Q) 4 wp

4-) O(1) )4 > 0 0 P (0 > 00 IO> 0 -10c 0
9 i (0o a- 0 m b) 10 m N xn Ci N M r 10 tr
P 0 () r-Cl rd 0 >4n >1 >4 %D 0 Wrl m 0 >4 (0-4 >1


r--l 9 l 0 Q) 0 0 00 (0 0) -1 M a 0 Q) 0


CM



-1
Er













n 4




CO
d4
0







oo !? i 0
o

"r I



0 0 0



0 C 0
) I "-I
oc



I I
-4 0





f~~3

II









"11
LS S




:3., 4 -Jo
o (
















0)U
)0 c0 0 0
4 C d U *O cO
-H1l r 0. 1 )H Ito10 0
,-i a) I ()1-I I LO
0 (n 1-1 N C 4- ';t 430 4-P
S0 o m 0 1 '0\ 00 *
S4-p r w- 41 HO o u
4) >i 0 >> U )
r- 4 -I NM l H (1) N 4J O
mA l r-4 0 0 0 (C r i o0 -O
0 r-4 -r-i U ) o o i0 u EnH o >io 0
a) M&J 0 rip m NrH0 4 4-i *\ H ,N (1) F p(v (a 3 0 c4 H M HHD rq- p 0 .. 0 *
H400 0~ O I o r-m I r- tO H otp
0 a c) H Q) O o 0)r- 0o o ( c PI
r P 0 0 r-i >14- n0mo r-i N r-I 4 N 0 Hrl rCl4J PI

1 0) 0 ) I N a) m1 Od ( 0I ) r Q) (O N0
S0 4 0 D 0 C-4 1 I 1 >, -l o 4 r-i a)

a) r-i o w o ,. 0 0 > I %H C (ON 0d ro- m
4 ,,- 00 r-4 -4 m o I ( ( -4 (z N >o
) il Br N1- W >, a) oi ; o N
>4 m.ul 4- 4 .1o 0 0 oH .o (d H Oc 00 aO) :O
0J U 0 0 ) r-OHI 1 p 4- to w --4 UrH- 4J 0
0 0 r- HIM c r 4 O0 0 H r-I r- 4 -I c O4
0) -4OHQ4 H IHI 0HeH' H
S- E r-I I (o r-44 4J -- to 0 H 4'. i m '
a)l-i 0 r:l r-'i a)) C-l > rHl tp r-l m af >
-4 p 0 M H- I N 1 1 O d % 0 NO
04 .- --H ) c w H -I M 0 r-I N r ) ~ H w Or O
0) p -1 WU '- M ''- 0n 0 ( r O -O P l a o Q)
Q) u M > z > o H 0 V r-ilcm 0 M- a )0-O
0 3 -- 4 ( 0- CO ) .-r-l Ir-l rd 0 > 0 -P4 0
>. r-i 0 p d rA 4 co0 -H O O ,--4 tO
N wI i OH .1.r C wtrl O > d G) r P 4 0 ,4 ) >,
.-. ( 0)I= (m 1 0 HO Ur o O 0 H 0 -) % (a 0


me4- > 4 0 (o H tor H a )O o ao to Q)
0H-1 0 C 3 30 t -HH1 H oHH 41 m p 6 >H O 0
d'0 4- (Z r d 'OHI p Zu O (t a) 0 N E r.
M C z ) *-H r4 -ri cN 0) H (I) 10 1 M tP 9 4J C O (A 0 M3 Ok
CH4 gcH > > I p >4CH4 >I l e P C
M 04 *P--l-l-P -4JH (d3 ,O rM N t4 0 O0\
- e im O .n ~O -m m H a H* 0 m LO r.(0 HO
4J 0 Hrl : W 9 : 3Ww ru n p )n E %o
- ) a) X (a-r H a H fMN a) rON 0 Oa) E n l
m0 0U wa 3 P W -l H 0) H-O 0 P -r-l o
S- o >G crdu m M > M 4 W\ 0 Ln tp
(d0 0 -A >i a) H- 4-4 W H 0 0) UN a))0 AOH
Q.4-wS-l d* r* 0 M 4.JN -0)Cfl\N 0 to
0) r-H44 l >H r CM04 Un-Pr'd -4- 4-) 4. 'O U) -
U I-10) It 0 E 00 O 0)4J ) 0 OW 0 V-H ) OHH O Q)
9 0 Z '0 > P k-P 3 4J V)H +-P--I 4JM 0' O 4 & 4 1 (0 (d 4J P l
(zM-H -H10) a) E00>4 000>> -4> 0 04 0 0
4J e (0E H 4IJ4 WM N t OrN N-\ NHg r t0U2'dC do
*-H-r 0 0 m > i>0 >i i >i O 00 (0 >i0- --4 > N
P P4J Z .Q) W N N P NH N P PO CP N4 > > NH
0)1U0-o --' 0 0 0 Q) 1)0 O0H 0) O0 -4 H-
4 0 P ) E0 M O 4J 0 F )4J4J4P 0 o dro
C o Nam M0) C*H 0 0 0) m0 0 0 ) )'0 0) 0* O 0 Z 0 0
H OYM P4 H r. 43 W ri M r.03C H 4 U -3HU-14QM-HH 4 (d





-H


















C)






SA *
Id












4 --4 -- q
4b mm m
m 9














I I
-a










I I






<" *g B a>*^B
























0
0)



r4
0



CA
4-)

0
01

4)
0
aD
4
-4






o

0)
N)




r-i


am
4C


4



0:
UH
C)






.-4
ar


0 )


CO V Ea 0 tnp r- (L) 4) r-I >4
I) ) 4 >4 I r-I H cJ )> N
C -H M 1 0N MI I r- 4-1 >0 N

S O N COO 0 o) 0 U rl I
S(d )0 W ) 3 W 4-) r- r-o 4-4l 4 4 a) -q-
r +jo 1- g ( ) r-i (0 a P 4 a
-1) a O o o W mn r- ac) n EIn r
4 4- t r- 4l V 4 (0-lH r- I C :) a)
1- >i i >i tz' (0 O C44 1-4 r( Nc a)
( -lN+ N 4J ) tI C C l O, l -) --0 co
0 44 0 0 0-A q (4 >i0 4-Q +O -- 4 -
O g 0C I rH1 oNJ N N 4J O4) W)C%
*H ) 4 0 C ) to ) (H (d 4-l r-0
> $4 4-m ) r. 4r-j rl : Z e 4 (1)-I o
H (v P (U 4J> td rd O W U) c% r-lr-l I -4
0 0 (o, >ir0--H () 44 ) 0 ) t -r-4 -H
Cr- o WH rHi u C >4 4 U' TO d1 aE
H I : W 44H (do o-1 4n En 0o c c )
c i- HHO 0 > j -4 '- 0(0 (J -IC14 0
I \'- l C O >i 4 (UM ) &0
-*4 4tH30 M OIH O N *r-lH Hrl >
O) 0 CO U 4- m 4-. >i -0 op co I (o N
t/3l r r 0) 9 l N () m -H --
(0o ( pH Q)W i uH -C ) 4 0r- 4 CO r
- 4J 4J (0D (d ( O4J H 4 W 04
0-4 0 o P H -r-ico n o H
(d C "A cW I(0 (a 0 >l m -H 4 0o a)
4n 4-) P W- N WU >f: O> 1d -) p
) Cp H)M > -H p Nm 0) -H ro
P0 ()M-H-H v O0-4 Fi f0o'-- 0) I r3 -1 P
pH r- (a u r 0W go. o C 0
P (d (a r. Z N 0 () H -4 Nt H
04 W t H0 H i r- OH O) m




O z -- tp (0 I o) (o ) )-H Nr-4 l
(0-H >io) H 0 0) 4-) 4-) r- 0 -
0 N r-4 (q N r-i O Z cr (OI I ro r-I H o
r 1 04 0 o ) o H 0 (x C
( rP 0 o r-I >I 1 ; 04 W 0 4 0
* X w ln ca rd d r-i N Ud-PUNU)-i MUc
!0 () 0) 1i r lo (o 0-rql rl 0 0) Mnr
r- ) C > M 0 0 r- r,-il -MHOD
0Q) (0(C 4 N -H CO () 0 34-H (0 H E



F.J 0 -ZOHM U (0 ) t C a) z I rI ) (A)W ) H4)
.4 0 -C ) H r- 0 a)-4 r E,- 4-4r-I a
O4- H- 0 0-H )u O r- I 0rH 0 >
:3 (d 0 ZPr-4 .e-r-i 0 n 4 ( tp r:r-i o aC ) N
r-i r- > (0 (0 ) > (0 4-) *- 0 0)
0 <1 N Z 9 P4 4 4r- + >0 t-4 H >+J Pr-I
a 1 I 0 g0 -H -*r-l 1 () *H ) (1) -
M 4 rc CI T rH 0' r O O > r-0
) rl 0 *0-H C) C) o0 ) c 0) ) M Z 0 W o)
4o 4 o r. ro O r-i t cz\ 4 H Z 4 Z 134H -4 O d


*M MOO** rlC OG)O aC O O*arb C O# r
dG O~mmdd MEHEADERH AM


LHH


H -(1
tic f U) 1





-H 4 -4 o
C) '0 C) z- 0
a4 r.4
OH(C) 1
C) E Q,-r1 0








C)40 4


-H 4-) H

m 0 En C 1
Q) Q) o1




- Lo o En
40 r .l




rd a It .o


-H4 1.4 C4 0



r-4 :H0 0
"D c











-- On 4-O '


HoQ NC
a)0 ) H O0)
-H4 ( 0 0


P r C OC
H C41N

d HC) 00




- H( ) (0
40) (0 OH
00r1( 00




a- r) k 4a
) a ) a, 0)

. )C C O
*0CC O
Q)CHEMAC

































-O




o <


4.'.**,


w w -






~0
soap














10
~ -
O:


N)

N) S

.3


S


ul

CR


~lra al
Ed









Table 2. Description of enzyme phenotypes and evidence for
polymeric structure of 11 of 20 loci in Anopheles
quadrimaculatus. NI = not investigated.



No. of electromorphs in gel phenotype

Locus Maximum per Present in Inferred
individual heterozygotes structure
of genetic cross


Acon 2 NI monomer

Adk 2 NI monomer

Est-1 2 2 monomer

Est-2 2 2 monomer

Est-3 2 2 monomer

Got-1 3 3 dimer

Got-2 3 3 dimer

a-Gpdh 3 NI dimer

HK-1 2 NI monomer

HK-2 2 NI monomer

Had 3 NI dimer

Idh-1 3 3 dimer

Idh-2 3 3 dimer

Mdh 3 3 dimer

Me 5 5 tetramer

Mpi-1 2 2 monomer

Pgm 2 2 monomer

Pgi 3 NI dimer

Sodh 3 NI dimer

Xdh 3 NI dimer









bands migrating to the same position on the gel as the

respective homozygotes and a denser, hybrid band at a

position between the two homodimers (see especially figure

3B). One enzyme Me presented a five banded heterozygote

(Figure 2D) phenotype indicating a tetrameric structure.

Observations concerning enzyme structure are only inferences.

Definitive determinations require enzyme purification and

dissociation-reassociation studies.



Unusual and Epigenetic Effects

Some banding patterns were observed which either did not

appear consistently or which failed to give predicted results

when studied by genetic crossing. In every gel stained for

Adk two rows of bands appeared cathodally to the major Adk

bands (Figure 1B). By comparing these to gels containing the

same material and stained for HK, it was determined that

these bands represented the two HK loci (Figure 2C). In

addition, a band located anodally to the Adk bands was

sometimes present (Figure 1B).

The most interesting effects were observed in three

enzymes, Had, Acph, and 6-Pgd and were related to blood

feeding in females. Figure 5 illustrates the effect of blood

feeding on the electrophoretic mobility of these three

enzymes. In Had the mobility and intensity of the band were

increased in females 24 hours after blood feeding (Figure

5A). Mobility returned to normal by 72 hours after blood












Figure 5.


The effect of blood feeding on the phenotype
of certain enzymes in Anopheles
quadrimaculatus. All individuals used were
from the Q2 strain and were genotypically
identical for the three enzyme loci
illustrated. The individuals in the gels
shown were treated as follows: individuals 1-
3 = non-bloodfed females; 4-6 =females 24
hours after taking a blood meal; 7-9 = 48
hours post blood meal; 10-12 = 72 hours post
blood meal; 13-15 = 96 hours post blood meal;
16-18 following oviposition; 19-21 = males.
A. Hydroxyacid dehydrogenase. Individuals 1-3
normal position of the Had100 allele in non-
blood fed females, 4-9 increased mobility and
intensity of staining in females analyzed 24-
48 hours following a blood meal; 10-18 return
to normal mobility, but with increased
intensity by 72 hours following a blood-meal,
16-18 normal mobility with some smeariness in
females following oviposition, 19-21 normal
mobility and faint bonding in adult males.
B. Acid phosphatase. Individuals 1-3 normal
mobility for the Acph100 allele in non-blood
fed females, 4-6 increased mobility and
staining intensity in females 24 hours after
taking a bloodmeal, 7-15 mobility increased
further and bands smeary in females 48-96
hours post-bloodmeal, 16-18 mobility
decreased, but not at normal position and
bands compact in females following
oviposition, 19-21 normal mobility and weak
banding in adult males.
C. 6-Phosphogluconate dehydrogenase.
Individuals 1-3 normal mobility of the 6-
Pgd100 allele in non-blood fed females, 4-6
increased mobility and staining intensity in
females 24 hours after taking bloodmeal, 7-15
increased mobility, stain intensity and
smeariness in females 48-96 hours post-
bloodmeal, 16-18 return to normal mobility
with increased staining intensity in females
following oviposition, 19-21 normal mobility
and weak staining in adult males.












* i


."*e tp


HAD


# an


B


1 2 4 S 6 g 10 1112 131415 16 1718 192021



4.X.


ACPH


13 460


* *


urn 16181 162021
S *


6-PGD









feeding, although staining intensity was higher than in non-

bloodfed females. A similar effect was observed in 6-Pqd

(Figure 5C). In this case mobility and intensity increased

24 hours after blood feeding, by 48 hours mobility remained

higher, but the banding became more diffuse. The diffuse

banding persisted through 96 hours after blood feeding.

Following oviposition the banding pattern returned to normal,

but staining was still more intense. The most profound

effect was observed in Acph (Figure 5B). The pattern was

similar to that observed in 6-Pgd. Increased mobility at 24

hours after a bloodmeal with diffuse banding at 48-96 hours

post bloodmeal. However in this case the smeariness

disappeared after oviposition, but the mobility remained

higher than in females which never had a bloodmeal.

Discussion

Electrophoretic techniques for the analysis of twenty-

seven enzyme gene loci were developed. Results revealed

genetic variability at twenty of the twenty-seven loci.

Inheritance patterns were determined for nine of the

polymorphic loci.

Epigenetic effects on three loci (Acph, Had and 6-Pqd)

were shown to be related to blood feeding in females. These

effects alter the mobility of these isozymes and should be

considered when interpreting electromorphs.

These techniques can now be applied to studies on

genetic mapping and population genetics of A.

quadrimaculatus.















CHAPTER II

EXPERIMENTAL HYBRIDIZATION OF GEOGRAPHIC STRAINS OF

Anopheles quadrimaculatus (Say)



Introduction



Species in the genus Anopheles commonly evolve without

developing conspicuous morphological differences. An

increasing number of sibling species are being described in

this genus from throughout the world, as documented by

numerous authors (see Discussion section, this paper).

Hybridization studies have been widely used to establish

the true biological species status of suspected sibling

species (Davidson, 1964, Davidson and Hunt, 1973, Paterson et

al., 1963). In addition, hybridization experiments have been

used to assess the degree of relatedness between sibling and

morphologically distinct, but related species (Davidson et

al., 1967, Kitzmiller et al., 1967).

The first sibling species complex described in the genus

Anopheles, was the Anopheles maculipennis complex. The

complex contains both Palearctic (Old World) and Nearctic

(New World) species. The Palearctic members include the nine








sibling species, Anopheles atroparvus Van Thiel, A.

beklemishevi Stegnii and Kabanova, A. labranchiae Falleroni,

A. maculipennis Meigen, A. martinius Shingarev, A. melanoon

Hackett, A. message Falleroni, A. sacharovi Farre and A.

sicaulti Roubaud (White, 1978). The Nearctic members of the

complex are morphologically distinct and include A. aztecus

Hoffmann, A. earlei Vargus, A. freeborni Aitken, A.

occidentalis Dyar and Knab and A. quadrimaculatus Say

(Buonomini and Mariani 1953, Kitzmiller et al., 1967).

Kitzmiller (1977) used polytene banding patterns to place A.

quadrimaculatus in a separate group that included Anopheles

walker Theobald and Anopheles artopos Dyar and Knab.

However, Joslyn (1978) recorded only nonviable eggs from

crosses with those species. On the contrary, viable eggs

were obtained from crosses of A. quadrimaculatus to A.

freeborni and A. aztecus (Kitzmiller, et al., 1967). These

results indicate a closer relationship between A.

quadrimaculatus and members of the Maculipennis complex,

instead of A. atropos or A. walker.

To date no sibling species have been described in the

Nearctic Anopheline fauna. A number of the Nearctic species

have broad distributions, and given what is known about other

species in this genus, make ideal subjects for studies on the

genetics of speciation. The purpose of the present study was

to assess the degree of cross fertility among nine field

populations of A. quadrimaculatus. This species has a broad







30

geographic distribution, ranging over the entire eastern half

of the United States.

Materials and Methods

The mosquitoes used in matings were offspring of females

collected from the following sites: in Florida at Ginnie

Springs (GIN), Gainesville at Kanapaha Botanical Gardens

(KBG), and Lake Panasofkee (PAN); in Alabama, 41 miles west

of Auburn on 1-85 (AUB), and Guntersville (GUN); in

Mississippi at Skene (SKE); in Arkansas at Stuttgart (ARK)

and Bebee (BEB); and in Louisiana at Lake Charles (LAC)

(Figure 6).

Field Collections

Both sexes were collected from daytime resting sites,

e.g. treeholes, farm buildings, and boxes placed in wooded

areas. Adults were put in Savage cages (Savage and Lowe,

1971) and provided with a 10% sucrose solution. The cages

were placed in styrofoam ice chests containing a small amount

of ice in plastic bags to keep the mosquitoes cool and

humidified. The chests were then air-mailed or transported

by car back to the laboratory.

Laboratory Procedures

On arrival, adults were transferred to larger cages (1

meter square). Gravid females from each collection were

transferred individually to 30 dram containers for

oviposition and non-blooded females were provided with

bloodmeals by placing a confined guinea pig in the cage

overnight. Blooded females were removed, held for ovarian

































0)

4-)

z
-Hl

En

(0 0
r-i -H-


04)



() f
Oa


r4 >




0 fd
-Pl










-1 4-) -P


0
Q)










~-4 En(
r.I 41r-







r-4 :5

0 0 -H
r14
'C,


0Q)

-H c c

















w









development and placed in containers for oviposition.

Frequently, females would not oviposit, even though they were

obviously gravid. Females were traumatized to induce

oviposition by tearing one wing from the thorax with a

jewelers forceps. Following oviposition, females were

removed and the eggs left in the containers to hatch, usually

1 to 2 days after they were laid. Newly hatched eggs were

infused with 1/2 ml. of a 2% aqueous suspension of 2 parts

Tetramin Baby "E" fish food and 1 part brewer's yeast.

One day after hatching the larvae were transferred to

large (40.6cm X 50.8cm X 7.6cm) plastic trays in 2 to 2.5

liters of tap water. Larvae from females collected from the

same site were pooled. Each tray contained about 300 larvae

and the larvae were fed daily on 20 ml of the mixture

described above. Larvae were reared at 27*C and pupation

usually began within a week of hatching. Pupae were removed

from trays and placed in 8 oz. plastic cups half-filled with

tap water. The cups were capped with 1 pint cardboard ice

cream containers covered with a mesh lid that provided a

place for emerging adults to collect. Adults emerged about

36 hrs post-pupation. Newly emerged adults were removed,

sexed, and placed in Savage cages. The adults were

maintained at 25"C and 70-80% RH and were provided with a 10%

sucrose solution. The numbers of F1 adults obtained from

field collected females were adequate for the completion of

all the crosses, so that further maintenance of stocks by









inbreeding was not necessary. The initial series of crosses

were all between the F1 adults and ORLANDO (ORL), a standard

laboratory strain maintained over forty years. The ORL

strain served as the standard against which all field strains

were compared. All crosses were accomplished using a

modification of the induced mating technique of Baker et al.

(1962). Females were held for 2 to 5 days prior to mating.

In order to avoid wasting time mating females which might

subsequently refuse to take a bloodmeal, females were

bloodfed on guinea pigs or humans immediately prior to

mating. Sterility in hybrid males was determined by

microscopic examination of the testes. The testes and the

distal portion of the vasa deferentia were dissected out and

transferred to a small drop of saline on a slide. A cover

slip was added and gentle pressure was applied. The

preparation was examined at 400x. Sterility could be

detected by the absence of normal spermatozoa, and could

usually be predicted by the gross appearance of the testes

which were greatly reduced in size in most sterile

individuals. Sterility in females was tested by crossing to

fertile males.



Development of strains of sibling species A and B.

Four strains from the AUB and KBG populations were

developed for further study. These strains were selected on

the basis of the fertility of the F1 progeny obtained in

crosses to the ORL strain (i.e., produced fertile or sterile









hybrid males). A series of isofemale lines were established

from field collected females from the AUB and KBG sites. A

sample of F1 males from each line was mated to ORL females

and the remainder of the Fl's were sib-mated. The adult

males produced from the crosses to ORL were scored for

fertility. Lines which produced fertile hybrid males were

pooled and maintained as the A-strains and those which

produced sterile hybrid males were combined to make the B

strains. Thus two pairs of sympatric lines, AUB-A and AUB-B

and KBG-A and KBG-B, were developed.



Results

Survey of Field Populations

The results from the first series of crosses, involving

matings between field strains and ORL mosquitoes, are

presented in Tables 3 and 4. These data represent only egg

batches which hatched. A significant number of females from

all crosses laid egg batches which failed to hatch. This

phenomenon is undoubtedly due, in part, to the use of the

induced mating technique since induced matings often result

in copulation without the transfer of sperm (Bryan, 1973).

These infertile matings appear normal, but the females are

not inseminated and lay only infertile eggs.

Results revealed the presence of two types of

individuals from the field, designated type A and type B.

Type A individuals were genetically compatible with ORL, type

B individuals were incompatible with ORL. The populations









Table 3. The percentage hatch, sex ratio, percent
survival to adult stage and F1 male fertility
in Type A population cross-matings.


Cross Number Percent
female of egg Percent Total Percent survival to Male
and male batches hatch adults males adult stage fertility


CONTROL

ORL x ORL 5 85.4 796 49.4 77.9 +

BEB x ORL 9 88.7 1332 52.5 78.3 +

ORL x BEB 9 85.9 870 51.5 90.0 +

ORL x GIN 8 91.7 883 56.3 91.4 +

GUN x ORL 11 86.0 1698 52.3 86.7 +

ORL x GUN 10 82.8 1088 48.2 82.9 +

LAC x ORL 10 91.7 2320 51.8 92.3 +

ORL x LAC 16 85.3 1359 52.4 88.1 +

ORL x PAN 8 78.7 700 55.7 77.4 +

SKE x ORL 7 87.7 968 51.7 86.1 +

ORL x SKE 8 80.8 1118 48.2 82.4 +









Table 4.


The percentage hatch, sex ratio, percent survival to adult stage
and F1 male fertility in Type A/B population cross-matings.


Cross Number Percent
female of egg Percent Total Percent survival to Male
X male batches hatch adults males adult stage fertility


A-ARK x ORL 12 71.4 1132 51.9 81.3 +

ORL x A-ARK 13 80.9 1530 51.0 75.3 +

B-ARK x ORL 2 66.4 125 54.4 78.1

ORL x B-ARK 12 74.5 799 27.8 55.5

ORL x A-AUB 20 nd 2723 55.0 nd +

B-AUB x ORL 10 70.1 982 51.4 70.3

ORL x B-AUB 12 71.3 526 12.2 34.9

A-KBG x ORL 6 83.3 519 48.8 66.1 +

ORL x A-KBG 7 85.8 643 50.2 63.0 +

ORL x B-KBG 7 nd 447 21.3 nd


nd: Data not determined.









were divided into two groups: type A populations, comprised

entirely of type A individuals and type A/B populations,

which were made up of a mixture of type A and type B

individuals. Crosses were made between all of the field

populations and ORL but all possible reciprocal crosses were

not achieved.

BEB, GIN, GUN, LAC, PAN and SKE were type A populations.

When crossed to ORL the F1 progeny were normal in every

respect and the results were consistent with the control ORL

x ORL crosses (Table 3). Crosses between individuals from

these six populations and ORL produced families with high

hatch (78.7 91.7%), high survival to adult stage (77.4 -

92.3%), 1:1 sex ratio (% males = 48.2 56.3) and fertile

male progeny. The percent survival to adult stage was

generally higher in the hybrid F1 than in the control; the

average for the ten hybrid crosses was 85.6% compared with

77.9% in the control. In outcrossing a longstanding colony

strain to field material one might expect increased vigor in

the F1 resulting from heterosis. The populations ARK, AUB

and KBG are type A/B populations. Two types of results were

obtained from matings to ORL. Some crosses were identical in

outcome to those from the A populations, while others

resulted in the production of sterile males in the Fl. These

data were grouped into A and B crosses, and are presented as

such in Table 4. Crosses in which B females were mated to

ORL males were completed for the ARK and AUB populations. In

these crosses, hatch was high, survival to adult stage was









high and sex ratio was normal. In both crosses all F1 males

were sterile. The reciprocal cross, ORL female X B male was

done for the ARK, AUB and KBG populations. Hatch was high in

the ARK and AUB crosses, but was not recorded for the KBG

crosses. Percent survival to the adult stage was

significantly lower in the ORL X B-ARK and ORL X B-AUB

crosses than in the respective reciprocal crosses. This was

due to heavy mortality in the male pupae. Consequently, the

sex ratio in the F1 was strongly distorted in favor of

females, 27.8% males in the ORL X B-ARK cross, 12.2% males in

the ORL X B-AUB cross and 21.3% males in the ORL X B-KBG

cross. The abnormal male pupae produced from these crosses

are illustrated in Figure 7. Typically, in these pupae the

wing buds lie outside the cephalothoracic capsule (Figure

7A). The wing buds became swollen with water and presumably

disrupted the pupa's ability to maintain buoyancy. In some

pupae, the head and thoracic appendages as well as the wing

buds were free (Figure 7B). In addition to these

abnormalities some of the male pupae had deformed genitalia.

Normally, the pupal genitalia lie in a genital pouch, and in

males this pouch is somewhat pointed and bifurcated distally,

with lobes being equal in size. In some of the hybrid male

pupae one or both lobes were not developed (Figure 7C). Many

of the pupae which did survive through the pupal stage died

during eclosion. Figure 7D shows a typical case where the

pupa has freed its abdomen from the puparium, but was unable

to free its head and thorax. No abnormalities were observed

















4-4
0


0) ,Q
1 r-4 0)
0 0
a M -4 4-%
E-1 0 0 0)
40 a)
X 00) 4, 0)
0 0 4 Q)

0 ,0) 4
-l (0 41 -- H
0 U) > n



o4 a O4
4- 4-) 0M 4 )
0 (aQ0 Q (

1- 4 4-0 En 4 4

0 0 4- 0

a) Q)M 0
o a) >4 P


i4 -1 0-4 p
p0 0 o d 0



,. 0 .
04 : 0. 1 0
0 ) 00




4 -H 4- 4P M (nO
S0 400 r






)H 4) H 0
P : 0 0 P 1 4-)
A P4 104 ( -- (0 :
>1 p C
S 9 0 d (0
r I4 -4 f 0) --
P l P 4-) 0 l 4O
A Q 0 r. p0








0 OC 0 M

ZH 4 4





I-1

--4
Qf
* 1 l ~ c
r^ a a
Pc36)~
a
O) C m l ,r
Cr> a d
*H~(r c
b )r( ((












among the female pupae. Without exception all the hybrid

males in each family from crosses between ORL and B males

were sterile. Figure 8 compares the gross appearance of the

normal and hybrid male reproductive systems. In normal

males, the testes were ovoid and larger than the accessory

glands (Figure 8A). The appearance of the hybrid male

reproductive system varied considerably. In many cases the

testes were completely atrophied and no wider than the vasa

deferentia. On the other extreme, some hybrid males had

testes which were normal in size, but contained no normal

spermatozoa. Figure 8B shows the reproductive system of a

hybrid male, the testes are smaller than the accessory

glands, and contained no sperm. Figure 9 shows the contents

of normal (9A) and hybrid (9B) testes. A large ball of

spermatozoa has been extruded through the vas deferens of the

normal testis (Figure 9A). A few abnormal sperm with short

tails can be seen in the hybrid testis (Figure 9B).



Hybridization of A and B Strains

A second series of crosses were undertaken using A and B

strains developed from both the AUB and KBG populations.

Crosses were done to define, more completely, the

relationship between type A and type B individuals. The

results from these crosses are presented in Tables 5 and 6.

Data from matings within each of the four strains were

collected to establish the integrity of each strain and to

provide data to which hybrid crosses could be compared. As



















Figure 8. The male reproductive system of A. quadrimaculatus
A. Normal male reproductive system
B. The male reproductive system, showing reduced
testes from a hybrid produced by crossing ORL
female and type B male.
t = testis; a.g. = accessory gland; v.d. = vas
deferens


I





















O x
0 UE
NmJ







N )
o r-i -:







4O (O
-4-0





oa)o
040


34i X-


0)




000


r40

.0 0
u i
S0! 0)
0 rd






e o Q)




41 4 01
) 41
4- l


















01 8
ro- 0 )-


*44-& C (
to Qo P


SE U)0




3a) p

4) 4 0








*r












Table 5. The percentage hatch from A and B strains
and cross-matings.



Cross
female Number of Total Percent
and male egg batches eggs hatch



AUB-A x AUB-A 5 1376 79.8

AUB-B x AUB-B 12 2030 73.9

AUB-A x ORL 7 1022 83.1

ORL x AUB-A 7 1136 89.1

AUB-B x ORL 13 2119 78.4

ORL x AUB-B 6 756 92.7

AUB-A x AUB-B 12 3087 78.6

AUB-B x AUB-A 15 3495 90.9

KBG-A x KBG-A 2 532 97.2

KBG-B x KBG-B 9 1592 94.9

KBG-A x KBG-B 9 1819 81.9

KBG-B x KBG-A 9 1790 90.0

AUB-B x KBG-B 8 927 66.2

KBG-B x AUB-B 4 813 73.9









Table 6. The sex ratio, percent survival to adult stage and
Fl male fertility in A and B strains and
cross-matings.




Cross Number of Percent
female first instar Total Percent survival to Male
and male larvae adults males adult stage fertility


AUB-A x AUB-A 1098 669 56.9 60.9 +

AUB-B x AUB-B 1501 1157 53.5 77.1 +

AUB-A x ORL 1230 760 51.1 74.4 +

ORL x AUB-A 1275 806 52.5 71.0 +

AUB-B x ORL 1662 1388 49.8 92.2

ORL x AUB-B 701 258 0 36.8 NA

AUB-A x AUB-B 2426 589 0 23.5 NA

AUB-B x AUB-A 3176 1387 48.4 43.7

KBG-A x KBG-A 517 371 45.3 71.8 +

KBG-B x KBG-B 1510 758 56.6 50.2 +

KBG-A x KBG-B 1489 692 40.8 39.7

KBG-B x KBG-A 1609 639 54.7 46.5

AUB-B x KBG-B 614 431 48.3 70.4 +

KBG-B x AUB-B 601 516 52.3 85.9 +









expected, the members of each strain were compatible among

themselves. As a second control, each of the AUB strains as

crossed to ORL. Both reciprocal crosses between AUB-A and

ORL resulted in progeny which were normal in every respect.

The AUB-B female X ORL male crosses gave results similar to

those obtained from the first series of crosses (Table 4),

that is, hatch and development of the F1 progeny appeared

normal, but all F1 males were sterile. The reciprocal cross,

ORL female X AUB-B male, produced results that were different

from the initial crosses, in that in contrast to the initial

cross where heavy mortality of the F1 male pupae was observed

(sex ratio of 12.2% males) (Table 4), this time all F1 male

pupae died (Table 6).

Crosses between the A and B strains of sympatric origin

were conducted. The cross AUB-A female X AUB-B male produced

Fl's with the same characteristics as those produced when

AUB-B males were mated to ORL. Hatch was high (78.6%), but

the % survival to adult was low (23.5%), and all of the F1

males died in the pupal stage. Results were different for

the KBG-A female X KBG-B male cross. Mortality in male pupae

was not pronounced; and therefore the sex ratio was closer to

normal (40.8% males). Survival to adult stage was also

higher (39.7%). In both the reciprocal crosses, AUB-B female

X AUB-A male and KBG-B female X KBG-A male produced progeny

which were normal in viability, and the sex ratio was normal;

but in both cases all F1 males were sterile.









The final pair of reciprocal crosses between AUB-B and

KBG-B established that these two strains were compatible.

Progeny resulting from these crosses were comparable in every

respect to the control (ORL X ORL). All F1 males were

fertile.

Backcrosses

Three of the four possible backcross combinations were

performed, using the AUB strains (Table 7). Hatch was lower

in the backcrosses than in the F1 crosses. In both the F1

(AUB-A female X AUB-B male) and the F1 (AUB-B female X AUB-A

male) backcrossed to AUB-A the % survival to adult stage was

comparable t the A female X B male crosses. Sex ratio was

skewed in favor of females, but to a lesser degree than

either of the crosses: AUB-B female X ORL male or AUB-B

female X AUB-A male. In the cross, F1 (AUB-A female X AUB-B

male) female X AUB-B male, % hatch was also lower than in the

F1 crosses, however % survival to the adult stage was

significantly lower. Sex ratio was also skewed in favor of

females. Sterility persisted through the backcross, and all

of the backcross males were sterile.



Hybridization in Nature

A X B hybrid males can be recognized by microscopic

examination of the testes (Figure 8B). Using this technique,

it was possible to examine field collected males and

determine if they were A X B hybrids. Males were collected









>1
.-P
Sl-I


0 0 HI I






4-%
1 ) r.. 4



H4 0 (0 r-i









C *
>0 :>3 0)


0 *r 0 U CM N









4-I
CC




4-i



01. 0) H





O W
4 -i













1 O r-
4--4






















*c *
0 Q)
) 0 4-O


r- Ui
> La






-HI I I


4-) -H N C
0 0 H








4-) U
g: .





) En 4 0
o4 0



Q)- (d 0











H-t m pq m
D D

Pm









from the AUB and KBG sites, returned to the laboratory, and

the testes were dissected and scored for sterility. Results

are presented in Table 8. Of the 143 males from the AUB

site and 185 from the KBG site all were normal, indicating

that all were either A or B types.

Discussion

A variety of different techniques have been applied to

the study of speciation. However, many of the measurable

species differences studied contribute little or nothing to

reproductive isolation. Hybridization experiments are

designed to measure post-mating reproductive isolation

directly, and as such have been recognized as the best method

available for the study of genic incompatibility (Templeton,

1981).

The hybridization experiments described in this study

began with the screening of nine geographic populations in an

attempt to detect genetic incompatibility. ORL served as a

standard to which all field strains were compared. This

strategy eliminated the need of making all possible crosses

between field strains. Sterility in F1 males was observed

for some of the crosses between ORL and three of the field

strains, ARK, AUB, and KBG while other crosses between these

strains and ORL produced normal F1 progeny. These results

established the existence of two sympatric sub-populations at

these sites. Crosses between ORL and the remaining six field

strains produced normal males in the Fl. It should be noted

that these results should not be interpreted as meaning that


















Table 8. Survey of AUB and KBG populations for presence of
sterile, hybrid males.




Number of Number of Number of
Population males examined fertile males sterile males




AUB 143 143 0

KBG 185 185 0









these populations are conspecific with ORL or each other.

Under the conditions of these experiments pre-mating

isolation between any of these strains would not be detected,

nor would post-mating isolation between specific field

strains. Crosses between A and B strains developed from the

AUB and KBG populations further confirmed the existence of

the two types. The incompatibility between the AUB-A X AUB-B

and KBG-A X KBG-B crosses proves the existence at these

sites, of two reproductively isolated and sympatric

populations. Crosses between AUB-B and KBG-B produced normal

progeny suggesting that these two populations are

conspecific.

Backcrosses of hybrid females to A and B males resulted

in a significantly lower hatch than was observed in any of

the control matings. The fact that male sterility persists

through the backcross indicates that potential gene flow

through F1 females is inhibited.

The examination of field collected males from both sites

provided no evidence of hybridization, indicating that some

form of pre-mating reproductive isolation separates the A and

B populations in nature.

Kitzmiller et al. (1967) used the results of

hybridization experiments to assess the degree of

relatedness between different species in the A. maculipennis

complex. Their results agreed well with phylogenetic

estimates based on chromosomal differences. Relatedness is

determined by the degree of genetic compatibility with









results ranging from failure of sperm to fertilize eggs to

the production of adults in a normal 1:1 ratio but with

varying degrees of sterility. The former case indicated a

distant relationship, the latter, a close relationship.

Applying these criteria to A. quadrimaculatus species A and

B, it can be seen that they represent two closely related

sibling species. In the cross, species A female X species B

male, adults were produced, but the sex ratio was usually

distorted in favor of females. Survival through the larval

stages was high, but heavy mortality of male pupae was

generally observed. In the reciprocal cross adults were

produced in a 1:1 sex ratio and survival through all stages

was high. All male progeny from both crosses were sterile.

Female progeny can be described as semi-sterile since when

backcrossed to species A or B, hybrid females produced

smaller egg batches and hatch was low. Sterility in male

progeny persisted in the backcrosses.

Relationships similar to that between A. quadrimaculatus

Species A and B have been described between other sibling

species in the genus Anopheles. Within the Palearctic

species of the A. maculipennis complex, several species show

relationships comparable to the one described here. The

cross, A. labranchiae X A. atroparvus, produced sterile males

and fertile females, but in this case male fertility was

recovered in the F1 backcross to A. atroparvus males.

Sterility in both sexes was observed in the F1 produced from

matings between A. maculipennis and A. atroparvus, whereas









only male progeny were sterile in the cross A. subalpinus

Hackett and Lewis X A. gambiae. All possible crosses between

members of the A. gambiae Giles complex have been made

(Davidson, 1964, Davidson and Hunt, 1973). Some crosses

produced only males. Without exception all of the males were

sterile, and when produced, the females were fertile.

Crosses between A. merus Doenitz females to A. gambiae s.s.

or A.arabiensis Patton males produced all male progeny.

Likewise, matings between A. melas Theobald females and A.

gambiae s.s or A. arabiensis males produced only males. On

the other hand, the crosses A. melas female X A. gambiae

species D males and A. gambiae species D females X A. merus

males produced sex ratios strongly in favor of females (25

and 16.7% males respectively). Mahon and Meithke (1982)

report the results of crosses between the three sibling

species of A. farauti Laveran. The relationship between

these three species parallels that between A.

auadrimaculatus A and B. All crosses between A. farauti

species 1, 2 and 3 produced sterile male progeny. Sex ratio

distortion in favor of females was observed for the crosses

A. farauti species No. 1 female X No. 3 male (5% males) and

A. farauti no. 3 female X No. 2 male (9% males), but the

reciprocals of each produced normal sex ratios. The general

pattern of sterile males and fertile females in the F1 have

been reported for a number of other species, including the

crosses A. balabacensis Baisas X A. dirus Peyton and Harrison

(Baima and Harrison 1980) and A. sinensis Wiedemann X A.









engarensis Kanda and Oguma (Kanda and Oguma 1978).

Unidirectional male sterility has been reported in A.

culicifaces Giles, where the cross A. culicifaces species A

females X species B males results in sterile male progeny but

the reciprocal produces fertile males (Miles 1981). In A.

coustani Laveran species A and B a similar unidirectional

effect has been observed (Coetzee 1983). In this case when

A. coustani species B is the female parent, the cross

produces sterile male progeny. However, the reciprocal cross

results in the production of non-viable eggs.

In conclusion, hybridization studies involving nine

geographic strains of A. quadrimaculatus revealed the

existence of two reproductively isolated sympatric

populations. No evidence of natural hybridization between

the two forms was found. These results support the

conclusion that A. quadrimaculatus actually exists as two

sibling species, provisionally designated A. quadrimaculatus

Species A and A. quadrimaculatus Species B.















CHAPTER III

ENZYME POLYMORPHISM AND GENETIC STRUCTURE OF POPULATIONS

OF Anopheles quadrimaculatus SPECIES A AND B



Introduction

The European Anopheles maculipennis complex stands as a

classic example of sibling species and is cited in almost

every written account of the sibling species phenomenon

(Mayr, 1963; 1969; 1982; Dobzhansky, 1970; White,1973;

Wright, 1978). The entire complex consists of fourteen

Holarctic species. Interestingly, the nine Palearctic

species are all morphologically identical, or nearly so

(sibling species), whereas the Nearctic fauna was,

heretofore, thought to be made up of five, closely related,

but morphologically distinct species. Recently,

hybridization studies have revealed that A. quadrimaculatus

Say, one of the Nearctic species, actually consists of two

sibling species (Chapter II). These studies demonstrated

that the two species exist sympatrically at three of nine

localities sampled.

When gene flow is restricted between two populations,

differences in the composition of alleles and in their









frequencies within each population may develop. Such

differences can be measured by determining the allelic

frequencies at a number of loci within each population and

comparing them. One way to accomplish this is to measure

allozyme frequencies.

The purpose of this study was to measure the genetic

variability of A. quadrimaculatus in the southeastern United

States, in an attempt to answer several questions. First,

how much genetic differentiation exists between the two

species and between local populations of each? Second, does

the pattern of genetic differences confirm the existence of

two species? Third, do allozyme phenotypes occur which can

be used to distinguish reliably the two species? Finally,

what inferences can be made concerning the phylogenetic

relationships between the two species?



Materials and Methods

Adult A. quadrimaculatus were collected from the same

nine sites that were sampled in the previous hybridization

experiments: in Florida at Ginnie Springs (GIN), at Lake

Panasofkee (PAN), (Gainesville) and Kanapaha Botanical

Gardens (KBG), and at Lake Panasofkee (PAN); in Alabama, 41

miles west of Auburn on 1-85 (AUB), and at Guntersville

(GUN); in Mississippi at Skene (SKE); in Arkansas at

Stuttgart (ARK) and Bebee (BEB) and in Louisiana at Lake

Charles (LAC, Figure 6, Chapter II). One additional site,

not sampled in the hybridization experiments, was included,








Lake Seminole in the Florida panhandle at the Florida-

Georgia-Alabama state line (Figure 6, Chapter II). The

collecting techniques employed were identical to those

described in Chapter II. On return to the laboratory

collections were sorted on a cold table and stored at -600C

until prepared for electrophoresis. All mosquitoes included

in this study were field collected adults.

Determination of species by hybridization was achieved

by crossing field collected males to species A females (ORL

strain, see Chapter II). If the resulting male progeny were

sterile, or no male progeny were produced the parental male

was identified as species B, if normal male progeny were

produced the male parent was identified as species A.

Electrophoretic techniques for 27 loci in 20 enzyme

systems were described in Chapter I. Of these, the following

20 loci were included in this study: Aconitase (ACON, 1

locus), Adenylate kinase (ADK, 1 locus), Catalase (CAT, 1

locus), Glutamate oxaloacetate transaminase (GOT, 2 loci),

alpha-Glycerophosphate dehydrogenase (alpha-GPDH, 1 locus),

Hexokinase (HK, 2 loci), Hydroxy acid dehydrogenase (HAD, 1

locus), Isocitrate dehydrogenase (IDH, 2 loci), Lactate

dehydrogenase (LDH, 1 locus), Malic dehydrogenase (MDH, 1

locus), Malic enzyme (ME, 1 locus), Mannose phosphate

isomerase (MPI, 2 loci), Peptidase (PEP, 1 locus),

Phosphoglucose isomerase (PGI, 1 locus), Phosphoglucomutase

(PGM, 1 locus), and Sorbitol dehydrogenase (SODH, 1 locus).

Techniques used for the visualization of the enzymes,







61

including buffer systems, staining procedures and locus and

allele nomenclature are described in Chapter I. In addition,

Chapter I contains a description of the genetic basis of

isozymes at nine loci. The banding patterns of the remaining

isozymes were consistent with a genetic interpretation and

agree with previously described phenotypes in other

Anopheline species. Thus banding phenotypes (electromorphs)

could be scored as genotypes. To insure identity of alleles

between populations, a series of gels were run with samples

representing each population run concurrently on the same gel

in combination with all other populations.

Analyses of allele frequency data were performed using

the BIOSYS-1 computer program of Swofford and Selander

(1981).



Results

Initially, allele frequencies were calculated under the

assumption that A. quadrimaculatus consisted of a single,

randomly mating population at each of the ten sites sampled.

This assumption was tested by calculating chi-square tests

for goodness of fit to Hardy-Weinberg equilibrium for each

polymorphic locus. A locus was considered polymorphic if the

frequency of the most common allele did not exceed 0.95

(Ayala, et al., 1974). This definition was used throughout

this report, unless otherwise stated. Significant deviation

from Hardy-Weinberg equilibrium was observed in two of the

tests. Chi-square values for Idh-1 and Idh-2 were highly













4C 0U


> -H
En t'4-l

( O4-4
Q)O)

-0 -H a
ua


kON
4-)






O
0 0 -0





Q)O -


4) 0
> i o








4) 0






S 0
:3:--- 0










>I


0. -
4 Cfl 0
a) U)









44 10









O
oc.


0-4 a)
>1 a)


M 'O Q






























0 r3 U
*-H



4 0
Ho I


On 0
drl o

o) a)

a)

























43


H LO m0 (N
0 (n co N>


om
0)
-)

a) t
-p >1
UON
0 N

) 0




0 o
Q)O

ao
a)

a) 0

>

4- >
) N
En o

-P
a)


z;


-4H r- 0"
'0 H-
v> n 1-1
U)i-i


-P-
0




4J
O







0



4-P


C
a)














E1
Q)














0l



d-
U

4-)


















S0
C
























0
14
0






























0
C
4)





0)






U)
.-















H
-P
(1)



U)
Qa)




















r-i ^
<-i
i< Q
(0 ^


















Figure 10.


Zymogram of gel stained for IDH (isocitrate
dehydrogenase) showing the positions of the
diagnostic alleles at the Idh-1 and Idh-2
loci. Q-2 = marker strain serving as
control; AUB-A = adult males of species A
from the AUB site; AUB-B = adult males of
species B from the AUB site.


















IDH-2


IDH-1









significant at four (ARK, AUB, KBG and SEM) of the ten sites

(Table 9). In addition, Selander's (1970) D coefficient is

negative in each case, indicating that a deficiency of

heterozygotes exists. These data strongly suggest at least

two populations at these sites.

The populations at ARK, AUB and KBG were known to be

composed of the two sibling species, designated A.

quadrimaculatus Species A and B. Differences between the two

species in allele frequencies at the IDH loci would account

for the deficiency in heterozygotes. To demonstrate this,

individual field collected males were positively identified

by hybridization, electrophoresed and the gels stained for

IDH. A total of 84 individuals from the AUB site and 22 from

the KBG site were tested. The Species B males were fixed for

a single allele at both the Idh-1 and Idh-2 loci, but species

A was polymorphic at these loci. Using the genotypes at

these two loci it was possible to correctly identify 32

individuals as species A and 74 as species B.

Figure 10 illustrates a typical IDH zymogram comparing

the two species. The diagnostic value of the IDH loci was

calculated after Ayala and Powell (1972). Using the genotype

at the Idh-1 locus, individuals could be correctly identified

as being species A or species B with a probability of 98.61%.

The Idh-2 locus provided correct identification at a

probability of 98.43%. Neither satisfies the definition of a

diagnostic locus, which has been defined by Ayala and Powell

(1972) as a locus which provides correct identification at a









probability of 99% or higher. When the two IDH loci are used

together, the probability of correct identification is

increased to 99.98%. Thus, a tool was provided for rapid

identification of individuals which could be grouped into

discrete populations whose genetic constitution could then be

defined and compared.

Chi-square tests for goodness of fit to Hardy-Weinberg

were repeated following grouping assuming that both species

represented single panmictic populations at each site. The

results for the species A populations are presented in Table

10; species B was, as mentioned, fixed for a single allele at

each locus. In two populations (SEM-A and AUB-A) the tests

indicate a significant departure from Hardy-Weinberg

expectations. Prior to grouping, the IDH genotypes departed

significantly from Hardy-Weinberg equilibrium, but after

grouping a close-fit to predicted genotypic frequencies was

apparent, except as noted for SEM-A and AUB-A.

Table 11 presents allele frequency data for the twenty

loci analyzed. Also included are the number of individuals

examined per locus for each population. Data for the

population of species B at the ARK site is not presented

because it was not possible to obtain an adequate sample size

representing this population.

Comparing populations of species A with those of species

B, the loci having the greatest differences in allele

frequencies were Idh-1 and Idh-2. At the Idh-1 locus the 100














C M
0





EC )
-H II

0)



M O
0 W
C)
0) 4-)



N)
OM

4 0




4-)
rC )



0 U






0 10
U 'd



QC 44
040 a)

O -Ii 0





S-4H CN

) I r-)
S41 (0











00




4O- 0
0 U
c 0 w










-H I 44
ao a











n0 0
C) -



-Q)
S 0
E0-


O C




H

OCH

O 1





.--I

E-i


OCl e CO N


(N
X



4-i
do 0

UN
0) 0
0
04 (
Q)







CUN
to
0 -P
N o

00
CN













(UN
0c o
























0 N
r0
UN
xC

X)
C)
X (1
0) 4


Hr-4 O'0
V.0 H
ro n r-
1-1


Q)




4-1
C
C












0
0
U
d








r4-1













a)

C)
0














Q)
-4













0
0



Q)
m
Ot
H












Co










-,--I
0m
aC









OH
Q)


r-4
0



Q)
H(
C)














Ifl4-












eC






























0



-P-
I















4l-




-H


-H




44-0
0





jl


OHO
000
O0.'O
L o o o


000
000
000
ooo
oooi


rOOOOO
N 0 'I o



(coloo
0o co o




.0 0 0 0 0
OHL O
iIOOOOO

H C o H 0
Krooooo
00000


000000





H c 0 0 0-0 0
000 00
*OO*OO
OOOa\oo




co oz n N o
0 00 000

OOOOO


0oN o














0 CZ O 0
000000
0 0
r100 00














0O .
'.00000







00 00 0
S ** r o


o cri (Y o o







S**0 0* 0
OMMOO
OmHoo




c00000 0




SM
OO


0 o- N

000
0 0)


LOcN
Sco
000)
oH.
HOO




WOO
o o
0 m0
0000


OO

00

OV
0001%



0 *
HOO
O %
0 00m

ooA

00
0 ON


0'100
0o m
O N
r *


o m
o C
o0oo


00)
00
**
co


o0
rAr


00
00
00
o0
L1 0 H


oo
00
00
Soo


00
00




00
00
%1000
6 1.4
HOH


00)
H00
00

0000
O C;
00



00
oOO
00
0 *0
HOH


000
000
000
co *
'OOHO


O o
0010
0000
HOOO

0 N
000
0 0* 0
r-A C;


0
'. .


Lo

On
n o


000
000
HOOO
L **


ONQ
0 CM 00
0c- co
000
H *
o0000


000
000
000
OHOd


coo
000
000
000
CO 0 ; 0

000
000
LAOoo
r- *
r- 0 HOHO- 0


coo o
000

CO 0
COOo
000

o r- o
moo r-

000



mao
000





000
MOOO

CO r- 0




000




%I0 LO 4
000






n N o
LON 0


OO S


000


000
***


000 o
ONO


e0n
00o
N*


00
oo

H
00





00


00
C LO






mO
OO




r- 0 0
mLo





00
Hoo


















m
rlOO
moo
r-' L


00


C0oo


NC.H

LOOO


M 1^

LA..
LAOO-

NH r
CNN*
N..o


r.
r11
0









LO 0
NO




N.
CA *
1-1
HO






0




L-O
0
'0








HO




il 0
NO


00
H *


C.
NO


00

H

i-lo


00



cnO


00
,'o
**o
uOO


OHN C C O
000 HHHH mNn H C
SNHO l l ii II
II II II


H! I


r-lrl




O co CM
NMOO


coo













NOOO
000








OOOr


000


HO
OH




HO
0-


HOO


000


MOO

000


SNH~

OO
66cu


r- HOC


0 %0
0o

O0

O)o

O


(NO
-HO
HO
0


HO
cN
r 1-
dTC


le*00
o00o
OO
00 r
OHi


o r
o00

o0


0000



toooO
000

OOO
*~ *


0 0o .q
000"

000


-4 0
r-O 0
- 0
0


00
NO O
T- O0
OO


0


WHO



WOO
r- O o








H.OO
co H OO




WHO
000
H LO o





HMOO

000
r-iin


,-1 o co
HON
H o 00




00

OWO
od

*n *
00


0000
6oo

HOO

000
***


0 C
%1 00 CM

HHn r- r-

z


(n 00
o D00
00
H o


o\o
00o


MOO

000


k0000
NooO
HOOO


HOO





SN OmO
00100
CHoOO
000


HO'000
COOO
000

co 0oo
000

1000
riooo
**OOO


rOO-
HOO
co; oo
HOO0
HOO0




(OOO

000


0o0
o


0r



0
.lo
Hlc

OB


oo
00
00
* .

00
oo
oo





* *
00


NOO
1000
HOO
-HO


0(No
O(N(
HHHlr
HHHlr


(1000
000
4 oo


r- kn

0
.
tO


HOQO
00- O
r- 0) 0


00
00
00
oo
rO0


1-4 CM r-


000
000 0




000

00oo


000
m00 0
600
LO00 O
m* 0
MOO


3) o0



00 k0 -Rr
H0(o





r-i tn O



0-,00
OOiO



00
HOO0




00


HO
r-10


H'00
HO0o
1000

(NOO
'0000
HOOO
HOOO


OH
N

CM I


(100
00
o0


000
000
000
ooo


00000
00000
00000
OHOOO
OOOOO*
OOOOO00


N 0
coo
NO
0


LOOO
000o


00

0) NLO

00o
cNiOH
00

HN (
LOOO



CM 0i
00



*
00


ON
0or
oo


%oo
00o
D 00
HO
0

W o
r-o

0o


000
000
000
*ooo
r-OO


nO nO 0
OHO
COO
000


Nmo
0 r, 0
coOO





00
000




o 0 o




OHNO
WOO
000
- o0









)OO
000
OHO





NOO



000
odd





HO 0
000


tn 0
(NNO
WOO




COO
000

68O
Woo


0
H
r-1



o
H
0
1-1




CM
H
.0

0


en
HO
S
O


CO -
WO

00


000
MOOH(N





r-1
II

H


4:

















mt

































































--I
r-

8


C LO
O \0
* *
00


r, 0
No
(Nd
* *
00


0000
0000
0000
\0000

0000
0000



0000
0000
0000



moooo



CN r-4 r o
000000
(n *0* *
r 'cOOOO







O 0 0 m 0
Q 00 10
S *
(N0000


LO c0 ZT
.- .LO

000


kD
,-I
ano
(N
r-o
HO

00
N d
HO1


0co n o


ooo


S(1i o

000oo
m N


NM % r- 0


C 00000
O *****


co U
HOOC



(1o
MO O
'00O


LON0




ooo
em co 0
000
C; 6


000
000
000
ri o 1 o


00%0
***


0
0
0
0

0
o
0




c;
0
0


0
0



0
0
0

0

o

o
0

o
o



0
o
o
0


000
000
000



000
000
000
H 0
Qo- oo


00
00
o *o
MOO
HOO

00
00




o o
1000
o *
HOH


oo

00
H *0



OO
0 0
r- *
H o
00
00
00
,,oOH


0
0
o;
o3


ScH- o

00 0-i o


Hdd
cn o o-t




0 000
000


000
000
0oo

o0e
000
000

S000
0 (Nr -
,r in o






MOOO


0 CHO
H000




0N On o
0 o Ho0




O)OC
0000


kD 0o

r
H 0


r-(



0


o
o
H *



o


oo


0en
Om
o00

dd


ONN>

0 ...
rln oo


0
0
o
Hl *
HO


0
01
Hn



HO



0
O


0HO CO
r-- ,- o



000
tn M -
moo



H' 0



000
6OO
000


0
0
0





CO ;
0n.
to


0


00
0


0


cO
0
o
0

o
o
o
0


o
(N
0
0


HOO
00
ooo

r-0

n o
00N
H ..



or-
00
o o
D *


o
0
o


0
)o
o
o

H.

H
0
o



o
c. .


o
o



0
o0
H-io


0'
o



0
In


o
d




.1



o




c;
0



0


0


o


oco
00t
00
00


NOO0
M0 0
oOO
6000

000
000
000

HOO

000

000
(100


moo
000
000






o o00
000
000
000




000


000




000



0N00
0)00
000

(OO







000

000
eninoo
0 CM


HHN 0 0n 00
Hr- C- CO O o 0 o
IN C N NcN 10 1 0 N O 0 l
II II II IIc c 4 H H m
ZHHHHH
(N


00
00
00



oo
oo
00
00



00
oo
oo
0OO
S*oo



0OO
00O


o
o



o
o
o
0
0
0



















Im





















Il


Es

iii
vl




I


00
o
0

'.o
0


coo
o




1 0
O 0
0





0
mco





O 0
Ho
0






No
*o
0
0O
too



oco



0







H 0
a\o
o




0

oo
0




0
0
0
00





0
o

oO
o
d






Loo
o




'n o
0


0


oo


o


IIIIIIII
IN-icmr- m-HooH


HHHHHHHH


NON
NM 0 >


000

CN*
OOO




OOr
000


SU)o





oom
000




0'4T0
ooo
000






ooo
OOCJ*



000
000
oo n

000

o o H
0*0 4








000






000













OOHo
000
o*o*




odd








000
000
ooo
*f n
00 oM


o 0 in
ONO
o iLO (

000


o co r-

000

%0 co r-


000

000 ( 0
000


,,o r- o




0 cn 0o
000
000







ooo
o-HO
000




OHO
oHo
ooo
0 r-1 0
000




OOO
000
000




S t
OHO
000







On.HO
000





0 r1 0












toNo
000
000




000
0 r-1 0


000












OOO
000




000




C;d


ONNO
0 V-4 0
odd
o00

000
01000
000

OHO

(000
HOOO




tnO co H
ooo



000




000
0 m 0
-i 0O0





000


000
H0O~O
HOOio
000
NO1 t.0


000


000
00 00


000



000




toN
coonoo



000

000



'0000
000

9OHO
So o
co000

















ION
rOO






0000
0O0



N N


*HH
NNN


oo0 0oo00
i -i4 0-4 -4 -4
z oHHH
S dif id i


HO
o
0
0


0 m
m r~


HO

*


0




c oo
o




0

coo

HO
e





r-l0

NO
LO
H0



o
Ho
0






HO
o








IAN


0
oo

,N-
0



0



*
o




o


0oo
m O

000




000


Hnoo








cooo
ooo
00n 00




000








NOOo
000
c- o o













So0o
000
HO C



MOO
co Na o










000
HOO

000
N 0 0





MOO



000













NOO
000





000
000






















m M 0


o O
coII H r-
II -1 -14 -4-
z KRKK


0000
000
OOH






0000N
n 0o coM
HOO0










000
n O l co











H000

0000
HOO0-t
000

0oco%
r-i o H r
000







' cr-i o.


NOOC


HOOD
H000
000

r o co co







000
0000








000

O* HH


000


HOOH C


000
(r o 6i \





































4s


SOO

00




00


(1 c L r


000


0


o

0%D-
mo
DH
0
0


vl


(1
co
0
0
o


0



r'-
o


d


o (n n
000


060


000

ddd
oim
omo
00C
OWO


o c^ ,o


000


0
0
0

d
0
0
0
0

o
0

0

0
o
o



d
0
0
0

0
o
o


0 n
o



0
0
no



NO
C0o


-0oo
00oo0



N 0-1 o





MOO
000


minoo

ddo


oMo00o
* ** *000


00000
on oo o o





WHOOO
00000


0r-l 0 0 0
zooo O

00000

CN r- 0 r


00000
6 6 6 C


00
oo
00
00
OO


OHO
0000



0000
O r C4O
0 a 0


t- 0
t--o
o
0
0



0
8


000
000
000


mo 0


000


00
H 0 0)
88OQ


Ino
ON"
00)
oCn
dd


H0 WC
COO
OCO



00
c-OO n


o a%
11oo


OIW
Sn 0 ,-l r,-l 0M ( 0 ,-I ,-l-
0 Ci i H N OH
IIWOHHHH II

z k kkk U2U2c1


t- on
o0
dd


o
0
0


o 0

0
,-o


00


000
000
000
ddd

o0o0
OHO
000



oioo
000


oLtC;


000
000
000
000


000








OOO
000
ooo







0000
ddd000





OOO
000
000
000
000
000



ddd000


000


000








allele predominated in populations of species A, whereas

populations of species B were fixed for the 85 allele. A

similar pattern was presented for the Idh-2 locus. The 136

allele was most frequent in species A populations while

populations of species B were fixed for the 173 allele.

There were four loci at which both species shared the

most common allele, but differed at alleles with intermediate

frequencies. At the Got-2 locus both species shared the 38

allele, but the average frequency of this allele in species A

was .335 while its frequency in species B was .049. There

were eight alleles at the Mpi-1 locus with both species

sharing the frequent allele. In populations of species A the

87 allele occurred with a frequency of .372, whereas, in

species B its frequency was .159. The 100 allele was the

second most common allele in species B with a frequency of

.324, the frequency of this allele in species A was .109.

With the exception of ARK-A and AUB-A, the 114 allele at the

Pgm locus was the second most common allele in populations of

both species, but occurred at very different frequencies. In

species B the 114 allele occurred at a frequency of .219, but

has a frequency of only .064 in species A. Likewise at the

Me locus the 100 allele was the most common allele in both

species, with the 94 allele being the next most frequent,

(absent in KBG-B) with an average frequency of .082 in

species A, but only .011 in species B.

Polymorphisms existed at six loci which showed little

differentiation between the two species. These included









Acon, Got-1, Had, Mdh, Mpi-2 and Pep. Eight loci: Adk, Cat,

Gpdh, Hk-l, Hk-2, Ldh, Sodh, and Pgi were not polymorphic by

the 0.95 criterion.

Measures of genetic variability, including mean numbers

of alleles per locus, percent polymorphic loci and mean

heterozygosity are presented for each population in Table 12.

These results indicate that populations of species B are less

variable genetically than those of species A. The number of

electrophoretically detectable alleles, occurring at a

frequency of at least 1%, varied from one (Cat, Hk-l, Hk-2,

Ldh) to eight (Mpi-l). The mean number of alleles per locus

over all populations averaged 2.9 for species A and 2.3 for

species B. Species A was polymorphic at 50.5% of the loci

studied and species B at only 31.7%. Species B had a lower

mean heterozygosity as well, with 10.3% of its genes, on

average, in the heterozygous condition, while species A had a

heterozygosity of 15.9%.

Estimates of genetic distance and similarity between

species and between populations within species, were made

using the I and D statistics as defined by Nei (1978). I and

D values for all pairwise comparisons are presented in Table

13. The average distance (D) between local populations of

species A was .005 ( .003) and between populations of

species B was .002 ( .014). Genetic identity and distance

coefficients demonstrate a high degree of differentiation

between the two species relative to that of local populations

within species. Genetic distance is much higher between











Table 12. Genetic variability in populations of Anopheles

quadrimaculatus species A and B.



Mean sample Mean number Percentage Mean
size per alleles per of loci hetero-
Population locus locus polymorphica zygosityb


SPECIES A

2.7
(0.3)
2.8
(0.3)
2.8
(0.3)
2.8
(0.3)
2.9
(0.3)
2.8
(0.3)
2.8
(0.3)
2.9
(0.2)
3.4
(0.3)
2.8
(0.3)
2.9

SPECIES B

2.5
(0.3)
2.3
(0.3)
2.2
(0.3)
2.3


50.0

60.0

50.0

45.0

55.0

50.0

50.0

45.0

55.0

45.0
50.5


40.0

25.0

30.0
31.7


0.127
(0.037)
0.180
(0.042)
0.143
(0.038)
0.162
(0.044)
0.137
(0.037)
0.159
(0.043)
0.162
(0.042)
0.155
(0.043)
0.175
(0.040)
0.148
(0.042)
0.159


0.103
(0.038)
0.102
(0.042)
0.105
(0.041)
0.103


a0.95 criterion
bHardy-Weinberg expected


ARK-A

AUB-A

BEB

GIN

GUN

KBG-A

LAC

PAN

SEM-A

SKE

MEAN


152.1
(19.9)
96.3
(7.2)
113.2
(9.4)
109.4
(6.5)
112.8
(6.7)
107.9
(14.3)
118.0
(8.0)
167.8
(13.7)
151.0
(13.4)
116.2
(9.5)
124.5


AUB-B

KBG-B

SEM-B

MEAN


134.6
(13.9)
96.6
(6.8)
53.0
(2.0)
94.7
















0 0 O 0


t A




















.( (0

















To o
O 6














.
0
































*H i
-2
-


*-


O

O




LO


C;


o





ak
0





















o



0







o
0r.



















0

*





O




o
0(.
.')
















en


O n -I M fN
4 0 H 0 0
.4 0 H 0 0
.4 0 0 0


o N H
H NM m 131 LA %. rN co 0)' H H H H


co LO 0a%
o\ o\ r
ONC ON (3)
o 0 0


a) a
C O O
O C*








0 0
0 K 0
4(,


(-1








species at the same locality than among the same species at

different localities.

A cluster analysis of the matrix of D values, using the

unweighted pair-groups method with arithmetic averages

(UPGMA, Sneath and Sokal, 1973), produced the dendogram

illustrated in Figure 11. The dendogram had two clusters at

a distance level of .092. One cluster contained the three

populations of Species B, and the other ten populations of

Species A.



Discussion

The data from this study confirm the existence of two

sibling species in what was formerly known as the single

species, A. quadrimaculatus. Analysis of genotypic

frequencies at two IDH loci revealed a highly significant

deficiency of heterozygotes at four of the sites sampled.

This phenomenon, known as the "Wahlund effect" (Crow and

Kimura, 1970), is interpreted as resulting when

reproductively isolated populations occur sympatrically and

are sampled as a single population. These data alone provide

very strong evidence for the existence of two species (Makela

and Richardson, 1977; Bullini and Coluzzi, 1982).

The presence of Species A individuals heterozygous at

the IDH loci suggested that a limited amount of gene flow

might occur between the two proposed species. In fact, it

was revealed that species B was fixed for a single allele at

both IDH loci and species A was polymorphic and included the























. 4 r-I

CO
O c
o I














tP 4
r-1 i (





UQ 0



MO
c cd





00.
O C


Q) W















0 0 0
d0 *
>tn




UO
(UQ)

10 E











6 -H
O rUICd
U










0 4-- 0




-H
0 0 '
r-l 0
e u -
(0
M 0
&> 0 ^
0 C 4-1
ao


C C
Q 0










0





0
0

-A




0


0
O4
0




O
0
0



0


0



0


0O
b
0

O


b
0


o0

b
0



o
b



o
0




o
b U, ___ m
mWm c mr c m' C
0 Z Q S Z m m Z
_, do > >W
i








same two alleles for which species B was fixed, albeit at

relatively low frequencies.

A comparison of populations of the two species yielded

an average value for the Nei's distance coefficient of 0.092

( .014). This value is much smaller than the value between

sibling species of Drosophila (D = 0.60) reported by Ayala

(1975). Average genetic distance between mosquito sibling

species are generally lower than those found in Drosophila.

The values reported in the literature for various Anopheline

sibling species are summarized in Table 14. The average

genetic distance between those members of the Palearctic A.

maculipennis complex which have been studied (excluding A.

melanoon x A. sacharovi) is 0.183. This is substantially

larger than the value between A. quadrimaculatus species A

and species B. Low values for genetic distance have been

reported in the A. gambiae complex (eg. A. gambiae x A.

arabiensis, D = 0.070). Extremely low values for D have been

reported between some members of the A. marshallii complex

(eg. A. marshallii species A x species B, D = 0.045 and A.

marshallii species A x species C, D = 0.029). These values

are comparable to the genetic distance between local

populations of A. maculipennis, D = 0.032 (Bullini and

Coluzzi, 1982). Bullini and Coluzzi (1973) observed that low

values for genetic distance, as in the A. gambiae complex,

are associated with higher levels of chromosomal divergence.

They suggest that the speciation process in such groups is









Table 14. Genetic distance (D) between sibling species of mosquitoes in the
genus Anopheles.



SPECIES COMPARISON D REFERENCE


Anopheles maculipennis complex

A. message x A. subalpinus

A. subalpinus x A. melanoon

A. subalpinus x A. maculipennis

A. melanoon x A. maculipennis

A. labranchiae x A. atroparvus

A. melanoon x A. sacharovi



Anopheles cambiae complex

A. gambiae x A. arabiensis


Bullini and Coluzzi, 1982


0.119

0.154

0.162

0.228

0.250

0.526


Bullini and Coluzzi, 1982


0.070


Anopheles marshallii complex

A. marshallii sp. A x sp. B

A. marshallii sp. A x sp. C

A. marshallii sp. A x sp. E

A. marshallii sp. B x sp. C

A. marshallii sp. B x sp. E

A. marshallii sp. C x sp. E



Anopheles quadrimaculatus complex

A. quadrimaculatus sp. A x sp. B


Lambert, 1983


0.045

0.029

0.118

0.107

0.220

0.128


0.092








different than in groups with higher genetic distances, and

lower levels of chromosomal divergence, as in members of the

European A. maculipennis complex (Bullini and Coluzzi,

19823). Templeton (1981), however points out that the

difficulty in such interpretations is the fact that it is

impossible to distinguish whether these differences are

responsible for the speciation event or are consequences of

evolution subsequent to speciation. Genetic distance values

may only indicate how recently a speciation event has

occurred, being smaller between species which have more

recently diverged (Avise, Smith and Ayala, 1975; Carson,

1976).

Conclusions can be drawn regarding the speciation

process in this case. There are several facts revealed in

the data in this dissertation which suggest that A.

quadrimaculatus species A is the ancestral species and that

species B evolved from it via a founder event. The genetic

distance between the two species is small, relative to the

distance values reported between mosquito sibling species,

which generally range from 0.10 to 0.30 (Bullini and Coluzzi,

1982). Low values for genetic distance have been observed

between even morphologically distinct species recently

separated by founder events (Sene and Carson, 1977). These

results conflict with conventional thinking which would

predict large genetic distances resulting from a "genetic

revolution" (Mayr, 1954) produced by the founder event.

Templeton (1980) suggests that founder events are more likely








to affect only a small number of genes, while the majority of

the genome is unaffected (Templeton, 1980a). In fact, there

is evidence that enzyme coding loci are relatively

insensitive markers of speciation (Templeton, 1980b).

Populations which go through a small bottleneck

experience a decline in genetic variability. The magnitude

of the reduction was thought to be substantial, with only a

small proportion of the original genetic variability left

(Mayr, 1963). Nei, et al. (1975) studied the problem

quantitatively and determined the loss of variability to be

much smaller when population size increases follow the

bottleneck. Table 12 summarizes the data on genetic

variability in species A and B. Species B is less variable

genetically than species A. Species A has a mean

heterozygosity of 15.9% whereas species B has a

heterozygosity of 10.3%. The difference is modest, but fits

the level of decline predicted by Nei, et al. (1975). The

reduction in heterozygosity associated with founder

populations is generally attributed to the loss of low

frequency alleles by drift. The allelic composition of

populations of species B lack many of the low frequency

alleles found in populations of species A. Whereas, with the

exception of one rare allele (Pep190, p = 0.004), populations

of species B contain no alleles not also present in

populations of species A.






84

In conclusion, the electrophoretic data confirm the

existence of a sibling species. The genetic composition of

this new species suggest that it evolved from the ancestral

population through a small bottleneck, the founding

population may have consisted of less than ten individuals.

















CONCLUSIONS

In summary, the results of this study prove the

existence of a new sibling species of Anopheles

quadrimaculatus (Say). This report represents the first

description of a sibling species in the Nearctic Anopheline

fauna. This discovery is consistent with findings from a

large number of workers that Anopheline species frequently

evolve without developing significant morphological

differences.

The proof given here, for the existence of the new

species, is two-fold. Hybridization experiments revealed

that three of the nine populations surveyed existed as two,

reproductively isolated, sympatric populations. Reproductive

isolation was determined by mating studies which identified

male hybrid sterility. Attempts at identifying naturally

occurring hybrids at two of the sites failed, indicating that

a pre-mating mechanism,probably behavioral, maintains

reproductive isolation between these two species. A survey

of allozymic variation at twenty gene loci produced data

which supported the existence of a sibling species complex.

At two loci, a significant deficiency of heterozygotes was

revealed in the same three populations identified as being

mixed in the hybridization experiments. The genotypes at








these two loci could be used in distinguishing individuals of

the two species. The two species were tentatively designated

A. quadrimaculatus Species A and B.

The patterns of the genetic makeup of each species were

compared and a hypothesis concerning the phylogenetic

relationship between them was made. The evidence indicated

that species A is the ancestral species and that species B

evolved from it through a founder event.

There are five species, in addition to A.

quadrimaculatus, which belong to the Nearctic branch of the

Anopheles maculipennis complex. The results of this study

indicate that each of the remaining four should be more

closely studied to determine if additional sibling species

exist in this interesting species group.














BIBLIOGRAPHY


Avise, J. C., J. J. Smith and J. F. Ayala. 1975. Adaptive
differentiation with little genic change between two
native California minnows. Evolution 29:411-426.

Ayala, F. J. 1975. Genetic differentiation during the
speciation process. Evolutionary Biology 8:1-78.

Ayala, F. J. and J. R. Powell. 1972. Allozymes as
diagnostic characters of sibling species of Drosophila.
Proc. Natl. Acad. Sci. USA. 69:1094-1096.

Ayala, F. J., J. R. Powell, M. L. Tracey, C. A. Mouras, and
S. Perey-Salas. 1972. Enzyme variability in the
Drosophila willistoni group. IV. Genic variation in
natural populations of Drosophila willistoni. Genetics
70:113-139.

Ayala, F. J., M. L. Tracey, L. G. Barr, J. F. McDonald, and
S. Perez-Salas. 1974. Genetic variation in natural
populations of five Drosophila species and the
hypothesis of the selective neutrality of protein
polymorphisms. Genetics 77:343-384.

Baima, V., and B. A. Harrison. 1980. Evidence of sibling
speciation in the balabancensis complex of Southeast
Asia (Diptera:Culicidae). Abstracts 10th Inter. Cong.
Trop. Med. Malair., Manila.

Baker, R. H., W. C. French and J. B. Kitzmiller. 1962.
Induced copulation in Anopheles mosquitoes. Mosquito
News 22:16-17.

Bryan, J. H. 1973. Studies on the Anopheles punctulatus
complex. II. Hybridization of the member species.
Trans. R. Soc. Trop. Med. Hyg. 67:70-84.

Bullini, L., and M. Coluzzi. 1982. Evolutionary and
taxonomic inferences of electrophoretic studies in
mosquitoes. In: Recent Developments in the Genetics of
Insect Disease Vectors, eds. Steiner, W. W. M., W. J.
Tabachnick, K. S. Rai, and S. Narang. Stipes Publishing
Co., Champaign.









Buonomini, G., and M. Mariani. 1953. World Anophelines
belonging to the subgenus Maculipennia Buonomini and
Mariani, 1946. Riv. Malariol. 32:173-188.

Carson, H. L. 1976. Inference of the time of origin of some
Drosophila species. Nature 259:395-396.

Coetzee, M. 1983. Chromosomal and cross-mating evidence for
two species within Anopheles coustani (Diptera:
Culicidae). Syst. Entomol. 8:137-141.

Crow, J. F., and M. Kimura. 1970. An Introduction to
Population Genetics Theory. Harper and Row, New York.

Commission on Biochemical Nomenclature. 1972. Enzyme
Nomenclature. Elsevier, New York.

Davidson, G. 1963. DDT-resistance and dieldrin-resistance in
Anopheles quadrimaculatus. Bull. Wld. Hlth. Org.
29:117-184.

Davidson, G. 1964. The five mating types in the Anopheles
gambiae complex. Riv. Malariol. 43:167-183.

Davidson, G., and R. H. Hunt. 1973. The crossing and
chromosome characteristics of a new, sixth species in
the Anopheles gambiae complex. Parassitologia 15:121-
136.

Davidson, G., and G. F. Mason. 1963. Genetics of mosquitoes.
Ann. Rev. Ento. 8:177.

Davidson, G., H. E. Paterson, M. Coluzzi, G. F. Mason, and D.
W. Micks. 1967. The Anopheles gambiae complex. In:
Genetics of Insect Vectors of Disease, eds. J. W. Wright
and R. Pal. Elsevier Publ. Ec., Amsterdam, pp. 211-250.

Dobzhansky, T. 1970. Genetics of the Evolutionary Process.
Columbia University Press, New York.

French, W. L., and J. B. Kitzmiller, 1963. Tests for
multiple fertilization in Anopheles quadrimaculatus.
Proc. New Jers. Mosq. Exterm. Assoc. 50:374.

French, W. L., and J. B. Kitzmiller. 1964. Linkage groups
in Anopheles quadrimaculatus. Mosq. News 24:32-39.

Harris, H. and D. A. Hopkins. 1976. Handbook of Enzyme
Electrophoresis in Human Genetics. North-Holland
Publishing, New York. 1976.

Joslyn, D. J. 1978. Evolutionary Genetics of Three
Anopheline Mosquitoes: Anopheles walker (Theobald),
Anopheles atropos (Dyar and Knab) and Anopheles









quadrimaculatus (Say). Ph.D thesis. Univ. Illinois,
Urbana-Champaign.

Kanda, T., and Oguma, Y. 1978. Anopheles engarensis, a new
species related to sinensis from Hokkaido Island, Japan.
Mosquito Syst. 10:45-52.

Kitzmiller, J. B. 1977. Chromosomal differences among
species of Anopheles mosquitoes. Mosquito Syst. 9:112-
122.

Kitzmiller, J. B., and W. L. French. 1961. Chromosomes of
Anopheles quadrimaculatus. Am. Zool. 1:366.

Kitzmiller, J. B., G. Frizzi, and R. E. Baker. 1967.
Evolution and speciation within the maculipennis complex
of the genus Anopheles. In: Genetics of Insect Vectors
of Disease, eds. J. W. Wright and R. Pal. Elsevier Publ.
Co., Amsterdam, pp. 151-210.

Kitzmiller, J. B., and G. F. Mason. 1967. Formal genetics
of Anophelines. In: Genetics of Insect Vectors of
Disease. eds. J. W. Wright and R. Pal. Elsevier Publ.
Co., Amsterdam, pp. 3-15.

Lambert, D. M. 1983. A population genetical study of the
African mosquito Anopheles marshallii (Theobald).
Evolution 37:484-495.

Makela, M. E., and R. H. Richardson. 1977. The detection of
sympatric sibling species using genetic correlation
analysis. I. Two loci, two gamodemes. Genetics
86:665-678.

Mayr, E. 1954. Change of genetic environment and evolution.
In: Evolution as a Process, ed. J. Huxley. Allen and
Unwin, London.

Mayr, E. 1963. Animal Species and Evolution. Belknap
Press. Cambridge, Mass.

Mayr, E. 1969. Principles of Systematic Zoology. McGraw-
Hill, New York.

Mayr, E. 1982. The Growth of Biological Thought:
Diversity, Evolution and Inheritance. Belknap Press.
Cambridge, Mass.

Mahon, R. J., and P. M. Meithke. 1982. Anopheles farauti No.
3, a hitherto unrecognized biological species of
mosquito within the taxon A. farauti Laveran
(Diptera:Culicidae). Trans. R. Soc. Trop. Med. Hyg.
76:8-12.









Miles, S. J. 1981. Unidirectional hybrid male sterility
from crosses between species A and B of the taxon
Anopheles (Cellia) culicifaces Giles. J. Trop. Med. Hyg.
84:13-16.

Mitchell, S. E., and J. A. Seawright. 1984a. Chromosome-
linkage correlation in Anopheles quadrimaculatus (Say).
J. Hered. 75:341-344.

Mitchell, S. E., and J. A. Seawright. 1984b. A red stripe
mutant and its relationship in an allelic series in
Anopheles quadrimaculatus. J. Hered. 75:421-422.

Nei, M. 1978. Estimation of average heterozygosity and
genetic distance from a small number of individuals.
Genetics 89:583-590.

Nei, M., T. Maruyama, and R. Chakraborty. 1975. The
bottleneck effect and genetic variability in population.
Evolution 2:1-10.

Paterson, H. E., J. S. Paterson, and G. J. Van Eeden. 1963.
A new member of the Anopheles gambiae complex. Med.
Proc. 9:414-418.

Savage, K. E., and R. E. Lowe. 1971. A one-piece aluminum
cage designed for adult mosquitoes. Mosq. News 31:111-
112.

Seawright, J. A., and D. W. Anthony. 1972. Black body, a
lethal mutant in Anopheles quadrimaculatus Say. Mosq.
News 32:47-50.

Selander, R. K. 1970. Behavior and genetic variation in
natural populations. Am. Zool. 10:53-66.

Selander, R. K., M. H. Smith, S. Y. Yang, W. E. Johnson, and
J. B. Gentry. 1971. Biochemical polymorphism and
systematics in the genus Peromyscus. I. Variation in the
old-field mouse (Peromyscus polionotus). Univ. Texas
Publ. 7103:49-90.

Sene, F. M., and H. L. Carson. 1977. Genetic variation in
Hawaiian Drosophila. IV. Allozymic similarity between
D. selvestris and D. heteroneura from the island of
Hawaii. Genetics 86:187-198.

Shaw, C. R., and R. Prasad. 1970. Starch gel
electrophoresis of enzymes: A compilation of recipes.
Biochem. Gen. 4:297-320.

Sneath, P. H. A., and R. R. Sokal. 1973. Numerical
Taxonomy. W. H. Freeman, San Francisco.








Steiner, W. W. M., and D. J. Joslyn. 1979. Electrophoretic
techniques for the genetic study of mosquitoes. Mosq.
News 39:35-54.

Swofford, D., and R. B. Selander. 1981. BIOSYS-1: A
FORTRAN program for the comprehensive analysis of
electrophoretic data in population genetics and
systematics. J. Hered. 72: 281-283.

Templeton, A. R. 1980a. The theory of speciation via the
founder principle. Genetics 94:1011-1038.

Templeton, A. R. 1980b. Modes of speciation and inferences
based on genetic distances. Evolution 34:719-729.

Templeton, A. R. 1981. Mechanisms of speciation a
population genetic approach. Ann. Rev. Ecol. Syst.
12:23-48.

White, G. B. 1978. Systematic reappraisal of the Anopheles
maculipennis complex. Mosq. Syst. 10:13-44.

White, M. J. D. 1973. Animal Cytology and Evolutionary
Process. Columbia University Press, London.

Wright, S. 1978. Evolution and the Genetics of Populations
vol. 4 Variability Within and Among Natural
Populations. University of Chicago Press, Chicago.








Biographical Sketch


Gregory Charles Lanzaro was born on October 2, 1950 in

New York City, New York. He graduated from Kansas State

University in 1972, with the degree of Bachelor of Science.

After graduation he served as a high school teacher of

biology at Omaha, Nebraska and New Haven, Connecticut. In

1975 he enrolled in graduate school at the University of

Arizona, where he obtained a Master of Science degree in

Entomology in 1978. In 1980 he began work for the Doctor of

Philosophy degree at the University of Florida. He is an

active member in four national scientific societies. At

present he serves as Assistant Medical Entomologist in the

Department of Entomology of Mississippi State University.







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.




ak A. Seawright, chairman
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.




Sudhir K. Narang
Adjunct 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.




Donald W. Hall
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.



Stanley C. Schank
Professor of Agronomy








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 1986


Dean, llege of lriculture




Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID EXFUABQ2V_JU6RNC INGEST_TIME 2012-02-17T16:44:09Z PACKAGE AA00003808_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES