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Microanatomical study on the eyes of the lone star tick and the screwworm fly with related electrophysiological studies

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Title:
Microanatomical study on the eyes of the lone star tick and the screwworm fly with related electrophysiological studies
Creator:
Phillis, William Avery, 1942-
Copyright Date:
1975
Language:
English
Physical Description:
xiv, 157 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Axons ( jstor )
Compound eyes ( jstor )
Cytoplasm ( jstor )
Eyes ( jstor )
Microvilli ( jstor )
Neurons ( jstor )
Ommatidia ( jstor )
Photoreceptors ( jstor )
Pigments ( jstor )
Ticks ( jstor )
Dissertations, Academic -- Entomology and Nematology -- UF
Entomology and Nematology thesis Ph. D
Screwworm ( lcsh )
Ticks ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 144-155.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by William Avery Phillis III.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
029891596 ( AlephBibNum )
02163948 ( OCLC )
ACF4523 ( NOTIS )

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MICROANATOMICAL STUDY ON THE EYES OF THE
LONE STAR TICK AND THE SCREWWORM FLY
WITH RELATED ELECTROPHYSIOLOGICAL STUDIES











By

WILLIAM AVERY PHILLIS III


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY





UNIVERSITY OF FLORIDA


1975


























To my two precious daughters,
Emily and Colby,
without whom my life would have little meaning.














ACKNOWLEDGMENTS


I sincerely wish to thank Dr. H. L. Cromroy, Chairman

of my Supervisory Committee, for his support, patience,

and guidance during the past four years. I also wish to

thank the other members of my supervisory committee,

Drs. H. R. Agee, H. C. Aldrich, J. F. Butler, and D. E.

Weidhass for their help and encouragement.

I am particularly grateful to Dr. H. C. Aldrich

for his help and generous use of the Biological Ultra-

structure Laboratory where much of this work was carried

out. I am indebted to Dr. H. R. Agee for his generous

help and for the liberal use of his electrophysiological

laboratory.

I wish to thank Dr. G. Holt, USDA, APHIS, Fargo,

North Dakota, and Dr. H. C. Hoffman, USDA, APHIS, Mission,

Texas, for supplying the screwworm flies used in this

study.

I am grateful to my parents and sisters for their

encouragement and understanding when it was most needed.

Financial support was provided by National Institute

of Health training grant No. T01 AI 00383-02. I am deeply

grateful to Dr. R. E. Bradley and the other members of

the Faculty of Parasitology for making this traineeship

available to me.














PREFACE


The experimental data presented in this dissertation

deal with two uniquely different photoreceptors, the tick

eye and the insect eye. For the purposes of clarity,

the current study has been subdivided into the following

three chapters: Chapter I, The Microanatomy of the Eye

of the "Lone Star Tick," Amblyomma americanum (L.);

Chapter II, The Microanatomy of the Eye of the "Screwworm

Fly," Cochliomyia hominovorax (Coquerel), and Chapter III,

The Spectral Sensitivity of the Compound Eye of Cochliomyia

hominovorax (Coquerel).















TABLE OF CONTENTS


ACKNOWLEDGMENTS . . . . . . .

PREFACE . . . . . . . .

LIST OF FIGURES . . . . . .

LIST OF PLATES. . . . . . .

ABSTRACT. . . . . . . .

CHAPTER

I THE MICROANATOMY OF THE EYE OF THE "LONE
STAR TICK," Amblyomma americanum L. .

Introduction . . . . .
Literature Review . . . .
Methods and Materials . . . .
Results . . . . . . .
Discussion . . . . . .

II THE MICROANATOMY OF THE EYE OF THE "SCRE1
WORM FLY," Cochliomyia hominovorax
(Coquerel). . . . . . .

Introduction . . .
Literature Review . . . .
Methods and Materials . . .
Results . . . . ... .
Discussion . . .

III THE SPECTRAL SENSITIVITY OF THE COMPOUND
EYE OF Cochliomyia hominovorax . . .

Literature Review . . . .
Methods and Materials . . .
Results . . . . ... .
Discussion . . . .

IV CONCLUSIONS . . . . . . .


Page

iii

iv

vii

viii

xiii












TABLE OF CONTENTS (continued)

Page

APPENDIX. .................... 141

LITERATURE CITED. . . . . . . .. 144

BIOGRAPHICAL SKETCH ... . . . . . 156















LIST OF FIGURES

Figure Page

1 Schematic diagram of an eye of Amblyomma
americanum . .. . . . .. . .. .... 12

2 Schematic diagram of 1 photoreceptor neuron of
Amblyomma americanum. .. . .... . 12

3 Schematic diagram of Cochliomyia hominovorax
eye showing the relative position of the
peripheral retina and lamina ganglionaris . 53

4 Schematic diagram of a longitudinal section of
an ommatidium of the compound eye of Cochliomyia
hominovorax . . . ... ... .... 53

5 Spectral sensitivity curve of the dark-adapted
compound eyes of Cochliomyia hominovorax. . 132

6 Weekly visual sensitivity of irradiated
Cochliomyia hominovorax . ... .. . 134
















LIST OF PLATES


Plate Page

I. Amblyomma americanum larva .... . . .14
A. Oblique section of an optic nerve
B. Oblique section of an eye

II. Amblyomma americanum larva . . . ... .17
A. Longitudinal section of an eye
B. Longitudinal section of a portion of an
eye

III. Amblyomma americanum adult . . . . . 19
A. Oblique cross section of the lens
B. Oblique section of lenticular pore canals
C. Longitudinal section of scutellar pore
canals

IV. Amblyomma americanum adult . . . . .. 21
A. Cross section of terminal microvilli
bearing region of photoreceptor cell
B. Cross section of terminal microvilli at
higher magnification

V. Amblyomma americanum adult . . . ... .25
A. Longitudinal section through tips of
microvilli
B. Cross section of a peripheral portion
of an eye

VI. Amblyomma americanum adult . . ... . .27
A. Oblique section of photoreceptor neuron
through base of microvilli
B. Cross section of photoreceptor neuron
through region B below microvilli
C. Oblique section of neuron at base of
microvilli
D. Cross section of region B directly below
microvilli

VII. Amblyomma americanum adult . . . . .. 30
A. Cross section of photoreceptor neurons on
periphery of eye just under the hypodermis
B. Cross section of photoreceptor neurons in re-
gion C, the soma, at a higher magnification
C. High magnification of glycogen-like inclu-
sions in region C


viii










LIST OF PLATES (continued)


Plate Page

VII. D. High magnification of coated vesicles
(cont.) around the nucleus
E. Lower magnification of glycogen-like
and vesicular inclusions of neural soma

VIII. Amblyomma americanum adult. . . . ... 33
A. Cross section of a single neuron through-
out region C
B. Higher magnification of the same section
C. Cross section of glial cell mesaxons,
extracellular sheath, bundles of fibrils,
and glial cell cytoplasm
D. Cross section of glial cell sheath fibril
bundles

IX. Amblyomma americanum. . . . . . ... 36
OTlTTque section of larval optic nerve
B. Cross section of 2 axons, a glial nucleus,
mesaxons, extracellular glial sheath, and
glial cell cytoplasm
C. Cross section of axons, glial nucleus, and
mesaxonal sheath surrounding the axons
D. Cross section of a small portion of the
axoplasm of a single axon showing charac-
teristic microtubules and mitochondria

X. Cochliomyia hominovorax unirradiated. .. 63
A. Cross section of lens
B. Off center longitudinal section of lens
and pseudocone

XI. Cochliomyia hominovorax unirradiated. .. 66
A. Cross section of pseudocone and primary
pigment cells
B. Longitudinal section of pseudocone and
Semper cells

XII. Cochliomyia hominovorax unirradiated. ... . 69
A. Cross section through a junction of the
pseudocone, Semper cells, and primary
pigment cells
B. Higher magnification of the same cross
section of a junction between pseudocone
and Semper cells










LIST OF PLATES (continued)

Plate Page

XIII. Cochliomn ia hominovorax unirradiated. ... . 71
A. Cross section of an ommatidium near junc-
tion of the pseudocone and 4 Semper cells
B. Cross section of the 4 Semper cells show-
ing the rhabdomere caps and the ommatidial
cavity

XIV. Cochliomyia hominovorax unirradiated. ... . 74
A. Cross section of the Semper cell junction
and 7-armed ommatidial cavity
B. Cross section of the Semper cell junction
and 7 arms of the ommatidial cavity
C. Cross section of Semper cell cytoplasm
junction near the ommatidial cavity
D. Higher magnification cross section of
Semper cell cytoplasm

XV. Cochliomyia hominovorax unirradiated. .. 77
A. Section through several pigment filled
vacuoles in a large pigment cell
B. Cross section of a junction between
Semper cells and retinular cells
C. Higher magnification of retinular cell R3

XVI. Cochliomvia hominovorax unirradiated. .. 80
A. Cross section of an ommatidium, primary
pigment cells, and large pigment cells
B. Cross section of a portion of retinular
cells R1, R6, and R7
C. Cross section through distal ommatidium

XVII. Cochliomyia hominovorax unirradiated. ... . 83
A. Cross section of an ommatidium midway
in the peripheral retina
B. Cross section of rhabdom at the junction
of the superior central cell and the
inferior central cell

XVIII. Cochliomyia hominovorax unirradiated. ... . 86
A. Cross section of ommatidium just below
the transition from superior central cell
to inferior central cell orthogonall
microvilli)
B. Cross section of a portion of an ommatid-
ium just below the transition from the
superior central cell to the inferior
central cell










LIST OF PLATES (continued)


Plate Page

XIX. Cochliomyia hominovorax unirradiated. ... . 90
A. Oblique section through the basement
membrane
B. Cross section of a single pseudocartridge

XX. Cochliomyia hominovorax unirradiated. .. . 94
A. Cross section of an optical cartridge
B. Synaptic loci or T-shaped synaptic ribbons
C. Single synaptic ribbon
D. Cross section of an optical cartidge
adjacent epithelial glial cell and glial
nucleus

XXI. Cochliomyia hominovorax irradiated. ... . 97
A. Oblique section through the distal portion
of an ommatidium
B. Cross section through distal portion of
an apparently normal irradiated omma-
tidium
C. Cross section of an apparently normal
R7-R8 central cell transition of an
irradiated ommatidium

XXII. Cochliomyia hominovorax irradiated. ... 100
A. Cross section of an aberrant irradiated
ommatidium (with obconical rhabdomeres)
B. Cross section of a portion of an abnormal
irradiated ommatidium (obconical
rhabdomeres)
C. Cross section of an abnormal irradiated
ommatidium

XXIII. Cochliomyia hominovorax irradiated. ... . 103
A. Cross section of 8 cell ommatidium
(parallel microvilli)
B. Cross section of 8 cell ommatidium
orthogonall microvilli)
C. Cross section of several ommatidia through
the transition zone of superior and in-
ferior central cells

XXIV. Cochliomyia hominovorax irradiated. ... .. 106
A. Cross section of an ommatidium with an
abnormal superior-inferior central cell
transition (6 rhabdomeres)
B. Cross section of an ommatidium with an
abnormal superior-inferior central cell
transition (8 rhabdomeres)

xi









LIST OF PLATES (continued)

Plate Page

XXV. Cochliomyia hominovorax irradiated. ... . 108
A. Cross section of an optical cartridge
B. Higher magnification cross section of 2
photoreceptor axons









Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment
of the Requirements for the Degree of Doctor of Philosophy

MICROANATOMICAL STUDY ON THE EYES OF THE
LONE STAR TICK AND THE SCREWWORM FLY
WITH RELATED ELECTROPHYSIOLOGICAL STUDIES

By

William Avery Phillis III

August, 1975

Chairman: Harvey L. Cromroy
Major Department: Entomology and Nematology


The electron microscope was used to detail the

microanatomy of the eyes of the "lone star tick," Amblyomma

americanum (L.), and the screwwormm" fly, Cochliomyia

hominovorax (Coquerel).

The eyes of the "lone star tick" consist of a cuticu-

lar lens and 30-40 underlying photoreceptor neurons. The

lens contains bundles of specialized lenticular pore canals

believed to function as light or wave guides. The photo-

receptor neurons possess the microanatomical structures

common to all rhabdomeric photoreceptors. The simplicity

of the tick eye is believed to be a primitive condition and

is the first arhabdomate eye described for the phylum

Arthropoda. The photoreceptor neurons of Amblyomma show

affinities with the arhabdomate eyes of the flatworms and

snails.

The microanatomy of the peripheral retina and

lamina of the screwwormm" fly, Cochliomyia hominovorax,


xiii









is similar to the other genera of cyclorraphan Diptera

published. The eyes of irradiated and unirradiated flies

were examined with electron microscopy. Irradiated flies

showed a number of abnormalities not encountered in the

unirradiated flies. Irradiated flies showed a large

increase in retinular cell vacuolation, increased numbers

of trachea in the peripheral retina, abnormally shaped

rhabdomeres, decreased numbers of rhabdomeric microvilli,

and abnormal central cell transitions. The function and

origin of the Semper cells are discussed. The Semper cells

are believed to have several functions. The most important

function is to maintain the optically important trapezoidal

pattern of the rhabdomeres and central cavity of the open

rhabdom. The central cells (R7 and R8) have two types of

rhabdomeres: The microvilli of the first type are orthogonal

and the microvilli of the second are parallel. The pigment

"granules" of previous authors are actually vacuoles filled

with pigment crystals.

The electrophysiological method of equal response

was used to determine the spectral sensitivity of the eye

of irradiated screwworm flies. The eye was maximally

sensitive at 490 nm with a secondary peak at 350 nm and a

small "pseudopeak" at 625 nm. The visual sensitivity of

irradiated mass reared screwworm flies exhibited consider-

able weekly variation, some weeks flies were as much as

10 times more sensitive than other weeks, when measured

with the ERG at 530 nm.















CHAPTER I


THE MICROANATOMY OF THE EYE OF THE
"LONE STAR TICK," Amblyomma americanum L.


Introduction


Ticks are extremely important pathopherous agents.

The lone star tick, Amblyomma americanum (L.), is an eco-

nomically and medically important pest of wildlife, live-

stock and man. It ranges from central Texas throughout

southcentral and southeastern United States north to Mary-

land and Pennsylvania (Cooley & Kohls, 1944; Bishopp &

Trembley, 1945). A. americanum is an important vector of

Rocky Mountain spotted fever (Rivers & Horsfall, 1959), Q

fever,and is known to produce tick paralysis in man.

In general, tick physiology and biology have received

considerable attention from Hoogstral (1970) in Africa and

Sonenshine, Hair and Semtner in the United States. Studies

by Sonenshine (Sonenshine et al., 1966; Sonenshine & Levy,

1971) and by Semtner et al. (Semtner et al., 1971a; Semtner

et al., 1971b; Semtner & Hair, 1973a; Semtner & Hair,

1973b; Semtner et al., 1973) on the biology and ecology of

Amblyomma americanum are particularly valuable. However,

very few studies have included work on the photobiology of










ticks. This is difficult to understand in view of several

studies indicating the importance of photoperiod in diapause,

oviposition and questing behavior. McEnroe and McEnroe

(1973) studied the questing behavior of Dermacentor variabilis

(Say) and found that a photostimulus is necessary to initiate

questing behavior. The ovipositional pattern of two ticks,

Anocenter nitens Neumann and A. maculatum Koch,were shown to

be highly sensitive to photoperiod (Wright, 1969; Wright,

1971). Photoperiod was also shown to be the critical factor

in initiation of diapause in Dermacentor albipictus

(Packard) (Wright, 1969) and Dermacentor variabilis (Smith &

Cole, 1941). In addition most taxonomic keys to the hard

ticks contain couplets that separate genera on the basis of

the presence or absence of eyes.

No adequate research is available on the function and

morphology of the eye of Ixodid ticks. Part of the problem

with prior anatomical studies has been that they were done

with light microscopy. Many of the characteristic structures

of photoreceptor cells are beyond the resolving power of the

light microscope and therefore fail to detail adequately

the fine structure and make a determination possible as to

whether or not a neuron could function as a photoreceptor.

This research was done with the electron microscope

to investigate and detail the structure of the eye of

Amblyomma americanum and to use the microanatomical analysis

for determination of the eye as a functioning photoreceptor.










Literature Review


Two types of eyes are present in the arthropods:

compound eyes consist of from several to several thousand

repeating units known as ommatidia and small unicorneal eyes

termed ocelli. The eyes of Amblyomma americanum are of the

second or unicorneal type. Unicorneal eyes have received

little attention in the literature due in part to the diffi-

culty encountered in working with such small structures. The

size and simplicity of these structures however make them of

potential importance in neurophysiological vision studies

and well suited to electron micrographic techniques.

With the exception of the compound eyes of Limulus

chelicerate eyes are typically unilenticular ocelli. The

anatomy and microstructure of these ocelli are very similar

throughout the chelicerates. The lens is biconvex with an

underlying vitreous body composed of a single layer of

transparent cells. The photosensitive or retinular cells

are organized into a cup-shaped retina. The closely packed

microvilli characteristic to all arthropod retinular cells

are always orientated perpendicular to the light path

(Miller, 1960; Eakin, 1965). The microvilli of each cell

form isolated units known as rhabdomeres or when combined

with the microvilli from one or more other retinular cells

they form a rhabdom.










Limulus has paired compound eyes as well as a pair of

unicorneal ocelli and a rudimentary median eye. The ocelli

consist of a biconvex lens and an underlying cup-shaped

retina. Three types of retinular cells are present in the

retina. The microvilli of the first type form single-layered

rhabdomeres and the microvilli of the other two form double-

layered rhabdoms. Rhabdoms consisting of two layers of

microvilli are of two types: (1) "self-rhabdoms" in which

both layers of microvilli arise from a single cell; (2)

those rhabdoms in which the microvilli of two cells form a

rhabdom (Nolte & Brown, 1971).

The phalangids are probably closely related to the

acarines (van der Hammen, 1968). They have typical unilen-

ticular ocelli consisting of a lens and an underlying

retina. The photoreceptor cells of the retina are organized

into units consisting of 4 retinula cells surrounding a

central rhabdom. Each retinula cell contributes microvilli

to the central rhabdom. The cytoplasm of the retinular cells

contains many mitochondria, prominent Golgi, multivesicular

bodies and endoplasmic reticula. The rhabdoms are highly

ordered and repetitive with an organization very character-

istic of the pattern found in compound eyes (Curtis, 1970).

Machan (1966) studied the structure of the lateral

and median ocelli of three species of scorpions. The ocelli

of scorpions consist of a biconvex corneal lens and a reti-

nal layer of photoreceptor cells forming a cup-shaped










retina. This study was conducted with the light micro-

scope and provides little information on the microstructure

of the rhabdomeres or rhabdoms.

The two families of spiders that have been studied in

detail with the electron microscope are the jumping spiders

(Eakin & Brandenberger, 1971; Land, 1969) and the wolf

spiders, Lycosa (Melamed & Trujillo-Cenoz, 1966; Baccetti &

Bedini, 1964). The eyes of these two families of spiders

are very similar and typical of those of the other cheli-

cerates. The lenses are biconvex cuticular thickenings and

possess highly ordered cup-shaped retinas. The rhabdomeres

of both families are made up of microvilli and show a highly

ordered repetitive pattern. Eakin and Brandenberger (1971)

divided the sensory cells of the anterior median (AM) eyes

into 4 regions: (1) a distal portion bearing the rhabdomeric

microvilli, (2) an intermediate cytoplasmic segment, (3) a

basal soma containing the nucleus, and (4) the long neurite

that enters the optic nerve. The cell body or soma of the

sensory cells of the other 6 eyes lies directly behind the

vitreous body distad to the rhabdomeric microvilli.

The eyes of 2 acarine species have been anatomically

studied. The eyes of these 2 species, Trombicula autumnalis

Shaw and Tetranychus urticae Koch, show a similar anatomical

organization.

T. autumnalis has 2 pair of eyes, the anterior pair

with biconvex lenses and a posterior pair with simple convex

lenses (Jones, 1950).










The eyes of T. urticae were studied by Mills (1973).

The anterior pair have biconvex lenses consisting of a thin

stratified external layer and a thick interior made up of

25-30 cuticular layers. The simple convex lens of the poste-

rior pair of eyes is a thin "hemi-ellipsoidal shell" of

cuticular material. Both pair of eyes share a common "eye-

manifold" consisting of 15 retinular cells; five beneath the

anterior eyes and 10 beneath the posterior eyes. The micro-

villi of the retinular cells lie within cup-shaped invagi-

nations of the cell membrane termed "retinular-cups" by

Mills. Three retinular cells of the anterior eyes and

8 retinular cells of the posterior eye have double rows of

adjoining microvilli that form single fused rhabdoms similar

to the "self-rhabdom" of Limulus. The other retinular cells

have single rows of microvilli forming simple rhabdomeres.

Three anatomical studies of ticks have included work

done on the eye. These prior studies, however, provide

little information on the function of the eyes of ticks

since they utilized light microscopy. The first person to

detail the histology of the tick eye was Bonnet (1907).

He described perpendicular striae accentuated by black pig-

ment in the lens. Gossel (1935) studied the eyes of 6

species of Ixodid ticks and showed them to consist of a

convex lens with underlying unipolar neurons. The eye of

Dermacentor andersoni Stiles was treated briefly by

Douglas (1943).









Eight species of eyeless ticks were studied by Bin-

nington (1972). He found unipolar neurons in three of the

ticks and believed them to be photoreceptor cells. He also

found that removal of the lateral "eye" in Argas persicus

Oken impeded phototaxis. All eight species studied had

optic ganglia and optic nerves of similar morphology.

Horridge (1965) stated that the "aberrant eyes of

ticks" do not fit into the category of arthropod photo-

receptors.


Methods and Materials


Ticks utilized in this study were obtained from two

sources. Larval and adult ticks were collected near Otter

Creek, Florida, by dragging a 3-ft.-square "flag" over

infested vegetation. Larval, nymphal,and adult ticks were

also obtained from a colony maintained at the USDA-ARS,

Insects Affecting Man and Animals Laboratory, Gainesville,

Florida. These ticks were collected by personnel of the

laboratory as adults and subsequently fed on a dog. The

engorged ticks were held in a chamber maintained at high

relative humidity. Each generation was reestablished with

wild-caught adults (USDA Rearing Bulletin).

Ticks were fixed in gluteraldehyde-paraformaldehyde

prepared according to Karnovsky (1965) and were then sub-

merged in fixative and cut into three pieces. The opistho-

soma was cut off behind the third pair of legs and discarded.










The podosoma was then cut into two pieces along the midline.

This dissection facilitated the penetration of fixative and

subsequent solutions.

Pieces of podosoma containing the eyes were placed in

fresh fixative for 6-7 hours at room temperature to com-

plete fixation. The pieces were washed in three changes of

0.1 M cacodylate buffer (pH 7.2) and post-fixed for 12 hours

in 2% osmium tetroxide in 0.2 M cacodylate buffer at 4C.

Following post fixation the pieces were rinsed in 0.1 M

cacodylate buffer prior to dehydration. Dehydration was

accomplished at 5-minute intervals in a series of 25, 50,

75% ethanol at 4C. The tissue was held in 2% uranyl ace-

tate in 75% ethanol at 40C for 3 hours to improve contrast.

Two-lO minute changes of 100% ethanol and two subsequent 15

minute changes of acetone preceded infiltration with Spurr's

plastic (Spurr, 1969). Tissue was held in 50% plastic in

acetone for 1 hour and in 100% plastic for 24 hours at room

temperature prior to polymerization in a 600 oven.

Silver to gold sections were cut using a duPont diamond

knife on a Porter-Blum MT-2 ultramicrotome after thick

sectioning (1 micron) brought the region of the eye to the

block face. Thin sections were placed on 75-mesh copper

grids covered with a Formvar film. The sections were post-

stained with uranyl acetate for 10 minutes and lead citrate

for 2-4 minutes (Reynolds, 1963) prior to examination with










either a Hitachi HU11C or HU11E electron microscope at

75 kV.


Results


The eyes of Ixodid ticks are located on the lateral

margins of the scutum unlike other arthropods where the eyes

are located on the head. In Amblyomma americanum the eyes

consist of 30-40 unipolar photoreceptor neurons (Fig. 1).

This pattern is the same for the larva, nymph, and adult

tick. In each succeeding stage the eye becomes larger but

the anatomy and microstructure remain the same. The eye

of the larval tick contains approximately 25-30 neurons and

is approximately one-fourth the size of the adult. Sections

of a larval tick eye were used in Plates I and II to provide

an overall view of the eye and the individual photoreceptor

neurons. Plates I and II are electron micrographs of whole

eyes. The orientation of the section may be determined by

using the orientation lines provided on the plates them-

selves. One line (D-V) indicates the dorsal-ventral axis

and the perpendicular line (L) indicates the midline-

laterad aspect. The lenticular pore canals follow a curved

path and converge in an area above the photoreceptor cells.

Lines inscribed on the longitudinal axes of the pore canals

would converge on a point in the microvillar region

(region A) of the eye.










The cuticular lens is roughly biconvex and deviates

only slightly from a simple convex configuration (Fig. 1,

Plate I B). A slight internal bulge is present and is

located on the ventral portion of the inner lens and the

second convex curve of the lens. The internal bulge of

the lens is always located proximal to the microvilli of the

photoreceptor neurons (Plate I B). The pore canals of the

lens are always perpendicular to the longitudinal axes of

the photoreceptor neurons. The pore canals (PC) of the scutum

are oriented in the dorsal-ventral axis (compare with the

pore canals of the lens). The exocuticle of the lens (EXO)

is darker in appearance than the endocuticle (ENDO) (Plate

I B).

The photoreceptor neurons of the eye are connected to

the optic lobes of the brain by the optic ganglion (Plate

I A). Each photoreceptor neuron contributes a single axonal

neurite to the optic nerve. The number of photoreceptor

neurons per eye can therefore be determined by counting the

number of axons in the optic nerve (Plate I A).

The individual photoreceptor neurons do not vary in

structure with regard to sex, age, or stage. An isolated

neuron is indistinguishable from any other neuron within

a single eye. The photoreceptor neuron has been divided

into 4 regions for descriptive purposes: (1) a distal

segment, region A, characterized by the presence of numerous












Figure 1. Schematic diagram of an eye of Amblyomma
americanum. Ax, axon; GN, glial nucleus,
GS, glial sheath; H, hypodermis; L, lens;
LPC, lenticular pore canals; Mv, micro-
villi of photoreceptor neuron; N, nucleus;
S, scutum; SPC, scutellar pore canals.




















Figure 2. Schematic diagram of 1 photoreceptor neuron
of Amblyomma americanum. A, region charac-
terized by numerous microvilli; B, region
containing numerous mitochondria and intra-
cellular channels; C, soma containing the
nucleus; D, proximal axon; Ax, axon; CV,
coated vesicles; GC, glial cell; GL,
glycogen; GN, glial nucleus, Go, Golgi,
M, mitochondria; Mv, microvilli.






































IlI
I I
I I
I I
D I
DCC
I I
I I
I I


2.














B A













Plate

A.


I. Amblyomma americanum larva

Oblique section of an optic nerve. Receptor
cell axons (Ax) are invested by glial elements
(arrows). The optic nerve lies directly beneath
the scutum (Sc) and hypodermal cells (HC).
X5890


B. Oblique section of an eye. Beneath the lens (L)
is the hypodermis (H) and 4 photoreceptor cells.
Each photoreceptor cell has a prominent soma
containing the nucleus (N) and numerous terminal
microvilli (Mv). X3800 Note internal lenticular
bridge (B), the deviation from simple convex
configuration, and orientation of pore canals
(PC).










microvilli, (2) an intermediate region containing numerous

mitochondria and intracellular channels, region B, (3) a

basal soma, region C, containing the nucleus, and (4)

region D, a proximal axonal neurite that together with the

other axonal fibers forms the optic nerve (Fig. 2). Three

of these regions are shown in cross section in Plate V B.

In addition to the photoreceptor neurons the eye is invested

by a tunic of glial cells (Fig. 1, Plate V B). The membran-

ous windings of the glial cells, the mesaxons (MA), glial

cytoplasm (arrows), and an extracellular glial sheath (ES)

isolate the neurons of the eye from the haemocoel. Often

the cytoplasm of the glial cells contains electron dense

opaque bodies (OB) and multivesicular bodies (MVB) (Plate

V B).


Lens


The lenses of arthropod eyes contain very few struc-

tural features and in this respect the lens of Amblyomma

americanum is unique. Unlike other lenses it has many pore

canals in the transparent matrix of the scutellar cuticle.

The pore canals (Plate III A) of the lens are organized into

bundles of 30-60. These bundles of pore canals condense

and their diameter becomes smaller as they approach the

hypodermis (H). The number of pore canals per bundle also

decreases as they near the hypodermis. This decrease is














Plate II. Amblyomma americanum larva

A. Longitudinal section of an eye. Photoreceptor
neurons have 4 distinct regions: a distal
segment of microvilli, region A; an intermediate
cytoplasmic segment containing many mitochondria,
region B; a soma containing the nucleus (N),
region C; and region D, a long axon (Ax). Note
the orthogonal orientation of microvilli shown
by arrows (CX and LX). X3800















B. Longitudinal section of a portion of an eye
(post-stained with barium permanginate).
Microvilli of several cells are oriented ortho-
gonally (arrows CX and LX). Four regions of
the eye are shown (A, B, C, and D). X3800
















Plate III. Amblyomma americanum adult

A. Oblique cross section of the lens. The pore
canals (PC) of the lens are organized into
bundles. X3800 The arrows indicate fusion
of pore canals.


B. Oblique section of lenticular
X3800


pore canals (PC).


C. Longitudinal section of scutellar pore canals
(PC). X3800







to )1 3 c (Yy~ 1 **9 *

aa



b b,


-0 .- O,
tl P .,





I d
0a ~0(



-r-



we'













IV. AmhljyRma americanum adult

Cross section of terminal microvilli (Mv)
bearing region of photoreceptor cell. Glial
investiture of the photoreceptor cells consists
of mesaxons (Ma) and an extracellular sheath
(arrows). Neural cytoplasm contains numerous
mitochondria (M) in this area. X11970


B. Cross section of terminal microvilli at higher
magnification. Photoreceptor cytoplasm (C)
and glial cell cytoplasm (arrows) shown. X22800


Plate
A.







lwhO


4-


?FY



,A
V,

i; _


z ^.









due to the regular fusion of pore canals (arrows). In Plate

III A, pore canal bundles are demonstrated in cross section.

This electron micrograph is oriented so the hypodermis under-

lying the lens is on the bottom and the top of the micro-

graph is laterad. The pore canal bundles diminish markedly

in diameter as they near the hypodermis and several pore

canals are sectioned at the point of fusion (arrows).

The lenticular pore canals and scutellar pore canals

differ radically in size in the adult and the nymph. The

lenticular pore canals are considerably larger (Plate III B)

than the scutellar pore canals (Plate III C). Plate III B

illustrates the curvilinear path taken by the pore canals

of the lens.


Hypodermis


A cellular epidermis, the hypodermis (H), lies directly

beneath the lens of the eye (Plate I B). The hypodermis is

one cell layer thick and rests upon an amorphous basal base-

ment membrane. The scutellar hypodermis (H) (Plate IX A)

and the lenticular hypodermis (H) (Plate I B) are indis-

tinguishable and no apparent lenticular hypodermal modifica-

tions were observed.









Retinular Cells


Region A. Microvilli

The distal portion of the photoreceptor neurons bears

thousands of parallel microvilli (Fig. II, Plate II A & B).

By counting the number of microvilli per square unit on a

micrograph estimates of the number of microvilli per photo-

receptor were calculated. These estimates ranged between

7,000 and 13,000 per photoreceptor cell. The microvilli

are oriented perpendicular to the path of light as in all

photoreceptors studied. The microvillar-bearing membrane

of the neuron is dome-shaped (Fig. 2) and cross sections of

this region often show a central portion of cytoplasm with

microvillar cross sections encircling it (Plate IV A).

The microvilli are independent and free within the glial

investment of the neurons (Plate IV A). The microvilli are

tightly packed within the mesaxonal investment (Plate VI B,

V A) but are not bonded to one another by tight junctions

as in other chelicerate eyes. The microvilli are typically

blind-ended evaginations of the distal membrane of the neu-

ron. Plate V A shows the tips of the photoreceptor micro-

villi.


Region B. Intermediate region of cytoplasm

The neural zone directly proximad the terminal micro-

villi is designated Region B, the intermediate zone of













Plate

A.


V. Amblyomma americanum adult

Longitudinal section through tips of microvilli.
X22800


B. Cross section of a peripheral portion of an eye.
Axons (Ax) are surrounded by glial cell membranes,
the mesaxons (Ma), glial cytoplasm (arrows),
and an extracellular sheath (ES). Glial nuclei
(GN) are located on the periphery of the eye.
Cytoplasm of the axons and glial cells often
contain electron opaque bodies (OB) and multi-
vesicular bodies (MVB). X17100











Plate VI. Amblyomma americanum adult

A. Oblique section of photoreceptor neuron through
base of microvilli (Mv). Deep invaginations,
intracellular channels (arrows), and numerous
mitochondria (M) between membranes are charac-
teristic of this zone (II). X11970

B. Cross section of photoreceptor neuron through
region B below microvilli (Mv). Numerous mito-
chondria, here in cross section, are character-
istic of this region. X8740













C. Oblique section of neuron at base of microvilli
(M). Pinocytotic vesicles (arrows) form between
microvillar bases. Mitochondria (M) lie in
cytoplasm between intracellular channels. X15750

D. Cross section of region B directly below micro-
villi (Mv). Mitochondria (M) are located between
intracellular channels (arrows). X38000









cytoplasm. It is characterized by numerous elongate

mitochondria, intracellular channels, and pinocytotic (or

exocytotic) vesicles. The numerous sausage-shaped mito-

chondria (M) present in this region are associated with a

system of intracellular channels (arrows (Plate VI A & D).

Mitochondria are present in all parts of the neuron but are

most prevalent in region B. These mitochondria lie between

cytoplasmic sheets formed by intracellular membranous chan-

nels. Plate VI D is a photomicrograph of a cross section

of this region and shows the mitochondria (M) between intra-

cellular channels (arrows). The intracellular channels

originate as inpocketings of the terminal membrane between

the bases of the microvilli (Plate VI C). Vesicles (arrows)

prevalent in this region appear to arise at the end of these

membranous channels between the microvillar bases (Plate VI).

This combination of microvilli, mitochondria and membrane-

lined intracellular channels is very characteristic of trans-

port cells such as Malpighian tubule cells, secretary, or

glandular cells.


Region C. Nucleus-bearing portion of the soma

The nucleus of the photoreceptor neuron is the most

prominent feature of zone C (Plate VII, VIII). Plate VII

A & B are electron micrographs of cross sections through this

region and show the relationship between the prominent

nucleus (N) and the other organelles characteristic of the











Plate VII. Amblyomma americanum adult

A. Cross section of photoreceptor neurons on
periphery of eye just under the hypodermis (H).
Two axons (Ax) and cross section of several
neural soma, in zone C, showing nuclei (N) and
rough endoplasmic reticulum (arrows). Glial
nucleus (GN), mesaxons (Ma), and extracellular
glial sheath (GS) cover the neurons. X5400






B. Cross section of photoreceptor neurons in region
C, the soma, at a higher magnification. The
cytoplasm of the neuron in region C contains a
prominent nucleus (N) and cisternae of rough
endoplasmic reticulum (RER) but fewer mitochon-
dria (white arrows) than region B. Note axons
(Ax), glial nucleus (GN), mesaxons (Ma), and
extracellular glial sheath (black arrows).
X8740









C. High magnification of glycogen-like (G) inclusions
common in the cytoplasm of neural soma in region
C. X57500


D. High magnification of coated vesicles (CV) associ-
ated with Golgi complex in the cytoplasm around
the nucleus. X57500


E. Lower magnification of glycogen-like (G) and
vesicular (CV) inclusions in cytoplasm of neural
soma (region C). X43700










perikaryon. The nucleus is located in the center of the

cell and is surrounded by conspicuous cisternae of endo-

plasmic reticulum (RER). Ribosomes are attached to the

surface of the endoplasmic reticulum. The cisternae of this

rough endoplasmic reticulum form concentric layers around

the nucleus. The number of mitochondria in this zone is

greatly reduced when compared to the preceding zone B

(Plate VII B).

Two inclusions of similar size are common in the cyto-

plasm around the nucleus (Plate VII E). One type of inclu-

sion appears to be glycogen (G) and the second, coated

vesicles (CV). Plate VII C is an electron micrograph of

alpha-glycogen rosettes present in the perikaryon in homoge-

neous masses (Plate VII E). Coated vesicles are elaborated

by Golgi complexes and are present throughout the perikaryon

in aggregates termed "Nebenkernen" (CV) by Fahrenbach in

Limulus (Fahrenbach, 1970) and as isolated vesicles in the

cytoplasm. One such "Nebenkern" (CV) is shown in Plate

VII E. These coated vesicles (DV), shown at higher magnifi-

cation in Plate VII D, are probably involved in the trans-

port or storage of synthetic products. The amount of

glycogen per cell is highly variable but the presence of

coated vesicles in the photoreceptor neurons is fairly

uniform.












Plate

A.


VIII. Amblyomma americanum adult

Cross section of a single neuron throughout region
C. Cisternae of rough endoplasmic reticulum
(ERE) and Golgi (Go) are always found near the
nucleus (N). Vesicular inclusions (arrows)
are associated with the golgi. Other cytoplasmic
inclusions include mitochondria (M) and multi-
vesicular bodies (MVB). (Note longitudinal
section of microvilli (M)). X17100


B. Higher magnification of the same section showing
the rough endoplasmic reticulum (RER), Golgi
(Go), and associated vesicles. Two types of
vesicles are formed by the golgi complex, small
coated vesicles (arrows), and larger vacuolate
vesicles (V). X28000







C. Cross section of glial cell mesaxons (Ma),extra-
cellular sheath (GS), bundles of fibrils (FB) often
in glial sheath, and glial cell cytoplasm (arrows).
X17100

D. Cross section of glial cell sheath fibril bundles
(arrows). X17100










The relationship of the nucleus (N), rough endoplasmic

reticulum (RER), Golgi (Go), and elaborated vesicles is

shown in Plate VIII A & B. Cisternae of rough endoplasmic

reticulum (RER) form concentric patterns around the nucleus

and are generally the most conspicuous organelles of the

perikaryon (Plate VIII A). Large numbers of coated vesicles

(arrows) are discharged by the Golgi apparatus. The first,

indicated by arrows in Plate VIII, are uniform in size and

are "coated" by a layer of electron dense material. The

second vesiculate type (V) are highly variable vaculoate

vesicles that have smooth membranous walls apparently

derived from the cisternal membrane of the golgi (Plate VIII

B). The Golgi apparatus in the photoreceptor neurons gen-

erally consists of between 5-7 cisternae regardless of its

size.


Region D. Axon

Each neuron attenuates rapidly behind the nucleus in

the direction of the central nervous system to form a long

axonal neurite that communicates directly with the optic

lobes of the brain. The optic nerve of A. americanum larvae

(Plate IX B & C, VII A) consists of 25-30 photoreceptor cell

axons and the optic nerve of the nymph and adult contains

30-40 axons. Only one type of photoreceptor axon (Ax)

is present in the optic nerve of A. americanum (Plate IX

A & B). However, bundles of small axons (X) of unknown















Plate IX. Amblyomma americanum

A. Oblique section of larval optic nerve. Optic
nerve contains 30-40 axons (Ax) from individual
photoreceptor neurons. The optic nerve is
located directly beneath the scutum (Sc) and
scutellar hypodermis (H). X11970


B. Cross section of 2
(GN), mesaxons (Ma),
(GS), and glial cell


axons (Ax), a glial nucleus
extracellular glial sheath
cytoplasm (arrows). X11500


C. Cross section of axons (Ax), glial nucleus (GN),
and mesaxonal (Ma) sheath surrounding the axons.
Two bundles of smaller axons of unknown origin
or function (X). X8740


D. Cross section of a -small portion of the axoplasm
of a single axon showing characteristic micro-
tubules (arrows) and mitochondria (M). X43700










































N,


AX
im


1


i
,
c


--


~" r- ~ ~~
rrrry L1:-14~

~~~C.I' r
' '~i~
i~3Li
\










function or origin were encountered in the optic nerve

of adult ticks (Plate IX C). The optic nerve consists

of photoreceptor axons, glial cells and a fibrous extra-

cellular perineural sheath.

The axoplasm contains numerous longitudinally

oriented microtubles (Plate IX arrows) that are orderly in

their distribution. The axonal mitochondria are located

more or less peripherally in the axoplasm adjacent to the

axonal surface (Plate IX B & D). The axoplasm also con-

tains infrequent dense opaque bodies (OB) (PlateV B, VIII D),

possible residual or autophagic lysosomes. No other inclu-

sions were encountered in the axonal region of the neuron.

The photoreceptor neurons are completely ensheathed

by glial cells that insulate the neurons from the environ-

ment of the haemocoel (Plate VII A & B). The glial invest-

ment of Amblyomma is intermediate between the condition

encountered in the myelinated and unmyelinated nerves of

vertebrates. The glial cytoplasm of myelinated nerves is

obliterated leaving a glial membranous sheath termed myelin.

In Amblyomma and other arthropods glial cytoplasm and glial

cell membrane cover the axon. This condition is common in

the arthropods and nerves of this type are called tunicated

nerves. In electron micrograph V B the glial cytoplasm

(arrows) can be seen between the glial cell membranes (Ma).

The neurons lie within invaginations of the glial cells.









These invaginations of the glial cell membrane form long

double membrane mesaxons (Ma) that wind around the axons

and neural cell somata (Plate XI C, VII B). Each neuron is

generally surrounded by two or three mesaxonal membranes.

The mesaxons often bifurcate (Plate VII B) and encompass

several axons. Glial cytoplasm contains few organelles or

inclusions but mitochondria, multivesiculate bodies,and

opaque inclusions are sometimes present (Plate V B).

Glial cell nuclei (GN) are located on the periphery of the

neurons (Plate IX B, C; Plate V B) and conform to the outline

of the axons.

A fibrous extracellular perineural sheath (GS) covers

the glial cells, axons, and photoreceptor neurons (Plate

IX B, VII B). Embedded in the glial sheath, termed the

neuralemma by some authors, are bundles of fibrils (Plate

VIII C & D). These fibril bundles are not encountered in a

predictable manner. In Plate VII B the glial sheath does not

contain any fibrils. Plate VIII C and Plate VIII D are

sections of these fibril bundles sectioned obliquely and in

cross section respectively. The extracellular glial sheath

is composed of an amorphous material very similar in appear-

ance to the basement membrane (compare BM and GS in VII A).

The optic nerve, upon leaving the eye, is located

directly beneath the scutellar hypodermis (H) and remains in

close proximity to the hypodermis until it enters the brain

laterally.










Discussion


Horridge (1965) stated that the eyes of ticks are

"aberrant" and "improbable" as "efficient sense organs."

The eyes of ticks do not appear aberrant or improb-

able as sense organs when studied microanatomically with

the electron microscope. The eyes of Amblyomma americanum

must be considered in the context of all rhabdomeric photo-

receptors, particularly those of the Mollusca and the

Platyhelminthes, and not just the highly advanced arthropod

compound eyes. They differ anatomically from any of the

other arthropod eyes that have been studied to date. The

principal difference is their simplicity. Despite this sim-

plicity, the eyes of Amblyomma possess all the structures

necessary to make them fully functional photoreceptors. The

eyes have a lens, well-developed photoreceptor neurons, and

an optic nerve that communicates directly with the optic

lobes of the brain. The microanatomy of the neural cells

beneath the lens identifies them with little doubt as

photoreceptor cells. Electron microscopy was necessary to

visualize the structures, in particular the microvilli which

are characteristic of rhabdomeric photoreceptors of light

microscopy.

The most unusual aspect of the tick eye is the corneal

lens. Bonnet (1907) described perpendicular striae accentu-

ated by black pigments in the lens and Gossel (1935, Fig. 36)










noted the chitin of the lens is pierced by fine "canals."

The "striae" they described were probably the bundles of

lenticular pore canals described in this study (Plate III A).

These bundles of pore canals appear as bright streaks when

thick sections are viewed with the light microscope. This

light-conducting property of the pore canal bundles indicates

that they may act as light or wave guides. The lenses of all

other arthropod eyes studied are devoid of pore canals or

other structures and the lens of Amblyomma is the first

described with such structures. In Limulus the unmodified

cuticle between the corneal facets of the compound eye has

pore canals (Fahrenbach, 1969) and may represent an inter-

mediate condition between the lens of Amblyomma and the typi-

cal ommatidial facet or unicorneal lens in the chelicerates.

The pore canals of insects have been studied in detail

(Locke, 1959, 1961) as have the pore canals in the integument

of ticks (Nathanson, 1967; Beadle, 1972) and mites (Wharton

et al., 1968). The pore canal bundles in the lens of Ambly-

omma have not been described from any other arthropods and

represent a completely different adaptation of cuticular

pore canals in the arthropods. The pore canals follow a

curved path through the lenticular cuticle and converge on

a point directly above the photoreceptor neurons. The di-

ameter of the pore canal bundles becomes progressively

smaller and the pore canals converge as they approach the










inner surface of the lens. If the pore canal bundles

actually function as light or wave guides their convergence

and reduction in diameter may serve to intensify the light

impinging on the photoreceptor neurons. Light energy

gathered on the outer surface of the lens where the surface

area is greatest would be condensed and concentrated on the

smaller inner lenticular surface over the photoreceptor

neurons.

The lenses of other chelicerate eyes are strongly bi-

convex (Horridge, 1965; Curtis, 1970; Eakin and Brandenberger,

1971; Melamed and Trujillo-Cenoz, 1966; Fahrenbach, 1963).

The lenses of ticks described with light microscopy are

simple convex or very slightly biconvex (Gossel, 1935).

The lens of Amblyomma americanum is slightly biconvex. It

has an off-center bulge on the internal ventral surface of

the lens.


Region A. Microvilli

All photoreceptors, rhabdomeric and ciliary, have

parallel membranous arrays. Arrays of membranes are charac-

teristic of all photoreceptors and it is assumed that their

function is to provide an ordered plano-arrangement of

membrane-bound photochemicals (Eakin, 1965, 1968). Two

lines of evolution are apparent in the evolution of multiple

membrane systems, the ciliary line and the rhabdomeric line

(Eakin, 1965, 1968; Varela, 1971). The rhabdomeric line,










characterized by the microvillus, is believed to have

arisen as an early offshoot of the ciliary line and de-

veloped independently (Eakin, 1965). Varela (1971) postu-

lates that the molluscs and arthropods do not belong in the

same evolutionary line and suggests that the Mollusca

constitute a third line of evolution.

The microvilli of the photoreceptor neurons of

Amblyomma americanum are typical of those encountered in

other arthropod photoreceptors. One important aspect of

the microvilli is the absence of any microvillar interaction.

The microvilli are closely packed (Plate IV A, B; V A) with-

out the development of a highly ordered hexagonal "honey-

comb" relationship characteristic of the insects such as

the cockroach (Smith, 1968),dipterans (Trujillo-Cenoz,

1972; Wolken, 1971), beetle (Meyer-Rochow, 1973), hemipterans

(Burton & Stockhammer, 1969), ants (Wolken, 1971), and

many crustacea (Eguchi & Waterman, 1966; Wolken, 1971)

or the formation of tight junctions between adjacent

microvilli as in Limulus (Fahrenbach, 1969; Lasansky, 1967;

Nolte & Brown, 1971), Lycosa (Melamed & Trujillo-Cenoz,

1966); Phalangids (Curtis, 1970), Octopus (Moody & Robert-

son, 1960), and the larval mosquito eye (White, 1967). The

microvilli are completely free within the glial sheath and

tight packing of the microvilli of Amblyomma americanum

produces neither the hexagonal honeycomb configuration

noroccluded intercellular bridges (Plate IV B). Tight









packing of microvilli in the eye of mandibulate arthropods

(Crustacea and Insecta) produces hexagonal packing with a

uniform intervillar space of constant dimensions. Tight

packing of microvilli in chelicerate eyes produces an

occluded intervillar space with tight junctions at points

where adjacent microvilli touch. In this respect the photo-

receptor microvilli of Amblyomma americanum show affinities

with the microvilli borne on the photoreceptor neurons of

planarians (Rohlich & Tdrdk, 1961; MacRae, 1964) and snails

(Rohlich & TorBk, 1963) whose microvilli exhibit neither

occluded intervillar space or hexagonal packing. Hexagonal

packing and occlusion of the intervillar space will be con-

sidered in this study as advanced evolutionary characteris-

tics. Free microvilli as encountered in the ticks, plan-

arians, and snails are considered primitive.

The longitudinal planes of the microvilli of several

neurons in Plate II A and B are perpendicular. This ortho-

gonal arrangement of microvilli provides the anatomical

basis of polarized light perception (Waterman & Horch, 1966).

This perpendicular or orthogonal pattern was encountered

only in larval ticks and not in nymphal or adult ticks.

This aspect of the tick eye deserves further investigation

to determine if the eye functions as a polarizer.









Region B. Intermediate zone of
intracellular channels

The system of membrane-bound intracellular channels

and vesicles originating at the base of the microvilli is

very characteristic of rhabdomeric photoreceptor neurons.

A system of subrhabdomeric cisternae and vesicles occurs in

Limulus (Fahrenbach, 1968), Lycosa (Melamed & Trujillo-

Cenoz, 1966) 7 species of the phalangids (Curtis, 1970),

5 species of decapod crustaceans (Eguchi & Waterman, 1966,

1968), Drosophilia (Waddington & Perry, 1960), the toad

bug, Gelastocoris (Burton & Stockhammer, 1969), a mosquito

larva (White, 1967), 4 genera of dipterans (Trujillo-Cenoz,

1965a, 1972), snail Helix (Rdhlich & Tbrdk, 1963), and 3

species of turbellarian platyhelminthes (Rdchlich & Torik,

1961; MacRae, 1964). These are common structures associ-

ated with rhabdomeric photoreceptor neurons and are found in

all the eyes studied to date. The presence of these or-

ganelles in the submicrovillar portion of the photoreceptor

neurons of Amblyomma americanum adds credence to their

identification as retinular cells. Long oval mitochondria

are oriented longitudinally and occur in large numbers

between the membrane-lined channels. Mitochondria are pres-

ent throughout the neural cell body and axoplasm but are

very abundant in region B (Plate VI A, B, & D) and rather

sparce in other zones (Plate VII A & B). The large numbers

of mitochondria located in this region indicate an intense

rate of metabolic activity and energy utilization.









Most authors interpret the presence of membranous

intracellular channels, associated mitochondria,and numerous

vesicles in this region as indicating protein uptake by

pinocytosis (Waddington & Perry, 1960; Melamed & Trujillo-

Cenoz, 1966, 1968; Burton & Stockhammer, 1969; Curtis,

1970). An alternate interpretation was provided by Fahren-

bach (1968). He believes the coated and vacuolate vesicles

are part of a secretary sequence originating with the rough

endoplasmic reticulum and Golgi that ends with secretion

into the rhabdom of the eye. Whether this region repre-

sents an area of pinocytotic uptake or exocytotic secretion

will require further study. The large number of mitochon-

dria (energy utilization) indicatespinocytosis but no known

material is present in the rhabdom other than haemolymph.

The microvilli of the tick eye are separated from the haemo-

coel by their glial investiture only and materials present

in the haemocoelic fluid would be more available than in

the rhabdomate eyes.

The photoreceptor neurons of Amblyomma americanum

have several important features in common with other rhab-

domeric eyes. These shared features include terminal

microvilli perpendicular to the light path, similar cyto-

plasmic organelles and organization, and an axonal neurite

that communicates with the optic lobes of the brain. The

important differences that separate it from other arthropod

photoreceptors are terminal microvilli oriented in










longitudinal axis of the neuron, the arhabdomate eye, the

unusual construction of the lens, and the total absence of

pigment in the neurons or associated cells.

The eyes of Amblyomma are the first arhabdomate or

arhabdomeric eyes described in the phylum Arthropoda. The

unicorneal eyes of chelicerates and the compound eyes of

the mandibulates are very similar in structure with the

exception of the lenticular structures. The retina of

chelicerates (Melamed & Trujillo-Cenoz, 1966; Curtis, 1970;

Eakin & Brandenberger, 1971) is composed of repeating units

very similar in structure and organization to the ommatidia

in the mandibulates. No such structures are present in the

arhabdomate eyes of Amblyomma. This arhabdomate condition

is not shared with the only other acarine eye to be studied,

Tetranychus urticae (Mills, 1973), that has both rhabdomeres

and rhabdoms.

The simplicity of the Amblyomma eye raises the ques-

tion of the secondary reduction. Is the eye of Amblyomma

primitive, possibly an archetype of arthropod eyes, or is it

secondarily reduced? I believe the eye to be very primitive

and not the product of secondary reduction. It could very

easily be an archetype of the arthropod eye since more

advanced retinular cell types could be derived from it by

merely changing the shape of the neuron. The tick eyes

represent a very plausible step in the evolution of the

compound eye of the mandibulates and unicorneal eye of the










chelicerates. It is an evolutionary step never before

recognized.

Two important considerations support this view of

simplicity rather than secondary reduction. First, the

cytoplasmic organelles, cell structures, and organization

are not reduced but are well developed. Organelles are

numerous and characteristic of a very generalized cell.

Advanced arthropodan photoreceptors have retinula cells that

appear metabolically inactive when compared with the photo-

receptor neurons of Amblyomma (see Chapter II of this study;

Trujillo-Cenoz, 1972; Boschek, 1971). Second, the tick eyes

exhibit important microanatomical affinities with the eyes

of two phylogenetically lower animals, the snails (Mollusca)

and flatworms (Platyhelminthes). The photoreceptor neurons

of all three (snails, flatworms, and ticks) are strikingly

similar. They all possess terminal microvilli parallel to

the longitudinal axis of the photoreceptor neurons, similar

organelles and cellular organization. The neurons are all

parallel and the microvilli are all oriented in the same

direction. In more advanced eyes the retinular cells are

located around a central rhabdom and the microvilli are

oriented in from two to eight different directions. No

microvillar tight junctions or highly ordered hexagonal

honeycomb pattern have developed in the snails, flatworms,

or ticks as a result of tight packing of microvilli. The

most advanced rhabdomeric eye, that of Octopus, has










microvilli joined by tight junctional bridges (Moody &

Robertson, 1960). The microvilli of flatworms, snails,

and ticks have the assumed primitive condition of no

intervillar interaction.

The archetypal eye of arthropodan stock was possi-

bly very similar to the eye of ticks. This primitive con-

dition probably persisted in the ticks primarily due to

their use of tarsal sensilli as the primary sensory re-

ceptors. The mites have well-developed tarsal sensilli

and in the ticks these sensilli are of particular impor-

tance in host location. Dependence on tarsal receptors

for host location could account for maintenance of the

primitive condition in tick eyes.

Varela (1971) postulates a separate rhabdomeric

evolutionary line for the Mollusca. The obvious affini-

ties of the Platyhelminthes, Mollusca, and tick eye place

them in the same evolutionary line of sensory receptors.














CHAPTER II


THE MICROANATOMY OF THE EYE OF THE
"SCREWWORM FLY," Cochliomyia hominovorax (Coquerel)


Introduction


The eradication of the screwworm fly, Cochliomyia

hominovorax (Coquerel), from Florida is one of the most

successful control programs instituted against a major pest

insect resulting in an estimated 14 million dollars a year

saving to cattlemen (Cromroy, 1971). The larva of the

screwworm fly is an obligate parasite and eats only the

living flesh of warm-blooded vertebrates.

The control program involved breaking the life cycle

of the fly by introducing overwhelming numbers of sterile

male flies into an area to mate with native fertile females.

The program was successful in Florida and eliminated the fly

from most of the southeastern U.S. after its initiation.

The sterile male technique has been used in the southwestern

United States along the Mexican border to prevent the usual

northward spread of the fly each year. A projected program

is now underway to use the sterile male technique in Mexico

in hopes of eliminating the fly from all of Mexico and

prevent its reintroduction into the United States.









The success of the sterile male technique depends

upon the production of large numbers of sterile male flies

that are competitive with wild flies. In order to be fully

competitive in the wild, they must be so close to their

wild counterpart behaviorally and physically that the

female flies will mate with them readily. Mass rearing of

the insects is therefore very important to the success of

this program. In all attempts of mass rearing certain

dietary and genetic problems are encountered because of the

requirements of the rearing program. Mass rearing tends to

cause either genetic deterioration of the breeding stock

used or poor quality flies due to inadequacies in the larval

diet.

One of the problems facing the screwworm project is

the lack of biological information about this fly. This study

was initiated to produce information on the microanatomy of

the eye of the adult fly. Irradiated and unirradiated flies

were used to study the possible effect of irradiation on the

eye of the fly. Several closely related genera have been

studied extensively with electron microscopy and this study

was also undertaken to confirm these prior studies and expand

the information available on the eyes of this important

group of flies.









Literature Review


Most adult insects have 2 large compound eyes located

on the lateral margins of the head. In general the com-

pound eyes of all insects are very similar. They all

possess a peripheral retina composed of repeating units

called ommatidia. Each ommatidium consists of the image-

forming dioptric apparatus and a variable number of unipolar

photoreceptor neurons, the retinular cells. The retinular

cells possess rhabdomeres composed of parallel arrays of

thousands of microvilli (Eakin, 1965). Photopigment molecules

are presumably located on the inner surface of the micro-

villar membrane. The rhabdomeres within a single ommatidium

form a central fused rhabdom in most arthropods (Trujillo-

Cenoz, 1972). Dipterans of the suborder Cyclorrapha have

rhabdomeres that project into a central extracellular omma-

tidial space. This arrangement is termed an open rhabdom.

In addition the dioptric apparatus of dipterans differs

anatomically in that it has a pseudocone beneath the cornea

(Trujillo-Cenoz, 1972; Bernhardt et al., 1972). These

anatomical differences combined with the quantity and

advanced nature of the research on dipteran eyes make it

appropriate to deal with it as an isolated unit.

Seven genera of cyclorraphan Diptera have been studied

in detail using electron microscopy: Musca domestic L.

(Boschek, 1971, 1972; Braitenberg, 1967, 1972; Kirschfeld,













Figure 3. Schematic diagram of Cochliomyia hominovorax
eye showing the relative position of the per-
ipheral retina (PR) and lamina ganglionaris
(LG).










Figure 4. Schematic diagram of a longitudinal section
of an ommatidium of the compound eye of
Cochliomyia hominovorax. BM, basement mem-
brane; BPC, basal pigment cell; L, lens;
LPC, large pigment cell; OC, ommatidial
cavity; PC, pseudocone; PP, primary pigment
cell; R1-7, retinula cells 1-7; R8, inferior
retinula cell.








































3 4

















3J 4


L


R1-7









1967, 1972; Kirschfeld & Francheschini, 1968, 1969; Kirsch-

feld & Reichardt, 1970; Campos-Ortega & Strausfeld, 1972),

Drosophilia melanogaster (Dannel, 1957; Fuge, 1967; Wadding-

ton & Perry, 1960), Anastrepha suspense (Loew) (Agee, in

prep.), the following 3 genera that were studied together

as a unit Chrysomia sp., Lucilia sp., and Sarcophaga sp.

(Melamed & Trujillo-Cenoz, 1968; Trujillo-Cenoz, 1965a,

1965b, 1969; Trujillo-Cenoz & Melamed, 1962, 1963, 1966a),

and Sympycnus lineatus Loew (Trujillo-Cenoz & Bernard,

1972). The eyes of these flies are structurally very similar

and I will review the structures common to the 7 genera

and introduce the terminology to be used in this study.

The dioptric apparatus is composed of a corneal lens

and an underlying gelatinous crystalline body termed

the pseudocone (Trujillo-Cenoz, 1972). The pseudocone is

a soft amorphous substance enclosed in a cup-shaped cavity

formed by 2 cells called the primary pigment cells by

Boschek (1971). The pseudocone is extracellular, contains

no inclusions or cellular organelles (Trujillo-Cenoz &

Melamed, 1966a) and has approximately the same refractive

index as the vitreous humor of the human eye (Bernhardt

et al., 1972). The proximal end of the pseudocone cavity

is closed by 4 wedge-shaped cells forming a plate-like

floor of the cavity (Trujillo-Cenoz, 1965a). Extracellular

amorphous prolongations of the rhabdomeres extend into an









invagination of the proximal membrane of the Semper cells

(Trujillo-Cenoz, 1965a, 1972). Boschek (1971) termed these

rhabdomeric prolongations, the rhabdomere caps. Boschek

(1971) and Trujillo-Cenoz (1965a, 1972) postulate that the

function of the Semper cells is to provide mechanical and

optical coupling between the dioptric apparatus and the open

rhabdom.

Three types of pigment cells are present in the

ommatidia: (1) the primary pigment cells, (2) the large

pigment cells located distally and containing a purple

pigment (Trujillo-Cenoz, 1972), and (3) small basal pigment

"cells" near the basement membrane of the peripheral retina

that contain a yellow-brown pigment (Trujillo-Cenoz, 1972).

The basal pigment "cells" in Aedes egyptii (L.) are actually

not cells but pigment filled bags at the end of thread-

like processes of the Semper cells (Brammer, 1970). Similar

Semper cell processes have been found in Musca (Boschek,

1971) but are not known to connect to the 4 basal pigment

cells.

Eight photoreceptor cells, the retinula cells (R1-R8),

make up the photosensitive portion of the ommatidium (Trujillo-

Cenoz, 1965a; Melamed & Trujillo-Cenoz, 1968; Boschek,

1971; Trujillo-Cenoz & Bernard, 1972). Six of these, R1-R6,

have rhabdomeres that are peripherally located around the

extracellular space forming the central ommatidial cavity.









The centrally located seventh rhabdomere consists of the

rhabdomeres of retinular cells R7 and R8. The rhabdomere of

R7, termed the superior central cell (SCC), forms the

distad portion of the central rhabdomere. The rhabdomere

of R8, termed the inferior central cell (ICC), forms the

proximad portion of the central rhabdomere. Rhabdomeres

R7 and R8 are subequal, the superior rhabdomere (R7) is

long and the inferior (R8) is relatively short. The axons

of R7-R8 do not synapse in the first visual ganglion, the

lamina, but pass directly into the second, the medulla

(Melamed and Trujillo-Cenoz, 1968; Trujillo-Cenoz, 1972).

The rhabdomere of R7-R8 differs from R1-R6 by being smaller

in diameter and cylindrical rather than a truncated cone

(Boschek, 1971).

The rhabdomeres are composed of tightly packed micro-

villi. The orientation of microvilli in rhabdomeres R1-R6

is such that the microvilli of the following are parallel:

R1 and R4, R2 and R5, and R3 and R6 (Boschek, 1971; Melamed

& Trujillo-Cenoz, 1968). The orientation of the rhabdomeric

microvilli in the central cells (R7 and RS) is orthogonal

in Musca, Crysomyia, Lucilia, and Sarcophaga (Boschek,

1971; Melamed & Trujillo-Cenoz, 1968; Trujillo-Cenoz,

1972; Bernhardt et al., 1972; Trujillo-Cenoz & Bernard,

1972).

In the species, Sympycnus lineatus, two types of omma-

tidia are present. Half the ommatidia, those with yellow










corneal facets, have the usual orthogonal or perpendicular

arrangement of the central rhabdomeric microvilli. In the

remaining half of the ommatidia, those with red corneal

facets, the microvilli, are parallel to one another (Trujillo-

Cenoz & Bernard, 1972).

The orthogonal arrangement of microvilli has been

postulated as a two-channel analyzer of plane-polarized

light (Waterman & Horch, 1966; Melamed & Trujillo-Cenoz,

1968; Trujillo-Cenoz, 1972). Rhabdomeres with parallel

arrangement of microvilli are postulated to diminish the

absorption of plane-polarized light in the opposite or

perpendicular plane (Trujillo-Cenoz & Bernard, 1972;

Trujillo-Cenoz, 1972). The parallel microvilli are oriented

in the vertical plane and are believed to minimize the

absorption of horizontally polarized light, i.e., reflected

light or "glare." A similar arrangement is found in the

ventral portion of the eye of the water strider, Gerris sp.

(Schneider & Langer, 1969), and is believed to allow a

better view into the water by differential screening of

surface reflected light.

Directly beneath the peripheral retina is the first

synaptic field of the eye, the lamina ganglionaris. The

lamina is divided anatomically into three layers: the

external fenestrated layer, an intermediate layer of uni-

polar cell soma,and the proximal plexiform layer (Trujillo-

Cenoz, 1965b; Boschek, 1971). The fenestrated layer










contains tracheoblasts, trachea, and bundles of eight retinu-

lar cell axons, the pseudocartridges. The somata of unipolar

second order neurons are located in the intermediate or

unipolar cell soma layer. The plexiform layer has two second

order axons termed L1 and L2 by Braitenberg (1967) which

synapse with the axonal fibers from retinular cells R1-R6

(Trujillo-Cenoz, 1965b) to form the optical cartridges.

The optical cartridges are surrounded by epithelial cells

that make intimate contact with retinular axons R1-R6 by

means of specialized glial projections called capitate

projections (Trujillo-Cenoz, 1965b; Boschek, 1971). These

structures were first believed to be synaptic in nature and

were termed synaptic buttons by Pedler and Goodland (1965).

The true synaptic loci however are formed by T-shaped

synaptic ribbons (Trujillo-Cenoz, 1965a, 1965b; Boschek,

1971, 1972).


Methods and Materials


The flies used in this study were obtained from two

sources. Unirradiated flies were reared by Dr. Gerald Holt,

USDA, APHIS, Fargo, North Dakota. These flies were fixed

and embedded in Fargo by Dr. Holt's laboratory personnel

following the same preparative technique used on irradiated

flies. Irradiated flies were reared in Mission, Texas, by

USDA, APHIS in their rearing facility and shipped via air

mail to Gainesville as pupae. The pupae were placed in a









shallow cup in a holding cage consisting of an aluminum

frame with tube gauze stretched over it. Cotton saturated

with a mixture of honey and water was provided as a source

of sugar and water for the adult flies. Flies from 3 to 6

days of age were utilized.

Living flies were submerged in paraformaldehyde-

gluteraldehyde fixative (Karnovsky, 1965) for dissection.

Following removal and bisection, the eyes were transferred

to fresh fixative for 4 hours at room temperature, rinsed

in 0.1 M cacodylate buffer (pH 7.2) for 20 minutes and post-

fixed in 2% osmium tetroxide for 20-24 hours at 40C. Rapid

dehydration at intervals of 5 minutes in 25, 50, and 75%

ethanol followed a second rinse in 0.1 M cacodylate buffer.

The eyes were held in 2% uranyl acetate in 75% ethanol for

4 hours at 4C. Dehydration was completed with 5-minute

changes of 95, 100% ethanol and two changes of 100% acetone

at room temperature. The eyes were infiltrated for 1 hour

with 50% Spurrs plastic (Spurr, 1969) and 24 hours in 100%

plastic prior to polymerization at 60C for 24 hours.

Silver and light gold sections were cut using a duPont

diamond knife on a Porter-Blum-MT-2 ultramicrotome, picked

up on 75-mesh copper grids covered with a Formvar film and

post-stained with uranyl acetate and lead citrate (Reynolds,

1963) prior to examination with either a Hitachi HU11C or

HU11E electron microscope at 75 kV.









Results


The Peripheral Retina


The peripheral retina of Cochliomyia hominovorax

consists of hexagonally packed ommatidia (Fig. 4). Each

ommatidium has a dioptric apparatus and 8 photoreceptor

cells, the retinular cells (R1-R8). The diptric apparatus

is composed of a corneal lens (L) and a gelatinous pseudo-

cone (PC). Surrounding the pseudocone and forming the

lateral walls of the pseudocone cavity are the primary pig-

ment cells (PP). The floor of the pseudocone cavity is

formed by a rectangular plate of wedge-shaped Semper cells

(S). In addition to the primary pigment cells, two other

types of pigment cells are found in the peripheral retina:

the large pigment cells (LPC) located laterally and the basal

pigment cells (BPC).

The receptor region of the peripheral retina is com-

posed of the 8 photoreceptor or retinula cells. Six of these

retinular cells (R1-R6) are located peripherally around the

central ommatidial cavity (OC). Retinular cells, R1-R6,

bear independent rhabdomeres made up of microvilli. The

rhabdomeres of retinular cells, R7 and R8, are centrally

located in the ommatidial cavity and form a single central

rhabdomere. The distal portion is formed by the rhabdomere

of R7, termed the superior central cell, and the proximal









portion by R8, the inferior central cell. The two central

retinular cells are subequal in length, R7 (SCC) is longer

being approximately 170 microns in length and R8 (ICC) is

approximately 60 microns in length.

Each retinular cell has an array of microvilli that

extends from the distal to the proximal end of the cell

body. The blind ends of the microvilli project into the

ommatidial cavity. The microvilli borne by one cell are

termed a rhabdomere and the rhabdomeres whether fused or

separate are termed a rhabdom. In most arthropods the rhab-

domeres are fused into a central rhabdom. Cochliomyia

hominovorax and the other members of the suborder Cyclor-

rapha have an ommatidial space or cavity (OC) that separates

the rhabdomeres and extends the length of the ommatidium.

This configuration is termed an open rhabdom.


The Dioptric Apparatus


The dioptric apparatus of the adult screwworm fly con-

sists of a corneal lens and an amorphous gelatinous pseudo-

cone. The lens (Plate X A) is a modification of the cuticle.

It has no pore canals or other structures usually associated

with the cuticle of insects. Plate X A is a cross section

of a lens showing the alternating "dense" and "rare" bands

believed by several authors to act as interference filters

(Trujillo-Cenoz, 1972; Bernard & Miller, 1968; Bernard














Plate X. Cochliomyia hominovorax unirradiated

A. Cross section of lens showing alternating dense
and rare bands believed to act as interference
filters. Note small round protubrences on
surface of lens (arrows). X8740



















B. Off center longitudinal section of lens (L) and
pseudocone (PC). Primary pigment cells IPP)
form the lateral walls of the pseudocone cavity.
Note microvilli-like projections of primary pig-
ment cell membrane (arrows) and alternating dense
and rare bands.









et a ., 1972). Plate X B shows these bands in longitudinal

section.

Beneath the corneal lens is the pseudocone cavity con-

taining the extracellular amorphous pseudocone (PC) (Plate

X B). The pseudocone cavity is formed by the primary pig-

ment cells (PP) (Plate X B, XI A & B). There are two primary

pigment cells that form the lateral walls of the pseudocone

cavity in Cochliomyia hominovorax (Plate X B; XI A & B).

Numerous irregular microvillar-type (Mv) evaginations of the

primary pigment cell membrane project into the pseudocone

cavity (Plate XI B). The pseudocone is not completely

homogeneous and contains material of greater electron density

irregularly concentrated toward the center of the pseudocone

(Plate XI A). Plate XI A is a micrograph of a cross section

through the pseudocone (PC), the primary pigment cells (PP),

and the primary pigment cell nuclei (N). The primary pig-

ment cells are tightly bound by spot desmosomes (SD), at

the edge of the cell that bounds the pseudocone, and gap

junctions (GJ) over the rest of the adjoining membrane.


The Semper Cells


The proximal end of the pseudocone cavity is closed by

4 flattened wedge-shaped cells that form a rectangular plate-

like floor (Plate XIII B, XI B). Plate XI B shows an off-

center longitudinal section (Fig. 5) of the pseudocone (PC)












Plate XI. Cochliomyia hominovorax unirradiated

A. Cross section of pseudocone (PC) and primary pigment
cells (PP). Two primary pigment cell nuclei (N)
and pigment filled vacuoles are most prominent
organelles in the primary pigment cells. Note
gap junction (GJ) and spot desmosome (SD) cell to
cell contact between primary pigment cells. X8740


















B. Off-center longitudinal section of pseudocone (PC)
and Semper cells (S). Semper cells have short un-
equal "microvilli" (black arrows) that project into
the pseudoncone cavity. Note Semper cell nuclei (N),
gap junction (GJ), and spot desomosome (SO). X11970










and the Semper cells (S). Plate XI B is an electronmicro-

graph of a cross section of the Semper cell plate. Short

irregular projections (arrows) of the Semper cells'distal

membrane project into the pseudocone cavity (PC) (Plate XI B).

The Semper cells are joined by cell junctions that are

very similar in structure to those of the primary pigment

cells. Plate XI B shows the cell junction between 2 Semper

cells (S). Their membranes are joined near the pseudocone

cavity by a spot desmosome (SD) or macula adherens and the

remaining membrane is joined by a gap junction (GJ) or zonula

occludens (Plate XI B). Spot desmosomes are cell contacts

that involve thickening of the cytoplasmic surface of the

cell membrane and gap junctions are cell contacts with a

partial obliteration of the intercellular space (Satir &

Gilula, 1973). The spot desmosome (SD) and gap junction

(GJ) between the Semper cells provide a close sealed appo-

sition and seal the bottom of the pseudocone cavity.

The distal membrane of the Semper cells is produced

into a network of ridges that project into the pseudocone

cavity. In cross section (Plate XI B) these ridges appear

to be irregular microvillar-type projections. Plate XII

shows two magnifications of the junction of the Semper cells

and the pseudocone. This network of Semper cell membrane

ridges projects into the pseudocone cavity presumably

holding the gel-like pseudocone in place.












Plate XII. Cochliomyia hominovorax unirradiated

A. Cross section through a junction of the pseudo-
cone (PC), Semper cells (SC), and primary pigment
cells (PP). Projections from the Semper cells
into the Pseudocone cavity form a network
(arrows). Note spot desmosome (SD) joining
Semper cell membranes. X5890



















B. Higher magnification of the same cross section
of a junction between pseudocone (PC) and Semper
cells (SC). X38000











Plate XIII. Cochliomyia hominovorax unirradiated

A. Cross section of an ommatidium near junction of
the pseudocone (PC) and 4 Semper cells (S). Note
presence of primary pigment cells IPP), primary
pigment cell nucleus (N), large pigment cells
(LPC), and distal end of the ommatidial cavity
(OC). Numerous granular inclusions (arrows)
present in the cytoplasm of the large pigment
cells and make it possible to distinguish them
from the primary pigment cells. Spot desmosomes
joining distal membranes of Semper cells are
indicated by arrows. X6650











B. Cross section of the 4 Semper cells (S) showing
the rhabdomere caps (RC) and the ommatidial
cavity (OC). Four Semper cell nuclei (SN) and
2 large pigment cell nuclei (PN) are shown.
Note that only gap junction (GJ) present.
X7600










The proximal membrane of the Semper cell plate is in-

vaginated; the ommatidial cavity (OC) and rhabdomere caps

(RC) project into this invagination. Plate XII A is a

cross section of the Semper cell plate showing 7 rhabdomere

caps and the distal portion of the ommatidial cavity. This

distal projection of the ommatidial cavity has 7 arms (Plate

XIV A & B). Between these arms of the ommatidial cavity

amorphous extracellular extensions of the rhabdomeres,

the rhabdomere caps, are situated (Plate XII B). There are

7 rhabdomere caps, one corresponding to each rhabdomere.

The trapezoidal configuration of the rhabdomere caps is

the same configuration as the distal rhabdomeres. Plate

XII is a cross section of the Semper cell plate. Four Semper

cell nuclei (SN) and 7 rhabdomere caps (RC) are present in

this section. The spot desmosomes that join the distal

membranes of the Semper cells (see Plate XII A or XII B)

are not present in this more proximal section. The spot

desmosomes of the Semper cells and belt desmosomes (BD)

that join the mesial face of the retinular cells (Plate

XVII) differ primarily in length. The spot desmosomes of

the Semper cells form localized plaques.

The tip of the extracellular rhabdomere caps (RC)

project distally between the arms of the ommatidial cavity

(OC) (Plate XIV A). The ommatidial cavity has 7 distal

arms; between these arms the rhabdomere caps end. The












Plate XIV. Cochliomyia hominovorax unirradiated

A. Cross section of the Semper cell junction and
7-armed ommatidial cavity (OC). The rhabdomere
caps (RC) appear first between the arms of the
ommatidial cavity. Note junction of Semper
cell (arrows). X22800

B. Cross section of the Semper cell (S) junction and
7 arms of the ommatidial cavity (OC). Note
tubules in the Semper cell cytoplasm. X3800

C. Cross section of Semper cell cytoplasm (S) junc-
tion near the ommatidial cavity (OC). Cytoplasm
completely filled with microtubules. X51400

D. Higher magnification cross section of Semper cell
cytoplasm (S). Tubules completely fill the cyto-
plasm. X57000









ommatidial cavity is located at the junction of the 4 Semper

cells (S) (Plate XIV A, B). Plate XIV A and B are cross

sections of the proximal portion of the junction of the

Semper cell plate. The ommatidial cavity (OC) is formed by

an invagination of the basal surface of the Semper cells.

Electron dense granular material fills the ommatidial cavity

(Plate XIV A, B, & D). The gap junctions (arrows, Plate

XIV A, B) joining the Semper cells separate at the rhabdomere

caps (RC) and reform on the other side of the cap (small

arrows, Plate XIV A).

The cytoplasm of the Semper cells is totally devoid

of organelles. Plate XIV has four different magnifications

of Semper cell cytoplasm. Microtubules completely fill the

Semper cells and no other organelles were observed. The

microtubules are randomly packed and have no apparent

orientation.

Plate XV B is an electron micrograph of a cross section

at the junction of the Semper cells and the retinular cells.

At this level the transition from rhabdomere cap (RC) to

rhabdomeric microvilli occurs. Rhabdomere R3 is sectioned

through the point of transition and shows both the amorphous

cap (RC) and rhabdomeric microvilli (arrows, Plate XV C).

The distal end of a retinular cell is attached to the proxi-

mal membrane of the Semper cell by a pointed evagination

of the retinular cell membrane with desmosomal contact

completely surrounding it (arrows, Plate XV B).












Plate XV. Cochliomyia hominovorax unirradiated

A. Section through several pigment-filled vacuoles
(PV) in a large pigment cell. Although generally
referred to as pigment "granules" a vacular
membrane (arrows) is present surrounding the
pigment. Note growth of pigment crystals in
vacuoles numbered 1-5. Note granular inclusions
(GI) in cytoplasm found only in large pigment
cells. X51300







B. Cross section of a junction between Semper cells
(S) and retinular cells (R). Four extracellular
rhabdomere caps (RC), R2, R4, and R5, are present
in the ommatidial cavity (OC). The rhabdomeres
(R) of R1 and R6 are present and R3 is at the
point of transition. Distal prolongations of
retinular cells project into Semper cells and are
joined by circular belt desmosome (arrows).
X15200






C. Higher magnification of retinular cell R3. Note
rhabdomeric cap (RC), microvilli (arrows),
mitochondria (M), and belt desmosome (BD).
X38000









Pigment Cells


Two types of pigment cells are present surrounding

the pseudocone and distal ommatidium (Fig. 4). These con-

sist of 2 primary pigment cells (PP) and 6 large pigment

cells (LPC) that extend from the middle of the pseudocone

proximally to near the basement membrane. These 2 pigment

cell types and their processes may be distinguished by dense

granular inclusions (GI) that occur only in the cytoplasm

of the large pigment cells (Plate XII, arrows; XV A). The

nuclei of the large pigment cells (PN) are situated near the

distal end of the ommatidium (Plate XIII B). There is a

third type of pigment cell located at the basement membrane

(Plate XIX A, Fig. 4). Four processes of these basal pigment

cells (BPC) occlude the ommatidial cavity at the basement

membrane (Plate XIX A).

Pigment-filled vacuoles are present in the cytoplasm

of the primary pigment cells, large pigment cells, and basal

pigment cells. The retinular cells also contain pigment-

filled vacuoles (arrows, Plate XVI A). Plate XVI A shows

both types of pigment'vacuoles, the small retinular cell

vacuoles (arrows), and the larger pigment cell vacuoles.

Referred to as pigment "granules" by previous authors, they

are actually vacuoles filled with pigment crystals. Plate

XV A is an electronmicrograph of several pigment vacuoles

(PV) in the cytoplasm of a large pigment cell (note the












Plate

A.


XVI. Cochliomyia hominovorax unirradiated

Cross section of an ommatidium, primary pigment
cells (PP), and large pigment cells (LP) just
below the Semper cells. Note the presence of
pigment filled vacuoles in the retinular cells
(arrows). X9500


B. Cross section of a portion of retinular cells
R1, R6, and R7. The rhabdomeric microvilli are
attached to the retinular cells by thin necks
creating an extracellular space at their base
(arrows). Note belt desmosomes (BD). X28500





C. Cross section through distal ommatidium (higher
magnification of plate XVI A). The retinular
cells are joined by belt desmosomes the entire
length of the ommatidial cavity (OC). Note
microvillar orientation and rotational asymmetry
of rhabdom. X22800









presence of granular inclusions that identify it as a large

pigment cell). The vacuolar membrane (arrows) is clearly

visible around pigment vacuoles that are incompletely filled

with pigment crystals (Plate XV A). Apparently the pigment

crystallizes within the vacuole and the vacuole fills with

these pigment crystals. Long needle-like crystals are

present in vacuoles 1 and 2. The vacuoles in Plate XV A

numbered from 1-5 indicate different states of maturation.

Vacuole 5 is considered to be a "mature" vacuole.


Retinular Cells


Each ommatidium has 8 retinular cells: 6 distributed

peripherally around the ommatidial cavity (R1-R6) and 2

(R7 and R8) that project into the central ommatidial cavity.

The retinular cells have two distinct regions: a soma or

cell body that bears the microvilli, and an axonal segment

that enters the first synaptic loci of the brain. The

peripheral retina is composed of the retinular cell somata.

The retinular cells are joined by belt desmosomes (BD)

that fuse the retinular cells for the entire length of the

ommatidium (Plate XVI B, XVII A, B). The 7 belt desmosomes

are the only points of attachment between the retinular cells.

Pigment cell processes are present between the nondesmosomal

membranes of the retinular cells.












Plate XVII. Cochliomyia hominovorax unirradiated

A. Cross section of an ommatidium midway in the
peripheral retina. The rhabdomere of the superior
central cell, R7, is round and centrally located.
The remaining rhabdomeres, R1-6, are conical.
Invaginations of the plasma membrane beneath the
microvilli form intracellular channels (arrows).
Retinular cell cytoplasm contains pigment vacu-
oles, mitochondria (M), multivesicular bodies
(MVB), and isolated cisternae of rough endoplas-
mic reticulum (RER). X8740


















B. Cross section of rhabdom at the junction of the
superior central cell (R7) and the inferior central
cell (R8). Note intracellular channels (arrows)
and multivesicular bodies (MVB). X8740









The arrangement of retinular cells Rl-R6 is fixed in

the distal portion of the rhabdom. The pattern is roughly

trapezoidal and is rotationally asymmetrical. Plate XVI

A and B are cross sections of an ommatidium passing directly

beneath the Semper cells. The trapezoidal pattern disappears

near the basement membrane (see Plate XVII B, XVIII A).

This pattern is also present in the rhabdomere caps (Plate

XIII B).

The configuration of this trapezoidal arrangement is

such that a line inscribed through R3, R2, and R1 will be

perpendicular to the horizontal plane of the eye and point

toward the midline of the eye. This line (R3-R2-R1) is

always parallel to the axis of the microvilli of the superior

central cell (R7). The blind ends of the microvilli of

R7 always point away from the midline of the eye. The supe-

rior cell microvilli in the dorsal portion of the eye point

upwards, and those in the ventral hemisphere of the eye

point down. This mirror image inversion has been found in

other dipterans (Trujillo-Cenoz, 1972). An electron

micrograph can be oriented using the microvilli of the supe-

rior central cell.

The ommatidial cavity (OC) (Plate XVI) extends the

entire length of the rhabdom and is filled with an unknown

material. This electron dense material completely fills the

ommatidial cavity distally (Plate XV B; XIV A, B; XIII B).

Directly beneath the Semper cells it forms clouds of













Plate XVIII. Cochliomyia hominovorax unirradiated

A. Cross section of ommatidium just below the transi-
tion from superior central cell (R7) to inferior
central cell (R8). Orientation of central cell
microvilli is orthogonal. X8740



















B. Cross section of a portion of an ommatidium just
below the transition from the superior central
cell (R7) to the inferior central cell (R8).
Microvillar orientation of central cell rhabdomeres
is parallel. X22800




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MICROANATOMICAL STUDY ON THE EYES OF THE LONE STAR TICK AND THE SCREWWORM FLY WITH RELATED ELECTROPHYSIOLOGICAL STUDIES By WILLIAM AVERY PHILLIS III A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1975

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To my without whom two precious daughters, Emily and Colby, my life would have little meam ng

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ACKNOWLEDGMENTS I sincerely wish to thank Dr. H. L. Cromroy, Chairman of my Supervisory Committee, for his support, patience, and guidance during the past four years. I also wish to thank the other members of my supervisory committee, Drs. H. R. Agee, H. C. Aldrich, J. F. Butler, and D. E. Weidhass for their help and encouragement. I am particularly grateful to Dr. H. C. Aldrich for his help and generous use of the Biological Ultrastructure Laboratory where much of this work was carried out. I am indebted to Dr. H. R. Agee for his generous help and for the liberal use of his electrophysiological laboratory . I wish to thank Dr. G. Holt, USDA, APHIS, Fargo, North Dakota, and Dr. H. C. Hoffman, USDA, APHIS, Mission, Texas, for supplying the screwworm flies used in this study. I am grateful to my parents and sisters for their encouragement and understanding when it was most needed. Financial support was provided by National Institute of Health training grant No. T01 AI 00383-02. I am deeply grateful to Dr. R. E. Bradley and the other members of the Faculty of Parasitology for making this traineeship ava i 1 abl e to me .

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PREFACE The experimental data presented in this dissertation deal with two uniquely different photoreceptors, the tick eye and the insect eye. For the purposes of clarity, the current study has been subdivided into the following three chapters: Chapter I, The Microanatomy of the Eye of the "Lone Star Tick," Ambl yomma americanum (L.); Chapter II, The Microanatomy of the Eye of the "Screwworm Fly," Cochl i omy i a ho minovorax (Coquerel), and Chapter III, The Spectral Sensitivity of the Compound Eye of Cochl i omyi a homi n ovorax (Coquerel ) .

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TABLE OF CONTENTS Pag e ACKNOWLEDGMENTS PREFACE LIST OF FIGURES LIST OF PLATES ABSTRACT CHAPTER I THE MICROANATOMY OF THE EYE OF THE "LONE STAR TICK," Amblyomma americanum L. . . . Introduction Literature Review Methods and Materials Results Discussion II THE MICROANATOMY OF THE EYE OF THE "SCREWWORM FLY," Cochl iomyi a hominovorax ( Coquerel ) Introducti on Literature Review Methods and Materials Resu Its Discussion Ill THE SPECTRAL SENSITIVITY OF THE COMPOUND EYE OF Cochl i omyi a hominovorax Literature Review Methods and Mater i al s Resu Its Discussion IV CONCLUSIONS l 1 VI vi i XT 1 1 1 3 7 9 39 49 49 51 58 60 104 121 121 125 130 130 137

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TABLE OF CONTENTS (continued) APPENDIX LITERATURE CITED BIOGRAPHICAL SKETCH Page 141 144 156 VI

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LIST OF FIGURES Figure 1 2 3 Schematic diagram of an eye of Ambl yomma americanum Schematic diagram of 1 photoreceptor neuron of Ambl yomma americanum Schematic diagram of Cochl i omyi a hominovorax eye showing the relative position of the peripheral retina and lamina ganglionaris . . . Schematic diagram of a longitudinal section of an ommatidium of the compound eye of Cochl i omyi a hom inovorax Spectral sensitivity curve of the dark-adapted compound eyes of Cochl i omyi a hominovorax . . . . Weekly visual sensitivity of irradiated Cochl i omyi a hominovorax Page 12 12 53 53 132 134

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LIST OF PLATES Plate I I III Amblyom ma americanutn A "' 1 arva Oblique section of an optic nerve Pag e 14 B. Oblique section of an eye Amblyomma americanum larva 17 A. Longitudinal section of an eye B. Longitudinal section of a portion of an eye Amblyomma a meri canum adult 19 A. Oblique cross section of the lens B. Oblique section of lenticular pore canals C. Longitudinal section of scutellar pore canal s IV V. VI VII. Amblyomma americanum adult A. Cross section of terminal microvilli bearing region of photoreceptor cell B. Cross section of terminal microvilli at higher magnification 21 25 A mblyomma am ericanum adult A. Longitudina"! section through tips of m i c r o v i 1 1 i B. Cross section of a peripheral portion of an eye Amblyomma americanum adult 27 A. Oblique section of photoreceptor neuron through base of microvilli B. Cross section of photoreceptor neuron through region B below microvilli C. Oblique section of neuron at base of mi crov i 1 1 i D. Cross section of region B directly below microvilli Amblyomma a mericanum adult 30 A. Cross section of photoreceptor neurons on periphery of eye just under the hypodermis B. Cross section of photoreceptor neurons in region C, the soma, at a higher magnification C. High magnification of glycogen-like inclusions in region C

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LIST OF PLATES (continued) Plate VII. D (cont . ) E Page High magnification of coated vesicles around the nucleus Lower magnification of gl ycogen1 i ke and vesicular inclusions of neural soma VIII. Arnblyom ma americanum adult A . Cro ss section of a single neuron throughout reg i on C B. Higher magnification of the same section C. Cross section of glial cell mesaxons, extracellular sheath, bundles of fibrils, and glial cell cytoplasm D. Cross section of glial cell sheath fibril bundles IX. Arnblyomma americanum AT Obi i q u e section of larval optic nerve B. Cross section of 2 axons, a glial nucleus, mesaxons, extracellular glial sheath, and glial cell cytopl asm C. Cross section of axons, glial nucleus, and mesaxonal sheath surrounding the axons D. Cross section of a small portion of the axoplasm of a single axon showing characteristic microtubules and mitochondria X . Cochl i omyi a homi novorax unirradiated JT. £ross s e ction of lens B. Off center longitudinal section of lens and pseudocone X I . Cochl i omyi a homi novorax unirradiated. . . . , A. Cross section of pseudocone and primary pigment cells B. Longitudinal section of pseudocone and Semper cells XII. Cochl i omyi a homi novorax unirradiated. . . . , A. Cross section through a junction of the pseudocone, Semper cells, and primary pigment cells B. Higher magnification of the same cross section of a junction between pseudocone and Semper cells 33 36 63 66 69 l x

PAGE 10

LIST OF PLATES (continued) Plate XIII. XIV. XV XVI Cochl iomyia hominovorax unirradiated A. Cross section of an ommatidium near junction of the pseudocone and 4 Semper cells B. Cross section of the 4 Semper cells showing the rhabdomere caps and the ommatidial cav i ty Cochl i omyi a homi novorax unirradiated A. Cross section of the Semper cell junction and 7-armed ommatidial cavity B. Cross section of the Semper cell junction and 7 arms of the ommatidial cavity C. Cross section of Semper cell cytoplasm junction near the ommatidial cavity D. Higher magnification cross section of Semper cell cytopl asm Coc h l i omyi a h omi novorax unirradiated A. Section through several pigment filled vacuoles in a large pigment cell B. Cross section of a junction between Semper cells and retinular cells C. Higher magnification of retinular cell R3 Cochl iomyia homi novorax unirradiated A. Cross section of an ommatidium, primary pigment cells, and large pigment cells B. Cross section of a portion of retinular eel Is Rl , R6, and R7 C. Cross section through distal ommatidium Page 71 74 77 80 XVII. Cochl i omyi a homi novorax unirradiated A. Cross section of an ommatidium midway in the peripheral retina B. Cross section of rhabdom at the junction of the superior central cell and the i nf er i or centra 1 cell XVIII. Cochl iomyia homi novorax unirradiated A. Cross section of ommatidium just below the transition from superior central cell to inferior central cell (orthogonal microvilli) B. Cross section of a portion of an ommatidium just below the transition from the superior central cell to the inferior central cell 86

PAGE 11

LIST OF PLATES (continued Plate XIX XX XXI XXII XXIII XXIV. Cochl i omyi a hominovorax unirradiated A. Oblique section through the basement membrane B. Cross section of a single pseudocartridge Cochl i omyi a homi novorax unirradiated A. Cross section of an optical cartridge B. Synaptic loci or T-shaped synaptic ribbons C. Single synaptic ribbon D. Cross section of an optical cartidge adjacent epithelial glial cell and glial nucleus Cochl i omyi a homi novorax irradiated A. Oblique section through the distal portion of an ommatidium B. Cross section through distal portion of an apparently normal irradiated ommatidium C. Cross section of an apparently normal R7-R8 central cell transition of an irradiated ommatidium Cochl i omyi a hominovorax irradiated A. Cross section of an aberrant irradiated ommatidium (with obconical rhabdomeres) B. Cross section of a portion of an abnormal irradiated ommatidium (obconical rhabdomeres) C. Cross section of an abnormal irradiated omma tid i urn Coc hl i omyi a homi novorax irradiated A. Cross section of 8 cell ommatidium (parallel microvilli) B. Cross section of 8 cell ommatidium (orthogonal microvilli) C. Cross section of several ommatidia through the transition zone of superior and inferior central cells Cochl i omyi a homi novorax irradiated A. Cross section of an ommatidium with an abnormal super i ori nf er i or central cell transition (6 rhabdomeres) B. Cross section of an ommatidium with an abnormal superior-inferior central cell transition (8 rhabdomeres) Page 90 94 97 100 103 106 x 1

PAGE 12

LIST OF PLATES (continued) Plate XXV Cochl i omy ia homino vorax irradiated A. Cross section of an optical cartridge B. Higher magnification cross section of 2 photoreceptor axons Page 108

PAGE 13

Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MICROANATOMICAL STUDY ON THE EYES OF THE LONE STAR TICK AND THE SCREWWORM FLY WITH RELATED ELECTROPHYSIOLOGICAL STUDIES By Wi 1 1 iam Avery Phil 1 is III August, 1975 Chairman: Harvey L. Cromroy Major Department: Entomology and Nematology The electron microscope was used to detail the microanatomy of the eyes of the "lone star tick," Amblyomma americanum (L.), and the "screwworm" fly, Cochl i omyia hominovorax (Coquerel). The eyes of the "lone star tick" consist of a cuticular lens and 30-40 underlying photoreceptor neurons. The lens contains bundles of specialized lenticular pore canals believed to function as light or wave guides. The photoreceptor neurons possess the microanatomical structures common to all rhabdomeric photoreceptors. The simplicity of the tick eye is believed to be a primitive condition and is the first arhabdomate eye described for the phylum Arthropoda. The photoreceptor neurons of Amblyomma show affinities with the arhabdomate eyes of the flatworms and snails. The microanatomy of the peripheral retina and lamina of the "screwworm" fly, Cochl i omyia hominovorax , x 1 1 1

PAGE 14

is similar to the other genera of cyclorraphan Diptera published. The eyes of irradiated and unirradiated flies were examined with electron microscopy. Irradiated flies showed a number of abnormalities not encountered in the unirradiated flies. Irradiated flies showed a large increase in retinular cell vacuolation, increased numbers of trachea in the peripheral retina, abnormally shaped rhabdomeres, decreased numbers of rhabdomeric microvilli, and abnormal central cell transitions. The function and origin of the Semper cells are discussed. The Semper cells are believed to have several functions. The most important function is to maintain the optically important trapezoidal pattern of the rhabdomeres and central cavity of the open rhabdom. The central cells (R7 and R8) have two types of rhabdomeres: The microvilli of the first type are orthogonal and the microvilli of the second are parallel. The pigment "granules" of previous authors are actually vacuoles filled with pigment crystals. The electrophysiological method of equal response was used to determine the spectral sensitivity of the eye of irradiated screwworm flies. The eye was maximally sensitive at 490 nm with a secondary peak at 350 nm and a small "pseudopeak" at 625 nm. The visual sensitivity of irradiated mass reared screwworm flies exhibited considerable weekly variation, some weeks flies were as much as 10 times more sensitive than other weeks, when measured with the ERG at 530 nm. xi v

PAGE 15

CHAPTER I THE MICROANATOMY OF THE EYE OF THE LONE STAR TICK," Amblyomma americanum L Introduction Ticks are extremely important pathopherous agents. The lone star tick, A mblyomma americanum ( L . ) , is an economically and medically important pest of wildlife, livestock and man. It ranges from central Texas throughout southcentral and southeastern United States north to Maryland and Pennsylvania (Cooley & Kohls, 1944; Bishopp & Trembley, 1945). A. americanum is an important vector of Rocky Mountain spotted fever (Rivers & Horsfall, 1959), Q fever, and is known to produce tick paralysis in man. In general, tick physiology and biology have received considerable attention from Hoogstral (1970) in Africa and Sonenshine, Hair and Semtner in the United States. Studies by Sonenshine (Sonenshine e_t aj_. , 1966; Sonenshine & Levy, 1971) and by Semtner et aj_. (Semtner e_t aj_. , 1971a; Semtner et aj_. , 1971b; Semtner & Hair, 1973a; Semtner & Hair, 1973b; Semtner et aj_. , 1973) on the biology and ecology of Amblyomma americanum are particularly valuable. However, very few studies have included work on the photobiology of

PAGE 16

ticks. This is difficult to understand in view of several studies indicating the importance of photoperiod in diapause, oviposition and questing behavior. McEnroe and McEnroe (1973) studied the questing behavior of Dermacentor variabilis (Say) and found that a photostimul us is necessary to initiate questing behavior. The ovi posi ti onal pattern of two ticks, Anocenter n j t e n s Neumann and A. macu 1 atum Koch, were shown to be highly sensitive to photoperiod (Wright, 1969; Wright, 1971). Photoperiod was also shown to be the critical factor in initiation of diapause in Derma centor a 1 b i p i c t u s (Packard) (Wright, 1969) and Dermacentor variabilis (Smith & Cole, 1941). In addition most taxonomic keys to the hard ticks contain couplets that separate genera on the basis of the presence or absence of eyes. No adequate research is available on the function and morphology of the eye of Ixodid ticks. Part of the problem with prior anatomical studies has been that they were done with light microscopy. Many of the characteristic structures of photoreceptor cells are beyond the resolving power of the light microscope and therefore fail to detail adequately the fine structure and make a determination possible as to whether or not a neuron could function as a photoreceptor. This research was done with the electron microscope to investigate and detail the structure of the eye of Amblyomma americanum and to use the mi croanatomical analysis for determination of the eye as a functioning photoreceptor.

PAGE 17

Literature Review Two types of eyes are present in the arthropods: compound eyes consist of from several to several thousand repeating units known as ommatidia and small unicorneal eyes termed ocelli. The eyes of Amblyomma americanum are of the second or unicorneal type. Unicorneal eyes have received little attention in the literature due in part to the difficulty encountered in working with such small structures. The size and simplicity of these structures however make them of potential importance in neur ophysi ol og i ca 1 vision studies and well suited to electron mi orographic techniques. With the exception of the compound eyes of L i m u 1 u s chelicerate eyes are typically uni 1 ent i cu 1 ar ocelli. The anatomy and mi cros tructure of these ocelli are very similar throughout the cheli cerates. The lens is biconvex with an underlying vitreous body composed of a single layer of transparent cells. The photosensitive or retinular cells are organized into a cup-shaped retina. The closely packed microvilli characteristic to all arthropod retinular cells are always orientated perpendicular to the light path (Miller, 1960; Eakin, 1965). The microvilli of each cell form isolated units known as rhabdomeres or when combined with the microvilli from one or more other retinular cells they form a rhabdom.

PAGE 18

L i m u 1 u s has paired compound eyes as well as a pair of unicorneal ocelli and a rudimentary median eye. The ocelli consist of a biconvex lens and an underlying cup-shaped retina. Three types of retinular cells are present in the retina. The microvilli of the first type form single-layered rhabdomeres and the microvilli of the other two form doublelayered rhabdoms. Rhabdoms consisting of two layers of microvilli are of two types: (1) " sel f -rhabdoms " in which both layers of microvilli arise from a single cell; (2) those rhabdoms in which the microvilli of two cells form a rhabdom (Nolte & Brown, 1971). The phalangids are probably closely related to the acarines (van der Hammen, 1968). They have typical unilenticular ocelli consisting of a lens and an underlying retina. The photoreceptor cells of the retina are organized into units consisting of 4 retinula cells surrounding a central rhabdom. Each retinula cell contributes microvilli to the central rhabdom. The cytoplasm of the retinular cells contains many mitochondria, prominent Golgi, multivesicular bodies and endoplasmic reticula. The rhabdoms are highly ordered and repetitive with an organization very characteristic of the pattern found in compound eyes (Curtis, 1970). Machan (1966) studied the structure of the lateral and median ocelli of three species of scorpions. The ocelli of scorpions consist of a biconvex corneal lens and a retinal layer of photoreceptor cells forming a cup-shaped

PAGE 19

retina. This study was conducted with the light microscope and provides little information on the mi crostructure of the rhabdomeres or rhabdoms. The two families of spiders that have been studied in detail with the electron microscope are the jumping spiders (Eakin & Brandenberger, 1971; Land, 1969) and the wolf spiders , Lycosa (Melamed & Trujillo-Cenoz, 1966; Baccetti & Bedini, 1964). The eyes of these two families of spiders are very similar and typical of those of the other chelicerates. The lenses are biconvex cuticular thickenings and possess highly ordered cup-shaped retinas. The rhabdomeres of both families are made up of microvilli and show a highly ordered repetitive pattern. Eakin and Brandenberger (1971) divided the sensory cells of the anterior median (AM) eyes into 4 regions: (1) a distal portion bearing the rhabdomeric microvilli, (2) an intermediate cytoplasmic segment, (3) a basal soma containing the nucleus, and (4) the long neurite that enters the optic nerve. The cell body or soma of the sensory cells of the other 6 eyes lies directly behind the vitreous body distad to the rhabdomeric microvilli. The eyes of 2 acarine species have been anatomically studied. The eyes of these 2 species, Trombicula autumnal is Shaw and Tetranychus u r t i c a e Koch, show a similar anatomical organization. T. autumnal is has 2 pair of eyes, the anterior pair with biconvex lenses and a posterior pair with simple convex lenses (Jones , 1 950 ) .

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The eyes of T, urti cae were studied by Mills (1973). The anterior pair have biconvex lenses consisting of a thin stratified external layer and a thick interior made up of 25-30 cuticular layers. The simple convex lens of the posterior pair of eyes is a thin "hemi -el 1 i psoidal shell" of cuticular material. Both pair of eyes share a common "eyemanifold" consisting of 15 retinular cells; five beneath the anterior eyes and 10 beneath the posterior eyes. The microvilli of the retinular cells lie within cup-shaped invaginations of the cell membrane termed "retinular-cups" by Mills. Three retinular cells of the anterior eyes and 8 retinular cells of the posterior eye have double rows of adjoining microvilli that form single fused rhabdoms similar to the " sel f -rhabdom" of Limul u s . The other retinular cells have single rows of microvilli forming simple rhabdomeres. Three anatomical studies of ticks have included work done on the eye. These prior studies, however, provide little information on the function of the eyes of ticks since they utilized light microscopy. The first person to detail the histology of the tick eye was Bonnet (1907). He described perpendicular striae accentuated by black pigment in the lens. Gossel (1935) studied the eyes of 6 species of Ixodid ticks and showed them to consist of a convex lens with underlying unipolar neurons. The eye of Dermacen tor andersoni Stiles was treated briefly by Douglas (1943).

PAGE 21

Eight species of eyeless ticks were studied by Binnington (1972). He found unipolar neurons in three of the ticks and believed them to be photoreceptor cells. He also found that removal of the lateral "eye" in Argas p e r s i c u s Oken impeded phototaxis. All eight species studied had optic ganglia and optic nerves of similar morphology. Horridge (1965) stated that the "aberrant eyes of ticks" do not fit into the category of arthropod photoreceptors . Methods and Materials Ticks utilized in this study were obtained from two sources. Larval and adult ticks were collected near Otter Creek, Florida, by dragging a 3-ft. -square "flag" over infested vegetation. Larval, nymphal, and adult ticks were also obtained from a colony maintained at the USDA-ARS, Insects Affecting Man and Animals Laboratory, Gainesville, Florida. These ticks were collected by personnel of the laboratory as adults and subsequently fed on a dog. The engorged ticks were held in a chamber maintained at high relative humidity. Each generation was reestablished with wild-caught adults (USDA Rearing Bulletin). Ticks were fixed in g 1 u teral dehyde-paraf orma 1 dehyde prepared according to Karnovsky (1965) and were then submerged in fixative and cut into three pieces. The opisthosoma was cut off behind the third pair of legs and discarded

PAGE 22

The podosoma was then cut into two pieces along the midline. This dissection facilitated the penetration of fixative and subsequent sol utions. Pieces of podosoma containing the eyes were placed in fresh fixative for 6-7 hours at room temperature to complete fixation. The pieces were washed in three changes of 0.1 M cacodylate buffer (pH 7.2) and post-fixed for 12 hours in 2% osmium tetroxide in 0.2 M cacodylate buffer at 4°C. Following post fixation the pieces were rinsed in 0.1 M cacodylate buffer prior to dehydration. Dehydration was accomplished at 5-minute intervals in a series of 25, 50, 75% ethanol at 4°C. The tissue was held in 2% uranyl acetate in 75% ethanol at 4°C for 3 hours to improve contrast. Two-10 minute changes of 100% ethanol and two subsequent 15 minute changes of acetone preceded infiltration with Spurr's plastic (Spurr, 1969). Tissue was held in 50% plastic in acetone for 1 hour and in 100% plastic for 24 hours at room temperature prior to polymerization in a 60° oven. Silver to gold sections were cut using a duPont diamond knife on a Porter-Blum MT-2 u 1 trami crotome after thick sectioning (1 micron) brought the region of the eye to the block face. Thin sections were placed on 75-mesh copper grids covered with a Formvar film. The sections were poststained with uranyl acetate for 10 minutes and lead citrate for 2-4 minutes (Reynolds, 1963) prior to examination with

PAGE 23

either a Hitachi HU11C or HU11E electron microscope at 75 kV. Results The eyes of Ixodid ticks are located on the lateral margins of the scutum unlike other arthropods where the eyes are located on the head. In Amblyomma americanum the eyes consist of 30-40 unipolar photoreceptor neurons (Fig. 1). This pattern is the same for the larva, nymph, and adult tick. In each succeeding stage the eye becomes larger but the anatomy and mi crostruc ture remain the same. The eye of the larval tick contains approximately 25-30 neurons and is approximately one-fourth the size of the adult. Sections of a larval tick eye were used in Plates I and II to provide an overall view of the eye and the individual photoreceptor neurons. Plates I and II are electron micrographs of whole eyes. The orientation of the section may be determined by using the orientation lines provided on the plates themselves. One line (D-V) indicates the dorsal-ventral axis and the perpendicular line (L) indicates the midlinelaterad aspect. The lenticular pore canals follow a curved path and converge in an area above the photoreceptor cells. Lines inscribed on the longitudinal axes of the pore canals would converge on a point in the microvillar region (region A) of the eye.

PAGE 24

10 The cuticular lens is roughly biconvex and deviates only slightly from a simple convex configuration (Fig. 1, Plate I B). A slight internal bulge is present and is located on the ventral portion of the inner lens and the second convex curve of the lens. The internal bulge of the lens is always located proximal to the microvilli of the photoreceptor neurons (Plate I B). The pore canals of the lens are always perpendicular to the longitudinal axes of the photoreceptor neurons. The pore canals (PC) of the scutum are oriented in the dorsal -ventral axis (compare with the pore canals of the lens). The exocuticle of the lens ( E X ) is darker in appearance than the endocuticle (ENDO) (Plate I B). The photoreceptor neurons of the eye are connected to the optic lobes of the brain by the optic ganglion (Plate I A). Each photoreceptor neuron contributes a single axonal neurite to the optic nerve. The number of photoreceptor neurons per eye can therefore be determined by counting the number of axons in the optic nerve (Plate I A). The individual photoreceptor neurons do not vary in structure with regard to sex, age, or stage. An isolated neuron is indistinguishable from any other neuron within a single eye. The photoreceptor neuron has been divided into 4 regions for descriptive purposes: (1) a distal segment, region A, characterized by the presence of numerous

PAGE 25

Figure 1. Schematic diagram of an eye of Amblyomma americanum . Ax, axon; GN, glial nucleus, GS, glial "sheath; H, hypodermis; L, lens; LPC, lenticular pore canals; M v , microvilli of photoreceptor neuron; N, nucleus; S, scutum; SPC, scutellar pore canals. Figure 2. Schematic diagram of 1 photoreceptor neuron o f Amb lyomma americanum . A, region characterized by numerous microvilli; B, region containing numerous mitochondria and intracellular channels; C, soma containing the nucleus; D, proximal axon; Ax, axon; CV, coated vesicles; GC, glial cell; GL, glycogen; GN, glial nucleus, Go, Golgi, M , mitochondria; M v , microvilli.

PAGE 26

2.

PAGE 27

Plate I. Amblyomma americanum larva A. Oblique section of an optic nerve. Receptor cell axons (Ax) are invested by glial elements (arrows). The optic nerve lies directly beneath the scutum (Sc) and hypodermal cells (HC). X5890 Oblique section of an eye. Beneath the lens (L) is the hypodermis (H) and 4 photoreceptor cells. Each photoreceptor cell has a prominent soma containing the nucleus (N) and numerous terminal microvilli (Mv). X3800 Note internal lenticular bridge (B), the deviation from simple convex configuration, and orientation of pore canals (PC).

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• *^7^^i^ #

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15 microvilli, (2) an intermediate region containing numerous mitochondria and intracellular channels, region B, (3) a basal soma, region C, containing the nucleus, and (4) region D, a proximal axonal neurite that together with the other axonal fibers forms the optic nerve (Fig. 2). Three of these regions are shown in cross section in Plate V B. In addition to the photoreceptor neurons the eye is invested by a tunic of glial cells (Fig. 1, Plate V B). The membranous windings of the glial cells, the mesaxons (MA), glial cytoplasm (arrows), and an extracellular glial sheath (ES) isolate the neurons of the eye from the haemocoel. Often the cytoplasm of the glial cells contains electron dense opaque bodies (OB) and multivesicular bodies (MVB) (Plate V B). Lens The lenses of arthropod eyes contain very few structural features and in this respect the lens of Amblyomma amer i canum is unique. Unlike other lenses it has many pore canals in the transparent matrix of the scutellar cuticle. The pore canals (Plate III A) of the lens are organized into bundles of 30-60. These bundles of pore canals condense and their diameter becomes smaller as they approach the hypodermis (H). The number of pore canals per bundle also decreases as they near the hypodermis. This decrease is

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Plate II. Amblyo mma americanum larva A. Longitudinal section of an eye. Photoreceptor neurons have 4 distinct regions: a distal segment of microvilli, region A; an intermediate cytoplasmic segment containing many mitochondria, region B; a soma containing the nucleus (N), region C ; and region D, a long axon (Ax). Note the orthogonal orientation of microvilli shown by arrows (CX and LX). X3800 Longitudinal section of a portion of an eye (post-stained with barium permanginate). Microvilli of several cells are oriented orthogonally (arrows CX and LX). Four regions of the eye are shown (A, B, C, and D). X3800

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Plate III. Amblyomma am ericanum adult A. Oblique cross section of the lens. The pore canals (PC) of the lens are organized into bundles. X3800 The arrows indicate fusion of pore canals. Oblique section of lenticular pore canals (PC) X3800 C. Longitudinal section of scutellar pore canals (PC). X3800

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'ii^-iwr™

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Plate IV. Amhl yomma a mericanum adult A. Cross section of terminal microvilli ( M v ) bearing region of photoreceptor cell. Glial investiture of the photoreceptor cells consists of mesaxons (Ma) and an extracellular sheath (arrows). Neural cytoplasm contains numerous mitochondria ( M ) in this area. XI 1 9 7 Cross section of terminal microvilli at higher magnification. Photoreceptor cytoplasm (C) and glial cell cytoplasm (arrows) shown. X22800

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\*r

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22 due to the regular fusion of pore canals (arrows). In Plate III A, pore canal bundles are demonstrated in cross section. This electron micrograph is oriented so the hypodermis under^ lying the lens is on the bottom and the top of the micrograph is laterad. The pore canal bundles diminish markedly in diameter as they near the hypodermis and several pore canals are sectioned at the point of fusion (arrows). The lenticular pore canals and scutellar pore canals differ radically in size in the adult and the nymph. The lenticular pore canals are considerably larger (Plate III B) than the scutellar pore canals (Plate III C). Plate III B illustrates the curvilinear path taken by the pore canals of the 1 ens . Hypodermi s A cellular epidermis, the hypodermis (H), lies directly beneath the lens of the eye (Plate I B). The hypodermis is one cell layer thick and rests upon an amorphous basal basement membrane. The scutellar hypodermis (H) (Plate IX A) and the lenticular hypodermis (H) (Plate I B) are indistinguishable and no apparent lenticular hypodermal modifications were observed.

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23 Reti nu 1 ar Cells R egion A . Microvilli The distal portion of the photoreceptor neurons bears thousands of parallel microvilli (Fig. II, Plate II A & B). By counting the number of microvilli per square unit on a micrograph estimates of the number of microvilli per photoreceptor were calculated. These estimates ranged between 7,000 and 13,000 per photoreceptor cell. The microvilli are oriented perpendicular to the path of light as in all photoreceptors studied. The mi crov i 1 1 ar-bear i ng membrane of the neuron is dome-shaped (Fig. 2) and cross sections of this region often show a central portion of cytoplasm with microvillar cross sections encircling it (Plate IV A). The microvilli are independent and free within the glial investment of the neurons (Plate IV A). The microvilli are tightly packed within the mesaxonal investment (Plate VI B, V A) but are not bonded to one another by tight junctions as in other chelicerate eyes. The microvilli are typically blind-ended evaginations of the distal membrane of the neuron. Plate V A shows the tips of the photoreceptor microvilli. Region B. Intermediate region of cytoplasm The neural zone directly proximad the terminal microvilli is designated Region B, the intermediate zone of

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Plate V. Ambl y omma ameri canum adult A. Longitudinal section through tips of microvilli. X22800 Cross section of a peripheral portion of an eye. Axons (Ax) are surrounded by glial cell membranes, the mesaxons (Ma), glial cytoplasm (arrows), and an extracellular sheath (ES). Glial nuclei (GN) are located on the periphery of the eye. Cytoplasm of the axons and glial cells often contain electron opaque bodies (OB) and multivesicular bodies (MVB). XI 7 1 00

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Plate VI. Amblyomma americanum adult A. Oblique section of photoreceptor neuron through base of microvilli (Mv). Deep invaginations, intracellular channels (arrows), and numerous mitochondria (M) between membranes are characteristic of this zone (II). X11970 B. Cross section of photoreceptor neuron through region B below microvilli (Mv). Numerous mitochondria, here in cross section, are characteristic of this region. X8740 C. Oblique section of neuron at base of microvilli (M). Pinocytotic vesicles (arrows) form between microvillar bases. Mitochondria (M) lie in cytoplasm between intracellular channels. X15750 D. Cross section of region B directly below microvilli (Mv). Mitochondria (M) are located between intracellular channels (arrows). X33000

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28 cytoplasm. It is characterized by numerous elongate mitochondria, intracellular channels, and pinocytotic (or exocytotic) vesicles. The numerous sausage-shaped mitochondria (M) present in this region are associated with a system of intracellular channels (arrows (Plate VI A & D). Mitochondria are present in all parts of the neuron but are most prevalent in region B. These mitochondria lie between cytoplasmic sheets formed by intracellular membranous channels. Plate VI D is a photomicrograph of a cross section of this region and shows the mitochondria (M) between intracellular channels (arrows). The intracellular channels originate as inpocketings of the terminal membrane between the bases of the microvilli (Plate VI C). Vesicles (arrows) prevalent in this region appear to arise at the end of these membranous channels between the microvillar bases (Plate VI), This combination of microvilli, mitochondria and membranelined intracellular channels is very characteristic of transport cells such as Malpighian tubule cells, secretory, or glandular cells. Region C. Nucleus-bearing portion of the soma The nucleus of the' photoreceptor neuron is the most prominent feature of zone C (Plate VII, VIII). Plate VII A & B are electron micrographs of cross sections through this region and show the relationship between the prominent nucleus (N) and the other organelles characteristic of the

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Plate VII. Amblyomma americanum adult A. Cross section of photoreceptor neurons on periphery of eye just under the hypodermis (H) Two axons (Ax) and cross section of several neural soma, in zone C, showing nuclei (N) and rough endoplasmic reticulum (arrows). Glial nucleus ( G N ) , mesaxons (Ma), and extracellular glial sheath (GS) cover the neurons. X5400 Cross section of photoreceptor neurons in region C, the soma, at a higher magnification. The cytoplasm of the neuron in region C contains a prominent nucleus (N) and cisternae of rough endoplasmic reticulum (RER) but fewer mitochondria (white arrows) than region B. Note axons (Ax), glial nucleus (GN), mesaxons (Ma), and extracellular glial sheath (black arrows). X8740 High magnification of g 1 ycogen-1 i ke (G) inclusions common in the cytoplasm of neural soma in region C. X57500 D. High magnification of coated vesicles (CV) associ ated with Golgi complex in the cytoplasm around the nucleus. X57500 E. Lower magnification of g lycogen1 i ke (G) and vesicular (CV) inclusions in cytoplasm of neural soma (region C). X43700

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31 perikaryon. The nucleus is located in the center of the cell and is surrounded by conspicuous cisternae of endoplasmic reticulum (RER). Ribosomes are attached to the surface of the endoplasmic reticulum. The cisternae of this rough endoplasmic reticulum form concentric layers around the nucleus. The number of mitochondria in this zone is greatly reduced when compared to the preceding zone B (Plate VII B) . Two inclusions of similar size are common in the cytoplasm around the nucleus (Plate VII E). One type of inclusion appears to be glycogen (G) and the second, coated vesicles (CV). Plate VII C is an electron micrograph of alpha-glycogen rosettes present in the perikaryon in homogeneous masses (Plate VII E). Coated vesicles are elaborated by Golgi complexes and are present throughout the perikaryon in aggregates termed " Nebenkernen" (CV) by Fahrenbach in Limulus (Fahrenbach, 1970) and as isolated vesicles in the cytoplasm. One such "Nebenkern" (CV) is shown in Plate VII E. These coated vesicles (DV), shown at higher magnification in Plate VII D, are probably involved in the transport or storage of synthetic products. The amount of glycogen per cell is highly variable but the presence of coated vesicles in the photoreceptor neurons is fairly uniform.

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Plate VIII. A mblyomma a meri canum adult A. Cross section of a single neuron throughout region C. Cisternae of rough endoplasmic reticulum (ERE) and Golgi (Go) are always found near the nucleus (N). Vesicular inclusions (arrows) are associated with the golgi. Other cytoplasmic inclusions include mitochondria (M) and multivesicular bodies (MVB). (Note longitudinal section of microvilli ( M ) ) . X17100 Higher magnification of the same section showing the rough endoplasmic reticulum (RER), Golgi (Go), and associated vesicles. Two types of vesicles are formed by the golgi complex, small coated vesicles (arrows), and larger vacuolate vesicles V). X 28 000 C. Cross section of glial cell mesaxons (Ma), extracellular sheath (GS), bundles of fibrils (FB) often in glial sheath, and glial cell cytoplasm (arrows). XI 7 1 00 D. Cross section of glial cell sheath fibril bundles (arrows). X17100

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34 The relationship of the nucleus (N), rough endoplasmic reticulum (RER), Golgi (Go), and elaborated vesicles is shown in Plate VIII A & B. Cisternae of rough endoplasmic reticulum (RER) form concentric patterns around the nucleus and are generally the most conspicuous organelles of the perikaryon (Plate VIII A). Large numbers of coated vesicles (arrows) are discharged by the Golgi apparatus. The first, indicated by arrows in Plate VIII, are uniform in size and are "coated" by a layer of electron dense material. The second vesiculate type (V) are highly variable vaculoate vesicles that have smooth membranous walls apparently derived from the cisternal membrane of the golgi (Plate VIII B). The Golgi apparatus in the photoreceptor neurons generally consists of between 5-7 cisternae regardless of its size. Region D. Axon Each neuron attenuates rapidly behind the nucleus in the direction of the central nervous system to form a long axonal neurite that communicates directly with the optic lobes of the brain. The optic nerve of A. amer i canum larvae (Plate IX B & C, VII A) consists of 25-30 photoreceptor cell axons and the optic nerve of the nymph and adult contains 30-40 axons. Only one type of photoreceptor axon (Ax) is present in the optic nerve of A. amer i canum (Plate IX A & B). However, bundles of small axons (X) of unknown

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Plate IX. Ambl yomma ameri canum A. Oblique section of larval optic nerve. Optic nerve contains 30-40 axons (Ax) from individual photoreceptor neurons. The optic nerve is located directly beneath the scutum (Sc) and scutellar hypodermis (H). XI 197 Cross section of 2 axons (Ax), a glial nucleus (GN), mesaxons (Ma), extracellular glial sheath (GS), and glial cell cytoplasm (arrows). XI 1 5 00 C. Cross section of axons (Ax), glial nucleus (GN), and mesaxonal (Ma) sheath surrounding the axons. Two bundles of smaller axons of unknown origin or function (X). X8740 D. Cross section of a small portion of the axoplasm of a single axon showing characteristic microtubules (arrows) and mitochondria (M). X43700

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37 function or origin were encountered in the optic nerve of adult ticks (Plate IX C). The optic nerve consists of photoreceptor axons, glial cells and a fibrous extracellular perineural sheath. The axoplasm contains numerous longitudinally oriented mi crotubl es (Plate IX arrows) that are orderly in their distribution. The axonal mitochondria are located more or less peripherally in the axoplasm adjacent to the axonal surface (Plate IX B & D). The axoplasm also contains infrequent dense opaque bodies (OB) (PlateV B, VIII D), possible residual or autophagic lysosomes. No other inclusions were encountered in the axonal region of the neuron. The photoreceptor neurons are completely ensheathed by glial cells that insulate the neurons from the environment of the haemocoel (Plate VII A & B). The glial investment of Ambl yomma is intermediate between the condition encountered in the myelinated and unmyelinated nerves of vertebrates. The glial cytoplasm of myelinated nerves is obliterated leaving a glial membranous sheath termed myelin I n Amblyomma and other arthropods glial cytoplasm and glial cell membrane cover the axon. This condition is common in the arthropods and nerves of this type are called tunicated nerves. In electron micrograph V B the glial cytoplasm (arrows) can be seen between the glial cell membranes (Ma). The neurons lie within invaginations of the glial cells.

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38 These invaginations of the glial cell membrane form long double membrane mesaxons (Ma) that wind around the axons and neural cell somata (Plate XI C, VII B). Each neuron is generally surrounded by two or three mesaxonal membranes. The mesaxons often bifurcate (Plate VII B) and encompass several axons. Glial cytoplasm contains few organelles or inclusions but mitochondria, mu 1 ti ves i cu 1 ate bodies, and opaque inclusions are sometimes present (Plate V B). Glial cell nuclei (GN) are located on the periphery of the neurons (Plate IX B, C; Plate V B) and conform to the outline of the axons. A fibrous extracellular perineural sheath (GS) covers the glial cells, axons, and photoreceptor neurons (Plate IX B, VII B). Embedded in the glial sheath, termed the neuralemma by some authors, are bundles of fibrils (Plate VIII C & D). These fibril bundles are not encountered in a predictable manner. In Plate VII B the glial sheath does not contain any fibrils. Plate VIII C and Plate VIII D are sections of these fibril bundles sectioned obliquely and in cross section respectively. The extracellular glial sheath is composed of an amorphous material very similar in appearance to the basement membrane (compare BM and GS in VII A). The optic nerve, upon leaving the eye, is located directly beneath the scutellar hypodermis (H) and remains in close proximity to the hypodermis until it enters the brain 1 atera 1 ly .

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39 Discussion Horridge (1965) stated that the eyes of ticks are "aberrant" and "improbable" as "efficient sense organs." The eyes of ticks do not appear aberrant or improbable as sense organs when studied mi croanatomi cal ly with the electron microscope. The eyes of Amblyomma americanum must be considered in the context of all rhabdomeric photoreceptors, particularly those of the Mollusca and the Platyhelmi nthes , and not just the highly advanced arthropod compound eyes. They differ anatomically from any of the other arthropod eyes that have been studied to date. The principal difference is their simplicity. Despite this simplicity, the eyes of Amblyomma possess all the structures necessary to make them fully functional photoreceptors. The eyes have a lens, we 1 1 -devel oped photoreceptor neurons, and an optic nerve that communicates directly with the optic lobes of the brain. The microanatomy of the neural cells beneath the lens identifies them with little doubt as photoreceptor cells. Electron microscopy was necessary to visualize the structures, in particular the microvilli which are characteristic of rhabdomeric photoreceptors of light microscopy . The most unusual aspect of the tick eye is the corneal lens. Bonnet (1907) described perpendicular striae accentuated by black pigments in the lens and Gossel (1935, Fig. 36

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40 noted the chit in of the lens is pierced by fine "canals." The "striae" they described were probably the bundles of lenticular pore canals described in this study (Plate III A). These bundles of pore canals appear as bright streaks when thick sections are viewed with the light microscope. This light-conducting property of the pore canal bundles indicates that they may act as light or wave guides. The lenses of all other arthropod eyes studied are devoid of pore canals or other structures and the lens of Amblyomma is the first described with such structures. In L i m u 1 u s the unmodified cuticle between the corneal facets of the compound eye has pore canals (Fahrenbach, 1969) and may represent an intermediate condition between the lens of Amblyomma and the typical ommatidial facet or unicorneal lens in the chel icerates . The pore canals of insects have been studied in detail (Locke, 1959, 1961) as have the pore canals in the integument of ticks (Nathanson, 1967; Beadle, 1972) and mites (Wharton e_t aJL , 1968). The pore canal bundles in the lens of Ambly omma have not been described from any other arthropods and represent a completely different adaptation of cuticular pore canals in the arthropods. The pore canals follow a curved path through the lenticular cuticle and converge on a point directly above the photoreceptor neurons. The diameter of the pore canal bundles becomes progressively smaller and the pore canals converge as they approach the

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41 inner surface of the lens. If the pore canal bundles actually function as light or wave guides their convergence and reduction in diameter may serve to intensify the light impinging on the photoreceptor neurons. Light energy gathered on the outer surface of the lens where the surface area is greatest would be condensed and concentrated on the smaller inner lenticular surface over the photoreceptor neurons . The lenses of other chelicerate eyes are strongly biconvex (Horridge, 1965; Curtis, 1970; Eakin and Brandenberger, 1971; Melamed and Tru j i 1 1 o-Cenoz , 1966; Fahrenbach, 1963). The lenses of ticks described with light microscopy are simple convex or very slightly biconvex (Gossel, 1935). The lens of Amblyomma americanum is slightly biconvex. It has an off-center bulge on the internal ventral surface of the lens. Region A . Microvilli All photoreceptors, rhabdomeric and ciliary, have parallel membranous arrays. Arrays of membranes are characteristic of all photoreceptors and it is assumed that their function is to provide an ordered piano-arrangement of membrane-bound photochemical s (Eakin, 1965, 1968). Two lines of evolution are apparent in the evolution of multiple membrane systems, the ciliary line and the rhabdomeric line (Eakin, 1965, 1968; Varela, 1971). The rhabdomeric line,

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42 characterized by the microvillus, is believed to have arisen as an early offshoot of the ciliary line and developed independently (Eakin, 1965). Varela (1971) postulates that the molluscs and arthropods do not belong in the same evolutionary line and suggests that the Mollusca constitute a third line of evolution. The microvilli of the photoreceptor neurons of Ambly omma amer icanum are typical of those encountered in other arthropod photoreceptors. One important aspect of the microvilli is the absence of any microvillar interaction. The microvilli are closely packed (Plate IV A, B; V A) without the development of a highly ordered hexagonal "honeycomb" relationship characteristic of the insects such as the cockroach (Smith, 1968),dipterans (Trujillo-Cenoz, 1972; Wolken, 1971), beetle (Meyer-Rochow , 1973), hemipterans (Burton & Stockhammer, 1969), ants (Wolken, 1971), and many Crustacea (Eguchi & Waterman, 1966; Wolken, 1971) or the formation of tight junctions between adjacent microvilli as in L i m u 1 u s (Fahrenbach, 1969; Lasansky, 1967; Nolte & Brown, 1971), Lycosa (Melamed & Trujillo-Cenoz, 1966); Phalangids (Curtis, 1970), Octopus (Moody & Robertson, 1960), and the larval mosquito eye (White, 1967). The microvilli are completelyfree within the glial sheath and tight packing of the microvilli of Amblyomma amer icanum produces neither the hexagonal honeycomb configuration nor occluded intercellular bridges (Plate IV B). Tight

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43 packing of microvilli in the eye of mandibulate arthropods (Crustacea and Insecta) produces hexagonal packing with a uniform intervillar space of constant dimensions. Tight packing of microvilli in chelicerate eyes produces an occluded intervillar space with tight junctions at points where adjacent microvilli touch. In this respect the photoreceptor microvilli of Ambly omma amer i canum show affinities with the microvilli borne on the photoreceptor neurons of planar ians (Rbhlich & Torb'k, 1961; MacRae, 1964) and snails (Rohlich & Torok, 1963) whose microvilli exhibit neither occluded intervillar space or hexagonal packing. Hexagonal packing and occlusion of the intervillar space will be considered in this study as advanced evolutionary characteristics. Free microvilli as encountered in the ticks, planarians, and snails are considered primitive. The longitudinal planes of the microvilli of several neurons in Plate II A and B are perpendicular. This orthogonal arrangement of microvilli provides the anatomical basis of polarized light perception (Waterman & Horch, 1966). This perpendicular or orthogonal pattern was encountered only in larval ticks and not in nymphal or adult ticks. This aspect of the tick eye deserves further investigation to determine if the eye functions as a polarizer.

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44 Region B. Intermediate zone of intracellular channel s The system of membrane-bound intracellular channels and vesicles originating at the base of the microvilli is very characteristic of rhabdomeric photoreceptor neurons. A system of subrhabdomer i c cisternae and vesicles occurs in L j m u 1 u s (Fahrenbach, 1968), Lycosa (Melamed & TrujilloCenoz, 1966) 7 species of the phalangids (Curtis, 1970), 5 species of decapod crustaceans (Eguchi & Waterman, 1966, 1968), D r o s o p h i 1 i a (Waddington & Perry, 1960), the toad bug, Gelastocoris (Burton & Stockhammer, 1969), a mosquito larva (White, 1967), 4 genera of dipterans (Tru j i 1 1 o-Cenoz , 1965a, 1972), snail Hel ix (Rohlich & T'drok, 1963), and 3 species of turbellarian pi atyhelmi nthes (Rdchlich & T'drok, 1961; MacRae, 1964). These are common structures associated with rhabdomeric photoreceptor neurons and are found in all the eyes studied to date. The presence of these organelles in the submi crov i 1 1 ar portion of the photoreceptor neurons of Amblyomma americanum adds credence to their identification as retinular cells. Long oval mitochondria are oriented longitudinally and occur in large numbers between the membrane-lined channels. Mitochondria are present throughout the neural cell body and axoplasm but are very abundant in region B (Plate VI A, B, & D) and rather sparce in other zones (Plate VII A & B). The large numbers of mitochondria located in this region indicate an intense rate of metabolic activity and energy utilization.

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45 Most authors interpret the presence of membranous intracellular channels, associated mi tochondri a , and numerous vesicles in this region as indicating protein uptake by pinocytosis (Waddington & Perry, 1960; Melamed & TrujilloCenoz, 1966, 1968; Burton & Stoc khammer , 1969; Curtis, 1970). An alternate interpretation was provided by Fahrenbach (1968). He believes the coated and vacuolate vesicles are part of a secretory sequence originating with the rough endoplasmic reticulum and Golgi that ends with secretion into the rhabdom of the eye. Whether this region represents an area of pinocytotic uptake or exocytotic secretion will require further study. The large number of mitochondria (energy utilization) i ndi cates pi nocytosi s but no known material is present in the rhabdom other than haemolymph. The microvilli of the tick eye are separated from the haemocoel by their glial investiture only and materials present in the haemocoelic fluid would be more available than in the rhabdomate eyes. The photoreceptor neurons of Amblyomma americanum have several important features in common with other rhabdomeric eyes. These shared features include terminal microvilli perpendicular to the light path, similar cytoplasmic organelles and organization, and an axonal neurite that communicates with the optic lobes of the brain. The important differences that separate it from other arthropod photoreceptors are terminal microvilli oriented in

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46 longitudinal axis of the neuron, the arhabdomate eye, the unusual construction of the lens, and the total absence of pigment in the neurons or associated cells. The eyes of Amblyomma are the first arhabdomate or arhabdomeric eyes described in the phylum Arthropoda. The uni corneal eyes of cheli cerates and the compound eyes of the mandibulates are very similar in structure with the exception of the lenticular structures. The retina of cheli cerates (Melamed & Trujillo-Cenoz, 1966; Curtis, 1970; Eakin & Brandenberger, 1971) is composed of repeating units very similar in structure and organization to the ommatidia in the mandibulates. No such structures are present in the arhabdomate eyes of Amblyomma . This arhabdomate condition is not shared with the only other acarine eye to be studied, Tetranychus u r t i c a e (Mills, 1973), that has both rhabdomeres and rhabdoms . The simplicity of the Amblyomma eye raises the question of the secondary reduction. Is the eye of Amblyomma primitive, possibly an archetype of arthropod eyes, or is it secondarily reduced? I believe the eye to be very primitive and not the product of secondary reduction. It could \/ery easily be an archetype of the arthropod eye since more advanced retinular cell types could be derived from it by merely changing the shape of the neuron. The tick eyes represent a very plausible step in the evolution of the compound eye of the mandibulates and unicorneal eye of the

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47 chel icerates. It is an evolutionary step never before recognized. Two important considerations support this view of simplicity rather than secondary reduction. First, the cytoplasmic organelles, cell structures, and organization are not reduced but are well developed. Organelles are numerous and characteristic of a \jery generalized cell. Advanced arthropodan photoreceptors have retinula cells that appear metabol ical ly inactive when compared with the photoreceptor neurons of Amblyomma (see Chapter II of this study; Trujillo-Cenoz, 1972; Boschek, 1971). Second, the tick eyes exhibit important microanatomical affinities with the eyes of two phylogenetically lower animals, the snails (Mollusca) and flatworms ( PI a tyhel mi n thes ) . The photoreceptor neurons of all three (snails, flatworms, and ticks) are strikingly similar. They all possess terminal microvilli parallel to the longitudinal axis of the photoreceptor neurons, similar organelles and cellular organization. The neurons are all parallel and the microvilli are all oriented in the same direction. In more advanced eyes the retinular cells are located around a central rhabdom and the microvilli are oriented in from two to eight different directions. Mo microvillar tight junctions or highly ordered hexagonal honeycomb pattern have developed in the snails, flatworms, or ticks as a result of tight packing of microvilli. The most advanced rhabdomeric eye, that of Octopus , has

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48 microvilli joined by tight junctional bridges (Moody & Robertson, 1960). The microvilli of flatworms, snails, and ticks have the assumed primitive condition of no intervillar interaction. The archetypal eye of arthropodan stock was possibly 'jery similar to the eye of ticks. This primitive condition probably persisted in the ticks primarily due to their use of tarsal sensilli as the primary sensory receptors. The mites have well-developed tarsal sensilli and in the ticks these sensilli are of particular importance in host location. Dependence on tarsal receptors for host location could account for maintenance of the primitive condition in tick eyes. Varela (1971) postulates a separate rhabdomeric evolutionary line for the Mollusca. The obvious affinities of the PI atyhel mi nthes , Mollusca, and tick eye place them in the same evolutionary line of sensory receptors.

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CHAPTER II THE MICROANATOMY OF THE EYE OF THE SCREWWORM FLY," Cochliomyia hominovorax (Coquerel Introduction The eradication of the screwworm fly, Cochl i omyi a homi nov orax (Coquerel), from Florida is one of the most successful control programs instituted against a major pest insect resulting in an estimated 14 million dollars a year saving to cattlemen (Cromroy, 1971). The larva of the screwworm fly is an obligate parasite and eats only the living flesh of warm-blooded vertebrates. The control program involved breaking the life cycle of the fly by introducing overwhelming numbers of sterile male flies into an area to mate with native fertile females. The program was successful in Florida and eliminated the fly from most of the southeastern U.S. after its initiation. The sterile male technique has been used in the southwestern United States along the Mexican border to prevent the usual northward spread of the fly each year. A projected program is now underway to use the sterile male technique in Mexico in hopes of eliminating the fly from all of Mexico and prevent its reintroduction into the United States. 49

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50 The success of the sterile male technique depends upon the production of large numbers of sterile male flies that are competitive with wild flies. In order to be fully competitive in the wild, they must be so close to their wild counterpart behaviorally and physically that the female flies will mate with them readily. Mass rearing of the insects is therefore yery important to the success of this program. In all attempts of mass rearing certain dietary and genetic problems are encountered because of the requirements of the rearing program. Mass rearing tends to cause either genetic deterioration of the breeding stock used or poor quality flies due to inadequacies in the larval diet. One of the problems facing the screwworm project is the lack of biological information about this fly. This study was initiated to produce information on the microanatomy of the eye of the adult fly. Irradiated and unirradiated flies were used to study the possible effect of irradiation on the eye of the fly. Several closely related genera have been studied extensively with electron microscopy and this study was also undertaken to confirm these prior studies and expand the information available on the eyes of this important group of flies.

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51 Literature Review Most adult insects have 2 large compound eyes located on the lateral margins of the head. In general the compound eyes of all insects are very similar. They all possess a peripheral retina composed of repeating units called ommatidia. Each ommatidium consists of the imageforming dioptric apparatus and a variable number of unipolar photoreceptor neurons, the retinular cells. The retinular cells possess rhabdomeres composed of parallel arrays of thousands of microvilli (Eakin, 1965). Photopigment molecules are presumably located on the inner surface of the microvillar membrane. The rhabdomeres within a single ommatidium form a central fused rhabdom in most arthropods (TrujilloCenoz, 1972). Dipterans of the suborder Cyclorrapha have rhabdomeres that project into a central extracellular ommatidial space. This arrangement is termed an open rhabdom. In addition the dioptric apparatus of dipterans differs anatomically in that it has a pseudocone beneath the cornea (Truji 1 lo-Cenoz, 1972; Bernhardt et aj_. , 1972). These anatomical differences combined with the quantity and advanced nature of the research on dipteran eyes make it appropriate to deal with it as an isolated unit. Seven genera of cyclorraphan Diptera have been studied in detail using electron microscopy: M u s c a domesti ca L. (Boschek, 1971, 1972; Braitenberg, 1967, 1972; Kirschfeld,

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Figure 3. Schematic diagram of Cochl iomyi a hominovo rax eye showing the relative position of the peripheral retina (PR) and lamina ganglionar is (LG). Figure 4. Schematic diagram of a longitudinal section of an ommatidium of the compound eye of Cochl i omy i a hominovorax . BM, basement membrane; BPC, basal pigment cell; L , lens; LPC, large pigment cell; OC, ommatidial cavity; PC, pseudocone; PP, primary pigment cell; Rl-7, ret inula cells 1-7; R8, inferior ret inula cell.

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Rl-7

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54 1967, 1972; Kirschfeld & Franchesch i ni , 1968, 1969; Kirschfeld & Reichardt, 1970; Campos-Ortega & Strausfeld, 1972), Drosop hilia m el anogas ter (Dannel, 1957; Fuge, 1967; Waddington & Perry, 1960), Anastrepha suspensa (Loew) (Agee, in prep.), the following 3 genera that were studied together as a unit Chrysomi a s p . , L u c i 1 i a s p . , and Sarcophaga s p . (Melamed & Tru j i 1 1 o-Cenoz , 1968; Tru j i 1 1 o-Cenoz , 1965a, 1965b, 1969; Tr uj i 1 1 o-Cenoz & Melamed, 1962, 1963, 1966a), and S ympycnus 1 ineatus Loew (Tru j i 1 1 o-Cenoz & Bernard, 1972). The eyes of these flies are structurally very similar and I will review the structures common to the 7 genera and introduce the terminology to be used in this study. The dioptric apparatus is composed of a corneal lens and an underlying gelatinous crystalline body termed the pseudocone (Tru j i 1 1 o-Cenoz , 1972). The pseudocone is a soft amorphous substance enclosed i n a cup-shaped cavity formed by 2 cells called the primary pigment cells by Boschek (1971). The pseudocone is extracellular, contains no inclusions or cellular organelles (Trujillo-Cenoz & Melamed, 1966a) and has approximately the same refractive index as the vitreous humor of the human eye (Bernhardt et a]_. , 1972). The proximal end of the pseudocone cavity is closed by 4 wedge-shaped cells forming a plate-like floor of the cavity (Trujillo-Cenoz, 1965a). Extracellular amorphous prolongations of the rhabdomeres extend into an

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55 invagination of the proximal membrane of the Semper cells (Truji 1 lo-Cenoz, 1965a, 1972). Boschek (1971) termed these rhabdomeric prolongations, the rhabdomere caps. Boschek (1971) and Tru j i 1 1 o-Cenoz (1965a, 1972) postulate that the function of the Semper cells is to provide mechanical and optical coupling between the dioptric apparatus and the open rhabdom . Three types of pigment cells are present in the ommatidia: (1) the primary pigment cells, (2) the large pigment cells located distally and containing a purple pigment (Truj i 11 o-Cenoz , 1972), and (3) small basal pigment "cells" near the basement membrane of the peripheral retina that contain a yellow-brown pigment ( Tru j i 1 1 oCenoz , 1972). The basal pigment "cells" in Aedes egyptii (L.) are actually not cells but pigment filled bags at the end of threadlike processes of the Semper cells (Brammer, 1970). Similar Semper cell processes have been found in Musca (Boschek, 1971) but are not known to connect to the 4 basal pigment cells. Eight photoreceptor cells, the retinula cells (R1-R8), make up the photosensitive portion of the ommatidium (TrujilloCenoz, 1965a; Melamed & Tru j i 1 1 o-Cenoz , 1968; Boschek, 1971; Trujillo-Cenoz & Bernard, 1972). Six of these, R1-R6, have rhabdomeres that are peripherally located around the extracellular space forming the central ommatidial cavity.

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56 The centrally located seventh rhabdomere consists of the rhabdomeres of retinular cells R7 and R8. The rhabdomere of R7, termed the superior central cell (SCC), forms the distad portion of the central rhabdomere. The rhabdomere of R8, termed the inferior central cell (ICC), forms the proximad portion of the central rhabdomere. Rhabdomeres R7 and R8 are subequal, the superior rhabdomere (R7) is long and the inferior (R8) is relatively short. The axons of R7-R8 do not synapse in the first visual ganglion, the lamina, but pass directly into the second, the medulla (Melamed and Tru j il lo-Cenoz , 1968; Trujillo-Cenoz, 1972). The rhabdomere of R7-R8 differs from R1-R6 by being smaller in diameter and cylindrical rather than a truncated cone (Boschek, 1971). The rhabdomeres are composed of tightly packed microvilli. The orientation of microvilli in rhabdomeres R1-R6 is such that the microvilli of the following are parallel: Rl and R4, R2 and R5, and R3 and R6 (Boschek, 1971; Melamed & Tru j i 1 1 o-Cenoz , 1968). The orientation of the rhabdomeric microvilli in the central cells (R7 and R8) is orthogonal i n Musca , Crysomyi a , L u c i 1 i a , and Sarcophaga (Boschek, 1971; Melamed & Trujillo-Cenoz, 1968; Trujillo-Cenoz, 1972; Bernhardt e_t aj_. , 1972; Trujillo-Cenoz & Bernard, 1972). In the species, Sympycnus 1 i n e a t u s , two types of ommatidia are present. Half the ommatidia, those with yellow

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57 corneal facets, have the usual orthogonal or perpendicular arrangement of the central rhabdomeric microvilli. In the remaining half of the ommatidia, those with red corneal facets, the microvilli, are parallel to one another (TrujilloCenoz & Bernard, 1972). The orthogonal arrangement of microvilli has been postulated as a two-channel analyzer of plane-polarized light (Waterman & Horch, 1966; Melamed & Tru j i 1 1 o-Cenoz , 1968; Tru j i 1 1 o-Cenoz , 1972). Rhabdomeres with parallel arrangement of microvilli are postulated to diminish the absorption of plane-polarized light in the opposite or perpendicular plane (Trujillo-Cenoz & Bernard, 1972; Trujillo-Cenoz, 1972). The parallel microvilli are oriented inthe vertical plane and are believed to minimize the absorption of horizontally polarized light, i.e., reflected light or "glare." A similar arrangement is found in the ventral portion of the eye of the water strider, Gerr i s sp. (Schneider & Langer, 1969), and is believed to allow a better view into the water by differential screening of surface reflected light. Directly beneath the peripheral retina is the first synaptic field of the eye, the lamina ganglionar is. The lamina is divided anatomically into three layers: the external fenestrated layer, an intermediate layer of unipolar cell soma, and the proximal plexiform layer (TrujilloCenoz, 1965b; Boschek, 1971). The fenestrated layer

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58 contains tracheoblasts, trachea, and bundles of eight retinular cell axons, the pseud ocartridges. The somata of unipolar second order neurons are located in the intermediate or unipolar cell soma layer. The plexiform layer has two second order axons termed LI and L2 by Braitenberg (1967) which synapse with the axonal fibers from retinular cells R1-R6 (Trujillo-Cenoz, 196 5b) to form the optical cartridges. The optical cartridges are surrounded by epithelial cells that make intimate contact with retinular axons R1-R6 by means of specialized glial projections called capitate projections (Trujillo-Cenoz, 1965b; Boschek, 1971). These structures were first believed to be synaptic in nature and were termed synaptic buttons by Pedler and Goodland (1965). The true synaptic loci however are formed by T-shaped synaptic ribbons (Trujillo-Cenoz, 1965a, 1965b; Boschek, 1971 , 1972). Methods and Materials The flies used in this study were obtained from two sources. Unirradiated flies were reared by Dr. Gerald Holt, USDA, APHIS, Fargo, North Dakota. These flies were fixed and embedded in Fargo by Dr. Holt's laboratory personnel following the same preparative technique used on irradiated flies. Irradiated flies were reared in Mission, Texas, by USDA, APHIS in their rearing facility and shipped via air mail to Gainesville as pupae. The pupae were placed in a

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59 shallow cup in a holding cage consisting of an aluminum frame with tube gauze stretched over it. Cotton saturated with a mixture of honey and water was provided as a source of sugar and water for the adult flies. Flies from 3 to 6 days of age were utilized. Living flies were submerged in paraf orma 1 dehydegluteraldehyde fixative (Karnovsky, 1965) for dissection. Following removal and bisection, the eyes were transferred to fresh fixative for 4 hours at room temperature, rinsed in 0.1 M cacodylate buffer (pH 7.2) for 20 minutes and postfixed in 2% osmium tetroxide for 20-24 hours at 4°C. Rapid dehydration at intervals of 5 minutes in 25, 50, and 75% ethanol followed a second rinse in 0.1 M cacodylate buffer. The eyes were held in 2% uranyl acetate in 75% ethanol for 4 hours at 4°C. Dehydration was completed with 5-minute changes of 95, 100% ethanol and two changes of 100% acetone at room temperature. The eyes were infiltrated for 1 hour with 50% Spurrs plastic (Spurr, 1969) and 24 hours in 100% plastic prior to polymerization at 60°C for 24 hours. Silver and light gold sections were cut using a duPont diamond knife on a Porter-Bl um-MT-2 u 1 trami crotome , picked up on 75-mesh copper grids covered with a Formvar film and post-stained with uranyl acetate and lead citrate (Reynolds, 1963) prior to examination with either a Hitachi HU11C or HU11E electron microscope at 75 kV.

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60 Resu Its The Peripheral Retina The peripheral retina of Cochl i omy i a hominovorax consists of hexagonally packed ommatidia (Fig. 4). Each ommatidium has a dioptric apparatus and 8 photoreceptor cells, the retinular cells (R1-R8). The diptric apparatus is composed of a corneal lens (L) and a gelatinous pseudocone (PC). Surrounding the pseudocone and forming the lateral walls of the pseudocone cavity are the primary pigment cells (PP). The floor of the pseudocone cavity is formed by a rectangular plate of wedge-shaped Semper cells (S). In addition to the primary pigment cells, two other types of pigment cells are found in the peripheral retina: the large pigment cells (LPC) located laterally and the basal pigment cells (BPC). The receptor region of the peripheral retina is composed of the 8 photoreceptor or ret inula cells. Six of these retinular cells (R1-R6) are located peripherally around the central ommatidial cavity (0C). Retinular cells, R1-R6, bear independent rhabdomeres made up of microvilli. The rhabdomeres of retinular cells, R7 and R8, are centrally located in the ommatidial cavity and form a single central rhabdomere. The distal portion is formed by the rhabdomere of R7, termed the superior central cell, and the proximal

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61 portion by R8, the inferior central cell. The two central retinular cells are subequal in length, R7 (SCC) is longer being approximately 170 microns in length and R8 (ICC) is approximately 60 microns in length. Each retinular cell has an array of microvilli that extends from the distal to the proximal end of the cell body. The blind ends of the microvilli project into the ommatidial cavity. The microvilli borne by one cell are termed a rhabdomere and the rhabdomeres whether fused or separate are termed a rhabdom. In most arthropods the rhabdomeres are fused into a central rhabdom. Cochl i omyia hominovor ax and the other members of the suborder Cyclorrapha have an ommatidial space or cavity (0C) that separates the rhabdomeres and extends the length of the ommatidium. This configuration is termed an open rhabdom. The Dioptric Apparatus The dioptric apparatus of the adult screwworm fly consists of a corneal lens and an amorphous gelatinous pseudocone. The lens (Plate X A) is a modification of the cuticle It has no pore canals or other structures usually associated with the cuticle of insects. Plate X A is a cross section of a lens showing the alternating "dense" and "rare" bands believed by several authors to act as interference filters (Trujil lo-Cenoz , 1972; Bernard & Miller, 1968; Bernard

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Plate A. X Cochl i omyi a homi novorax unirradiated Cross section of lens showing alternating dense and rare bands believed to act as interference filters. Note small round protubrences on surface of lens (arrows). X8740 Off center longitudinal section of lens (L) and pseudocone (PC). Primary pigment cells IPP) form the lateral walls of the pseudocone cavity. Note mi crovi 1 1 i -1 i ke projections of primary pigment cell membrane (arrows) and alternating dense and rare bands .

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64 et al., 1972). Plate X B shows these bands in longitudinal secti on . Beneath the corneal lens is the pseudocone cavity containing the extracellular amorphous pseudocone (PC) (Plate X B). The pseudocone cavity is formed by the primary pigment cells (PP) (Plate X B, XI A & B). There are two primary pigment cells that form the lateral walls of the pseudocone cavity in Cochl i omyia hominovorax (Plate X B; XI A & B). Numerous irregular mi crov i 1 1 artype (Mv) evaginations of the primary pigment cell membrane project into the pseudocone cavity (Plate XI B). The pseudocone is not completely homogeneous and contains material of greater electron density irregularly concentrated toward the center of the pseudocone (Plate XI A). Plate XI A is a micrograph of a cross section through the pseudocone (PC), the primary pigment cells (PP), and the primary pigment cell nuclei (N). The primary pigment cells are tightly bound by spot desmosomes (SD), at the edge of the cell that bounds the pseudocone, and gap junctions (GJ) over the rest of the adjoining membrane. The Semper Cells The proximal end of the pseudocone cavity is closed by 4 flattened wedge-shaped cells that form a rectangular platelike floor (Plate XIII B, XI B). Plate XI B shows an offcenter longitudinal section (Fig. 5) of the pseudocone (PC)

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Plate A. XI Cochl iomyia hominovorax unirradiated Cross section of pseudocone (PC) and primary pigment cells (PP). Two primary pigment cell nuclei (N) and pigment filled vacuoles are most prominent organelles in the primary pigment cells. Note gap junction (GJ) and spot desmosome (SD) cell to cell contact between primary pigment cells. X8740 Off-center longitudinal section of pseudocone (PC) and Semper cells (S). Semper cells have short unequal "microvilli" (black arrows) that project into the pseudoncone cavity. Note Semper cell nuclei gap junction (GJ), and spot desomosome (SD). X11970 (N),

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r'.'A-

PAGE 81

67 and the Semper cells (S). Plate XI B is an el ectronmicrograph of a cross section of the Semper cell plate. Short irregular projections (arrows) of the Semper cells' distal membrane project into the pseudocone cavity (PC) (Plate XI B) The Semper cells are joined by cell junctions that are very similar in structure to those of the primary pigment cells. Plate XI B shows the cell junction between 2 Semper cells (S). Their membranes are joined near the pseudocone cavity by a spot desmosome (SD) or macula adherens and the remaining membrane is joined by a gap junction (GJ) or zonula occludens (Plate XI B). Spot desmosomes are cell contacts that involve thickening of the cytoplasmic surface of the cell membrane and gap junctions are cell contacts with a partial obliteration of the intercellular space (Satir & Gilula, 1973). The spot desmosome (SD) and gap junction (GJ) between the Semper cells provide a close sealed apposition and seal the bottom of the pseudocone cavity. The distal membrane of the Semper cells is produced into a network of ridges that project into the pseudocone cavity. In cross section (Plate XI B) these ridges appear to be irregular mi crov i 1 1 artype projections. Plate XII shows two magnifications of the junction of the Semper cells and the pseudocone. This network of Semper cell membrane ridges projects into the pseudocone cavity presumably holding the gel-like pseudocone in place.

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Plate XII. Cochl iomyia hominovorax unirradiated A. Cross section through a junction of the pseudocone (PC), Semper cells (SC), and primary pigment cells (PP). Projections from the Semper cells into the Pseudocone cavity form a network (arrows). Note spot desmosome (SD) joining Semper cell membranes. X5890 Higher magnification of the same cross section of a junction between pseudocone (PC) and Semper cells (SC). X38000

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Plate XIII. Cochliomyia hominovorax unirradiated A. Cross section of an ommatidium near junction of the pseudocone (PC) and 4 Semper cells (S). Note presence of primary pigment cells IPP), primary pigment cell nucleus (N), large pigment cells (LPC), and distal end of the ommatidial cavity (OC). Numerous granular inclusions (arrows) present in the cytoplasm of the large pigment cells and make it possible to distinguish them from the primary pigment cells. Spot desmosomes joining distal membranes of Semper cells are indicated by arrows. X6650 Cross section of the 4 Semper cells (S) showing the rhabdomere caps (RC) and the ommatidial cavity (OC). Four Semper cell nuclei (SN) and 2 large pigment cell nuclei (PN) are shown. Note that only gap junction (GJ) present. X7600

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72 The proximal membrane of the Semper cell plate is invaginated; the ommatidial cavity (OC) and rhabdomere caps (RC) project into this invagination. Plate XII A is a cross section of the Semper cell plate showing 7 rhabdomere caps and the distal portion of the ommatidial cavity. This distal projection of the ommatidial cavity has 7 arms (Plate XIV A & B). Between these arms of the ommatidial cavity amorphous extracellular extensions of the rhabdomeres, the rhabdomere caps, are situated (Plate XII B). There are 7 rhabdomere caps, one corresponding to each rhabdomere. The trapezoidal configuration of the rhabdomere caps is the same configuration as the distal rhabdomeres. Plate XII is a cross section of the Semper cell plate. Four Semper cell nuclei (SN) and 7 rhabdomere caps (RC) are present in this section. The spot desmosomes that join the distal membranes of the Semper cells (see Plate XII A or XII B) are not present in this more proximal section. The spot desmosomes of the Semper cells and belt desmosomes (BD) that join the mesial face of the retinular cells (Plate XVII) differ primarily in length. The spot desmosomes of the Semper cells form localized plaques. The tip of the extracellular rhabdomere caps (RC) project distally between the arms of the ommatidial cavity (OC) (Plate XIV A). The ommatidial cavity has 7 distal arms; between these arms the rhabdomere caps end. The

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Plate XIV. Cochl i omyia hominovorax unirradiated A. Cross section of the Semper cell junction and 7-armed omnia ti d i a 1 cavity (OC). The rhabdomere caps (RC) appear first between the arms of the ommatidial cavity. Note junction of Semper cell (arrows). X22800 B. Cross section of the Semper cell (S) junction and 7 arms of the ommatidial cavity (OC). Note tubules in the Semper cell cytoplasm. X3800 C. Cross section of Semper cell cytoplasm (S) junction near the ommatidial cavity (OC). Cytoplasm completely filled with microtubules. X51400 D. Higher magnification cross section of Semper cell cytoplasm (S). Tubules completely fill the cytoplasm. X57000

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75 ommatidial cavity is located at the junction of the 4 Semper cells (S) (Plate XIV A, B). Plate XIV A and B are cross sections of the proximal portion of the junction of the Semper cell plate. The ommatidial cavity (OC) is formed by an invagination of the basal surface of the Semper cells. Electron dense granular material fills the ommatidial cavity (Plate XIV A, B, & D). The gap junctions (arrows, Plate XIV A, B) joining the Semper cells separate at the rhabdomere caps (RC) and reform on the other side of the cap (small arrows , Plate XIV A) . The cytoplasm of the Semper cells is totally devoid of organelles. Plate XIV has four different magnifications of Semper cell cytoplasm. Microtubules completely fill the Semper cells and no other organelles were observed. The microtubules are randomly packed and have no apparent orientation. Plate XV B is an electron micrograph of a cross section at the junction of the Semper cells and the retinular cells. At this level the transition from rhabdomere cap (RC) to rhabdomeric microvilli occurs. Rhabdomere R3 is sectioned through the point of transition and shows both the amorphous cap (RC) and rhabdomeric microvilli (arrows, Plate XV C). The distal end of a retinular cell is attached to the proximal membrane of the Semper cell by a pointed evagi nation of the retinular cell membrane with desmosomal contact completely surrounding it (arrows, Plate XV B).

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Plate XV. Cochl j omyi a homi novorax unirradiated A. Section through several pigment-filled vacuoles (PV) in a large pigment cell. Although generally referred to as pigment "granules" a vacular membrane (arrows) is present surrounding the pigment. Note growth of pigment crystals in vacuoles numbered 1-5. Note granular inclusions (GI) in cytoplasm found only in large pigment cells. X51300 Cross section of a junction between Semper cells (S) and retinular cells (R). rhabdomere caps (RC), R2, R4 in the ommatidial cavity (OC (R) of Rl and R6 are present point of transition. Distal retinular cells project into Semper cells and joined by circular belt desmosome (arrows). X 1 5200 Four extracel 1 u 1 ar and R5 , are present The rhabdomeres and R3 is at the prolongations of are C. Higher magnification of retinular cell R3. Note rhabdomeric cap (RC), microvilli (arrows), mitochondria (M), and belt desmosome (BD). X38000

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Pigment Cel 1 s Two types of pigment cells are present surrounding the pseudocone and distal ommatidium (Fig. 4). These consist of 2 primary pigment cells (PP) and 6 large pigment cells (LPC) that extend from the middle of the pseudocone proximally to near the basement membrane. These 2 pigment cell types and their processes may be distinguished by dense granular inclusions (GI) that occur only in the cytoplasm of the large pigment cells (Plate XII, arrows; XV A). The nuclei of the large pigment cells (PN) are situated near the distal end of the ommatidium (Plate XIII B). There is a third type of pigment cell located at the basement membrane (Plate XIX A, Fig. 4). Four processes of these basal pigment cells (BPC) occlude the ommatidial cavity at the basement membrane (Plate XIX A). Pigment-filled vacuoles are present in the cytoplasm of the primary pigment cells, large pigment cells, and basal pigment cells. The retinular cells also contain pigmentfilled vacuoles (arrows, Plate XVI A). Plate XVI A shows both types of pi gment' vacuo! es , the small retinular cell vacuoles (arrows), and the larger pigment cell vacuoles. Referred to as pigment "granules" by previous authors, they are actually vacuoles filled with pigment crystals. Plate XV A is an el ectronmi crograph of several pigment vacuoles (PV) in the cytoplasm of a large pigment cell (note the

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Plate XVI. Cochl i omyi a hominovorax unirradiated A. Cross section of an ommati di urn, primary pigment cells (PP), and large pigment cells (LP) just below the Semper cells. Note the presence of pigment filled vacuoles in the retinular cells (arrows). X9500 Cross section of a portion of retinular cells Rl , R6, and R7 . The rhabdomeric microvilli are attached to the retinular cells by thin necks creating an extracellular space at their base (arrows). Note belt desmosomes (BD). X28500 Cross section through distal ommatidium (higher magnification of plate XVI A). The retinular cells are joined by belt desmosomes the entire length of the ommati dial cavity (OC). Note microvillar orientation and rotational asymmetry of rhabdom. X22800

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.* %T W * 9 wm • ft ^*U miir;i#

PAGE 95

presence of granular inclusions that identify it as a large pigment cell). The vacuolar membrane (arrows) is clearly visible around pigment vacuoles that are incompletely filled with pigment crystals (Plate XV A). Apparently the pigment crystallizes within the vacuole and the vacuole fills with these pigment crystals. Long needle-like crystals are present in vacuoles 1 and 2. The vacuoles in Plate XV A numbered from 1-5 indicate different states of maturation. Vacuole 5 is considered to be a "mature" vacuole. Retinular Cells Each ommatidium has 8 retinular cells: 6 distributed peripherally around the ommatidial cavity (R1-R6) and 2 (R7 and R8) that project into the central ommatidial cavity. The retinular cells have two distinct regions: a soma or cell body that bears the microvilli, and an axonal segment that enters the first synaptic loci of the brain. The peripheral retina is composed of the retinular cell somata. The retinular cells are joined by belt desmosomes (BD) that fuse the retinular cells for the entire length of the ommatidium (Plate XVI B ,. XVII A, B). The 7 belt desmosomes are the only points of attachment between the retinular cells Pigment cell processes are present between the nondesmosomal membranes of the retinular cells.

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Plate XVII. Cochl i omyi a hominovorax unirradiated A. Cross section of an ommatidium midway in the peripheral retina. The rhabdomere of the superior central cell, R7, is round and centrally located. The remaining rhabdomeres, Rl-6, are conical. Invaginations of the plasma membrane beneath the microvilli form intracellular channels (arrows). Retinular cell cytoplasm contains pigment vacuoles, mitochondria ( M ) , multivesicular bodies (MVB), and isolated cisternae of rough endoplasmic reticulum (RER). X8740 Cross section of rhabdom at the junction of the superior central cell (R7) and the inferior central cell (R8). Note intracellular channels (arrows) and multivesicular bodies (MVB). X8740

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?*>' L^-S^k;

PAGE 98

84 The arrangement of retinular cells R1-R6 is fixed in the distal portion of the rhabdom. The pattern is roughly trapezoidal and is rotati onal ly asymmetrical. Plate XVI A and B are cross sections of an ommatidium passing directly beneath the Semper cells. The trapezoidal pattern disappears near the basement membrane (see Plate XVII B, XVIII A). This pattern is also present in the rhabdomere caps (Plate XIII B). The configuration of this trapezoidal arrangement is such that a line inscribed through R3, R2, and Rl will be perpendicular to the horizontal plane of the eye and point toward the midline of the eye. This line (R3-R2-R1) is always parallel to the axis of the microvilli of the superior central cell (R7). The blind ends of the microvilli of R7 always point away from the midline of the eye. The superior cell microvilli in the dorsal portion of the eye point upwards, and those in the ventral hemisphere of the eye point down. This mirror image inversion has been found in other dipterans (Tru j i 1 1 o-Cenoz , 1972). An electron micrograph can be oriented using the microvilli of the superi or central cell. The ommatidial cavity (OC) (Plate XVI) extends the entire length of the rhabdom and is filled with an unknown material. This electron dense material completely fills the ommatidial cavity distally (Plate XV B; XIV A, B ; XIII B). Directly beneath the Semper cells it forms clouds of

PAGE 99

Plate XVIII. Cochl j omyia homi novorax unirradiated A. Cross section of ommatidium just below the transi tion from superior central cell (R7) to inferior central cell (R8). Orientation of central cell microvilli is orthogonal. X8740 Cross section of a portion of an ommatidium just below the transition from the superior central cell (R7) to the inferior central cell (R8). Microvillar orientation of central cell rhabdomeres is parallel. X22800

PAGE 100

rrMl *.*&;

PAGE 101

electron dense material around the rhabdomeres (Plate XVI B, C). Near the midline of the eye this material persists around the rhabdomeres (Plate XVII A), but near the transition zone from the superior central cell (R7) to the inferior central cell (R8) it is evenly dispersed in the ommatidial cavity and shows no affinity for the rhabdomeres (Plate XVII B; XVIII A). The ommatidial cavity is sealed at the basement membrane by processes from the 4 basal pigment cells (Plate XIX A), laterally by the belt desmosomes fusing the retinular cells and distally by the occluded gap junction and spot desmosomes of the Semper cell membranes of the Semper cell plate. The rhabdomeric microvilli are borne on thin neck-like stalks (Plate XVI B). The central cells bear between 13-15 longitudinal rows of microvilli and the peripheral rhabdomeres (R1-R6) have between 22-24 rows of microvilli (Plate XVI). The rhabdomeric microvilli are oriented perpendicularly to the longitudinal axis of the rhabdom and therefore perpendicular to the light path. The long axes of the microvilli of Rl and R4 are parallel, as are the axes of R2 and R5 and R3 and R6 (Plate XVI C and XVII A). The rhabdomeres of retinular cells R1-R6 have the shape of a truncated cone in cross section (Plate XVI A, C; XVII A, B; XVIII). The peripheral rhabdomeres (R1-R6) retain this shape throughout their entire length. The central

PAGE 102

rhabdomere is round in cross section (Plate XVI A, B, C; SV II). The seventh centrally located rhabdomere actually consists of the rhabdomeres of the 2 retinular cells R7 and R8 (Plate XVII, B, XVIII). The distal cell (R7), the superior central cell (SCC), bears a rhabdomere approximately twice as long as the inferior central cell (R8, ICC). The rhabdomere of the inferior central cell (R8) is directly below the rhabdomere of the superior central cell (R7) forming a single central rhabdomere (Plate XVII B). The inferior central cell is located between the peripheral retinular cells Rl and R2 (Plate XVII B). The rhabdomeres can be divided into two populations by the orientation of the central rhabdomere microvilli. In one group the microvilli of the superior and inferior central cells are orthogonal (Plate XVII B) and in the other group the microvilli are parallel (Plate XVIII A). The retinular cells contain small pigment vacuoles (arrows, Plate XVI A) similar in structure to those of the primary, large, and basal pigment cells. Retinular cell pigment vacuoles are restricted to the distal portion of the superior central cell and are not present near or below the transition zone from the superior central cell to the inferior central cell (compare A & B of Plate XVII). Numerous mitochondria (M) are present in the cytoplasm of the retinular cells (Plate XVII A). Sparce and isolated cisternae of

PAGE 103

Plate XIX. Cochl i omy j a hominovorax unirradiated A. Oblique section through the basement membrane (BM). Near the basement membrane the ommatidial cavity (0) is filled by 4 processes of the basal pigment cells (1-4). Below the basement membrane are bundles of axons, the pseudocapsu 1 es (PC), trachea (T), tracheoblast nuclei (TN). X3800 Cross section of a single pseudocartridge directly beneath the basement membrane (BM). The retinular cell axons (Ax 1-8) contain mitochondria ( M ) and numerous microtubules (arrows). X7600

PAGE 105

91 rough endoplasmic reticulum (RER) are present in the cytoplasm but few ribosomes are attached to them. Mul ti vesicular bodies (MVB) are often encountered in the cytoplasm of the retinular cells (Plate XVII A, B ) . Deep invaginations of the plasma membrane (arrows) form long membranous channels and pinocytotic vesicles at the base of the rhabdomeric microvilli (Plate XVII A, B ; XVIII A). Lamina Near the basement membrane (BM) the retinular cells lose their rhabdomeres and pass through the basement membrane into the lamina ganglionar is. Plate XIX A is an electron micrograph of a section through the area of the basement membrane. The ommatidial cavity is filled by four processes (1-4) of the basal pigment cells or sacs. The external layer of the lamina, the fenestrated layer, contains both numerous tracheae (T), and bundles of retinula cell axons, the pseudocartridges (PC). Eight axonal fibers (Ax 1-8) derived from the retinular cells form the pseudocartridges (Plate XIX B) and contain numerous mitochondria ( M ) and longitudinally oriented microtubules (arrows) (Plate XIX B). The axons derived from the 6 peripheral retinular cells (R1-R6) separate below the pseudocartridges and enter the external plexiform layer of the lamina. The external plexiform layer consists of units containing 2 second order neurons surrounded by 6 photoreceptor axons, termed optic

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92 cartridges. Plate XX A is a cross section of an optic cartridge showing the 2 second order neurons (Ll and l_2) and 6 ret inula cell axons (Ax 1-6) surrounding them. The axons from the superior central cell and the inferior central cell (Ax 7 and Ax 8) are displaced laterally bypassing the lamina and synapse directly with the medulla. Each photoreceptor axon (Ax 1-6) is associated with a pair of axons (A & B) termed the centrifugal fibers (TrujilloCenoz, 1965b). The optical cartridges are separated by epithelial glial cells that surround the cartridges (Plate XX A & D). Processes from the glial cells penetrate between the peripheral retinula cell axons, Ax 1-6. In addition, there are specialized glial projections, the capitate projections (CP), which project into Ax 1-6. The capitate projections are formed by the evaginations of the glial cell process membrane into axons 1-6 (Plate XX A, B, & C). The capitate projections have a narrow "stalk" and up to 3 terminal masses. Plate XX B and C show sections of terminal masses (CP) composed of (1) an inner membrane or glial cell process membrane, (2) an outer membrane or the invaginated axonal membrane, and (3) an electron dense covering. A "grazing" section of the electron dense coat of a terminal mass (CP3) is shown in Plate XX B. The T-shaped synaptic ribbons identified by previous authors as chemical synapses (Tru j i 1 1 o-Cenoz , 1965a, 1965b; Boschek, 1971) are present in the retinula cell axons.

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Plate XX C o c h 1 i o rrr hominovorax unirradiated Cross section of an optical cartridge. The cartridge consists of 2 second order axons (LI and L2) surrounded by 6 retinular cell axons (Axl-6). Paired axons of the central cells (Ax7 and Ax8) are displaced laterally as are the centrifugal cell axons (A and B). Numerous mitochondria (M) and specialized glial processes, the capitate projections (arrows), derived from the epithelial glial cells (E) are prevalent in Axl-6. X15200 B. Synaptic loci or T-shaped synaptic ribbons (arrows Presynaptic axon (ax) is filled with synaptic vesicles (SV). Note trilaminate head piece of capitate projection (1, 2, 3). X50000 Single synaptic ribbon. Presynaptic vesicles (SV) and T-shaped synaptic ribbon (arrow) are in the presynaptic axon (Ax). Post synaptic fibers (F) contain dense plate-like structures (P). Note capitate projections (CP) through layered terminal head (1 , 2, 3) . X93100 Cross section of an optical cartridge adjacent epithelial glial cell (E) and glial nucleus (N). X22800

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95 They consist of an electron dense T-shaped synaptic ribbon and synaptic vesicles contained in the presynaptic fiber, a ret inula cell axon. In Plate XX B and C the retinula cell axons contain numerous synaptic vesicles and T-shaped synaptic ribbons (arrows). The post-synapt i c axonal fibers (F), usually LI or L 2 or processes of LI or L2, contain hollow plate-like structures surrounded by electron dense mater i a 1 . Irradiated Flies The screwworm flies that were mass reared on an artificial diet, sterilized, and shipped to Gainesville from Mission, Texas, showed a number of abnormalities. Three types of abnormalities were encountered: (1) highly vacuolate "thin" retinular cell cytoplasm, (2) abnormally shaped rhabdomeres, and (3) large vacuoles in the axoplasm of the retinular cell axons in the external plexiform layer. One striking aspect of the mass-reared flies was the extreme variability between weekly shipments and within a shipment. Many of the eyes examined appeared normal while others within the same group were abnormal. Plate XXI A is a cross section of an ommatidium from an irradiated fly. The rhabdomeres appear normal in both shape and relative position; however, the cytoplasm of the retinular cells is very thin, highly vacuolated, and devoid

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Plate A. XXI Cochl imyi a h omi novora x irradiated Oblique section through the distal portion of an ommatidium. The cytoplasm of the retinular cells contains large atypical vacuoles (V). Note mi r row image reversal in numbering. X3800 Cross section through the distal portion of an apparently normal irradiated ommatidium. XI 1 97 Cross section of an apparently normal R7-R8 central cell transition of an irradiated ommatidium. Note mi tochrondr ia (M), multivesicular bodies (MVB), and isolated cisternae of rough endoplasmic reticulum (RER). Trachea (T) numerous and impinge on the retinular cells. X8740.

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98 of small retinular cell pigment vacuoles. Plate XX B is a cross section of the distal end of an ommatidium just below the Semper cells. These irradiated rhabdomeres appear normal. Plate XXI C is an electron micrograph of an apparently normal central cell transition zone (R7-R8). The cytoplasm is thin and several tracheae (T) impinge on the ommatidium (compare with normal ommatidia in Plate XVII and XVIII and note the absence of trachea). Intracellular channels and pinocytotic vesicles generally present at the base of the microvilli are considerably reduced when compared to the unirradiated flies. Plate XXII illustrates the most common abnormality encountered in the irradiated flies examined. The rhabdomeres of these abnormal ommatidia are obconical in shape (compare with the normal truncated cone shape of normal rhabdomeres Plate XVI and XVII). The abnormal obconical rhabdomeres have fewer rows of microvilli than the normal unirradiated rhabdomeres. The peripheral rhabdomeres (R1-R6) have between 15-17 longitudinal rows of microvilli compared with a normal complement of 22-24 and the central rhabdomere has between 10-11 longitudinal rows of microvilli rather than the normal 13-15 rows. The rhabdomeres of the retinular cells shown in Plate XXII C are partially obconical and the rhabdomeric microvilli of R5 and R6 (arrows) are oriented in two planes. The microvilli appearing in the cross section appear to be bent since they are

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Plate XXII. Cochl iomyia homi novorax irradiated A. Cross section of an aberrant irradiated ommati di um, Note abnormal obconical shape of the rhabdomeres . X8740 Cross section of a portion (R7, 1, 2, and 3) of an abnormal irradiated omnia ti d i um. Rhabdomeres are obconical in shape and microvilli are greatly reduced in number. X22800 C. Cross section of an abnormal irradiated ommatidium. Rhabdomeres and microvilli are abnormally shaped. The microvilli of R5 and R6 (arrows) are bent. Note abnormal number of trachea present. X17100

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101 observed both longitudinally and transversely. Normal microvilli are straight and their longitudinal axis should always be perpendicular to the light path. Plate XXIII A and B are cross sections of two normal appearing ommatidia from an irradiated fly below the transition zone. These photomicrographs have been included since they show the relative orientation of the rhabdomeric microvilli of the superior and inferior central cells (R7 and R8). The central cell rhabdomere microvilli in XXIII A are parallel and the rhabdomeric microvilli in XXIII B are orthogonal . In Plate XXIII C two abnormal transition rhabdoms (AR) are indicated by arrows. This photomicrograph is of a cross section through the transition zone and shows an abundance of tracheae not present in the unirradiated flies. Plate XIV A and B are higher magnification photomicrographs of the two abnormal rhabdoms of Plate XXIII C. The rhabdomeres are obconical as well as showing abnormal R7-R8 central cell transition zones. Plate XXIII A shows a rhabdom with only 6 rhabdomeres, 2 of these are touching in the central part of the ommatidial cavity. The touching rhabdomeres should be numbers R7 and R8 , the central cells; however, since there are 2 rhabdomeres between the 2 central cells they cannot be R7 and R8. The abnormal rhabdom shown in Plate XXIV C has 8 rhabdomeres present, 1 more

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Plate XXIII. Cochl i otnyi a hominovorax irradiated A. Cross section of 8 cell ommatidium. Microvilli of the superior and inferior central cells are parallel. X8740 B. Cross section of 8 cell ommatidium. Microvilli of the superior and inferior central cells are orthogonal. X8740 C. Cross section of several ommatidia through the transition zone of superior and inferior central cells. Note 2 abnormal (AR) transition ommatidia (arrows). X3800

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104 than normal. The central cells lie side by side in the ommatidial cavity rather than the normal arrangement with the central cell rhabdomeres on top of one another. A and B of Plate XXIV show the presence of abnormal numbers of trachea (T) penetrating the space between the retinula cells and impinging on the rhabdom. One of the abnormalities encountered in the irradiated flies were large vacuoles (V) in the axoplasm of the retinula cell axons at the level of the capsules in the external plexiform layer of the lamina (Plate XXV A and B) The two central second order axons appear to be normal. Plate XXV B is a higher magnification electron micrograph of 2 abnormal axons containing large vacuoles. The other components of the lamina appear to be normal. Discussion The microstructure of the peripheral retina and lamina of unirradiated Cochl i omy i a homi novorax is very similar to that of other genera of cyclorraphan Diptera previously studied. It would be very difficult to distinguish electron micrographs of Cochl i omyi a homi novorax from those published of the eyes of Musca (Boschek, 1971) Drosophi 1 i a (Fuge, 1 967 ) , Chrysomi a , Luci 1 i a , and Sar cophaga (Melamed & Tru j i 1 1 o-Cenoz , 1968; Tru j i 1 1 o-Cenoz , 1965a, 1965b, 1969; Tr u j i 1 1 oCenoz & Melamed, 1962, 1963, 1966a). The eyes of irradiated screwworm flies reared in

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Plate XXIV. Cochl i o myi a hominovorax irradiated A. Cross section of an omnia ti d i urn with an abnormal superiorinferior central cell transition. Only 6 abnormal rhabdomeres are present and 2 of these apparently R7 and R8, are touching. Note the abnormal number of trachea (T) impinging on the rhabdom. X8740 Cross section of an ommatidium with an abnormal superior-inferior central cell transition. Eight rhabdomeres are present, 1 more than normal. Rhabdomeres R7 and R8 are joined and form a bridge. Note the abnormal number of trachea present. X8740

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Plate XXV. Cochliomyia h ominovorax irradiated A. Cross section of an optical cartridge. The optical cartridges of irradiated flies contain large vacuolar spaces in the axoplasm (V). Second order axons, LI and L2, appear normal. X5890 Higher magnification cross section of 2 photoreceptor axons (Ax) in the optical cartridge showing large abnormal vacuoles (V) in the axoplasm X 1 1 970

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109 Mission, Texas, for mass release differed markedly in a number of respects from the unirradiated flies and the other dipteran previously studied genera. Periphe ral retina The peripheral retina of Cochl i omyi a hominovorax i s composed of ommatidia characteristic of the cyclorraphan diptera. Each ommatidium has the characteristic dioptric apparatus and open rhabdom. The organization of the 8 retinula cells, 6 peripheral and 2 central, is very characteristic of the other genera studied. The open rhabdom of the dipterans has been studied in greater detail than any other compound eye because Musca and C a 1 1 i p h o r a are used in many electrophys i cl og ical studi es (Wehner, 1972). This research has verified important prior studies and will contribute new observations. Dio pt ric apparatus The corneal lens of the eye of Cochl i omyi a is very similar to that of Sympycnus 1 i neatus (Trujillo-Cenoz, 1972) and Cal 1 iphora (Seitz, 1968). The small rounded elevations that cover the surface of the lens are characteristic of dipterans previously studied but are not considered the equivalent of corneal nipples found in Depidoptera (Bernard & Miller, 1968; Bernhardt e^ aj_. , 1972). The lens has alternating dense and rare layers (Plate X) that have been described as quarter wavelength interference

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no filters in the horsefly (Bernard & Miller, 1968) and are common in many dip terans (Trujillo-Cenoz, 1972). I n Coc hi i omyi a and other diptera with pseudocone eyes, the cone is a gelatinous material described as being isotropic by previous authors (Seitz, 1968; TrujilloCenoz, 1972). The pseudocone of Cochl i omyi a is not entirely isotrophic and has a diffuse. area of greater electron density in the center of the pseudocone. This electron dense material may be analogous to the electron dense core at the junction of the 4 cone cells in the eucone eyes of insects (Fischer & Horstmann, 1971). The Semper Cells The function and origin of the Semper cells is not fully understood. The most obvious function is the closure of the bottom of the pseudocone cavity. The 4 Semper cells are wedge-shaped and form a transparent flattened plate (XIII B) that occludes the proximal end of the pseudocone cavity. The other postulated function is to "couple" the dioptric apparatus to the ret inula cells (Boschek, 1971; Trujillo-Cenoz, 1972) and maintain proper optical spacing of the distal rhabdomeres (Boschek, 1971). The cytoplasm of the Semper cells of Cochl i omyi a and Musca (Boschek, 1971) is devoid of organelles. This lack of organelles is interpreted by Boschek (1971) as meaning the Semper cells are metabol i ca 1 ly inactive. This is also the

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Ill case in Cochl i omyi a ; no mitochondria, endoplasmic reticulum, golgi, or other organelles indicative of metabolic activity are present. Microtubules completely fill the Semper cells. In M u s c a the microtubules are oriented at an angle of 30° off the longitudinal axis of the ommatidium (Boschek, 1971). The microtubules in Cochl i omyi a show no orientation and are randomly packed. The function of these microtubules is probably cytoskel etal . They provide rigidity and resistance to deformation of the Semper cell plate. This rigidity is important in maintaining the trapezoidal conformation and dimensions of the distal end of the open rhabdom of (3. hominovorax . Insecta with a closed rhabdom do not encounter the conformational maintenance problems of insects with open rhabdoms. The trapezoidal rhabdomeric configuration is maintained throughout the visual system (Braitenberg, 1972; Tru j i 1 1 o-Cenoz , 1969). Mirror image symmetry of the trapezoidal pattern is constant in the 4 quadrants of the eye (Braitenberg, 1972) and indicates the importance of maintenance of the trapezoidal configuration in the distal rhabdom. In an open rhabdom each rhabdomere functions as an independent wave guide. Varela (1971) postulates the open rhabdom of di pterans resembles the vertebrate eye in this regard and is the most advanced compound eye in the arthropods. The open rhabdom is probably an excellent analyzer of polarized light (Varela, 1971) and the

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112 trapezoidal rhabdomeric configuration probably represents an important part in the processing and analysis of visual stimul i , The trapezoidal pattern is only maintained in the distal portion of the ommatidia and disappears near the basement membrane indicating that a distal structure, such as the Semper cells, is responsi bl e. f or the conformation. The projection of the retinula cell axons over the lamina reproduces the trapezoidal pattern of the distal rhabdom in such a way that the axons from retinula cells whose microvilli are in the same plane end in the same optical cartridge (Trujillo-Cenoz, 1965b, 1972; Trujillo-Cenoz & Bernard, 1972; Kirschfeld, 1967). Several other structures appear to be very important in the maintenance of the rhabdomeric trapezoidal configuration. The distal ends of the retinula cells are firmly attached to proximal invaginations in the Semper cells (Plate XV B arrows). Pointed processes of the distal end of the retinula cells are anchored by desmosomes (Plate XV B arrows). The distal end of each rhabdomere is covered by an electron dense rhabdomere cap that extends distally from the rhabdomere along the side of the ommatidial cavity and into the rhabdomeric cap invaginations of the Semper cells between the cell junctions. These electron dense rhabdomere caps retain the trapezoidal pattern of the rhabdomeres (Plate XIII B) and probably assist in its maintenance.

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113 This optically important configuration of the ommatidium is probably in great measure also conserved by the desmosomal and gap junctions joining the retinular cells and Semper cells. Desmosomes are generally present in cells where structural stability of form or attachment is important (Satir & Gilula, 1973). The ret inula cells are joined their entire length by belt desmosomes and the Semper cells by spot desmosomes and gap junctions. These desmosomal connectives, Semper cell to Semper cell, retinula cell to retinula cell, and retinula cell to Semper cell help maintain the rigidity of the ommatidium plate. The ommatidial cavity is filled with fluid, probably haemolymph or a derivative of the haemolymph. The subrhabdomeric intracellular channels and vacuoles may secrete fluid into the ommatidial cavity. This fluid may help hydrostatical ly maintain the ommatidial cavity since the eyes of dehydrated flies collapse. An extracellular electron dense amorphous substance is present in the rhabdomic fluid. The material completely fills the ommatidial cavity at its distal end where the ommatidial cavity occupies the invaginated proximal membrane of the Semper cell plate. Beneath the Semper cells the electron dense material is associated with the rhabdomeres forming a halo around the tips of the microvilli (Plate XVI B, C). The halo or cloud of electron dense material is not associated with the rhabdomeric microvilli below the midpoint of the ommatidium.

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114 The distal membrane surface of the Semper cell plate is produced into a network of membranous projections or ridges (Plate XII). These ridges have been described by previous authors (Boschek, 1971; Trujillo-Cenoz, 1972) as short irregular microvilli. The function of these ridges is unknown but they may keep the gelatinous pseudocone from slipping or being displaced laterally. The ommatidial cavity occupies a 7-armed membranous invagination of the Semper cell plate's proximal membrane. TrujilloCenoz (1965a) and Boschek (1971) published electron micrographs of the distal end of the ommatidial cavity but failed to recognize it as such or mention it in the text of their articles. Pi gment Cells Pigment filled vacuoles are present in the pigment cells and ret inula cells of C. homi novorax . Pigment-filled vacuoles are referred to by prior authors as pigment "granules" (Trujillo-Cenoz, 1972; Boschek, 1971; Butler e_t aj_ . , 1970). In C. homi nvorax a distinct vacuolar membrane surrounds the pigment (Plate XV A arrows). This vacuolar membrane has been previously reported in the eye of the toad bug, Gel astocori s occul atus by Burton & Stockhammer (1969). The pigment vacuoles of Gelastocoris contain needle-like pigment crystals similar in appearance to those in the pigment vacuoles of the screwworm fly. A process of

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115 pigment crystal percipitation within the vacuoles and subsequent filling or maturation of the vacuole appears to occur. Plate XV A shows 5 pigment vacuoles, numbered 1-5, in various states of maturation. The origin of the basal pigment cells is unknown. Processes of the basal pigment "cells" fill the bottom of the ommatidial cavity (Plate XIX A) and are present between the basal ommatidia at the basement membrane. B rammer (1970) in Aedes a e g y p t i described processes from the 4 Semper cells as terminating near the basement membrane in pigment filled sacs. A detailed examination of numerous serial sections of the basal third of an ommatidium failed to produce any nuclei associated with the basal pigment "cells." It is probable that these are pigment filled sacs and until the cell body associated with these processes can be identified their origin will remain unknown . Retinular cells The cytoplasm of the ret inula cells of C_ . hominovorax contains few organelles other than numerous mitochondria indicating a high level of energy utilization. Rough endoplasmic reticulum and golgi are poorly developed and generally consist of single isolated cisternae. Subrhabdomeric membranous channels and vesicles common in all the rhabdomeric photoreceptors studied (see Discussion, Chapter I) are well developed in C^. hominovorax .

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116 The cytoplasm of the retinula cells appears so thin in many of the electron micrographs that the quality of the fixation was questioned. This condition is present in all the published micrographs of compound eyes (Wolken, 1971; Smith, 1968; Tru j i 1 1 o-Cenoz , 1965a, 1972; Boschek, 1971). The fixation and preparative procedures are not believed responsible for this condition since the pigment cells, asons, pseud ocartridges, glial cells, and cartridges were well fixed. It is probable that after the eye is formed the retinula cells play a passive role. The presence of large numbers of mitochondria indicate a high level of energy utilization probably required for photochemical processes. After the eye is fully developed the primary function of the retinular cells is probably to maintain the rhabdomeres and provide the energy required for microvillar membrane bound photochemical reactions. The relative orientation of the microvilli in the peripheral rhabdomeres of £. hominovorax is the same as that reported for Musca (Boschek, 1971), Chrysomi a , Sarcophaga , and L u c i 1 i a (Trujillo-Cenoz, 1965a, 1972), and C a 1 1 i p h o r a (Smith, 1968). The microvillar orientation of the central cells (R7 and R8) is similar to that of Sympycnus 1 i n e a t u s (Dolichopodidae) (Trujillo-Cenoz & Bernard, 1972). In S_. 1 i neatus and C. hominovorax two populations of ommatidia are present based on the orientation of the central cell microvilli. Some of the ommatidia

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117 have the typical orthogonal arrangement (Boschek, 1971; Melamed & Tru j i 1 1 o-Cenoz , 1968) while others have parallel microvilli (Tru j i 1 1 o-Cenoz & Bernard, 1972) (see Plates XVIII and XXIII A, B). The parallel arrangement of microvilli is believed to reduce the reception of polarized light in the plane perpendicular to the axis of the microvilli (Waterman et aj_. , 1969; Waterman and Horch, 1966; Eguchi & Waterman, 1966; Tru j i 1 1 o-Cenoz , 1972; TrujilloCenoz & Bernard, 1972). Open rhabdom--Hemi ptera and Diptera In addition to the higher dipterans, several aquatic and semiaquatic Hemiptera have open rhabdoms. Notonecta (Ludtke, 1953) and the toad bug, Gelastocoris ocul atus (Burton & Stoc khammer , 1969) have open rhabdom eyes. Hemipteran eyes are of the eucone type. In eucone eyes the dioptric apparatus consists of a lens and 4 cone cells. In the pseudocone eyes characteristic of higher dipterans an extracellular pseudocone replaces the 4 cone cells. In spite of this basic difference several striking similarities are present when the eyes of Cochl i omyia , and other higher dipterans, are compared with the eyes of Gel asto c o r i s and N otonecta . The cone cells of Hemiptera and the Semper cells of the dipterans have few organelles other than microtubules. In the dipterans the function of the cone cells is performed

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118 by an extracellular pseudocone. It is very possible that the Semper cells of Cochl i omyi a and higher dipterans were derived from cone cells in a eucone ancestor. The fact that the lower Diptera have eucone eyes (Brammer, 1970) tends to support this view. If this is correct the pseudocone developed as an evolutionary advance that paralleled the reduction of the 4 cone cells. The cone cells in Gelastocoris are surrounded by 2 primary pigment cells anatomically similar in structure to the primary pigment cells in the higher Diptera (TrujilloCenoz, 1972) that form the pseudocone cavity. I believe pseudocone eyes are a specialized type of eucone eye and the Semper cells are homologous with the cone cells. The organization of the ret inula cells in Gelastocoris (Hemiptera) and Cochl i omyi a (Diptera) is very similar. Both have 6 peripheral and 2 central rhabdomeres. The microvilli of the 2 central cells of G elastocoris are orthogonal as are those of most higher dipterans (Trujillo-Cenoz, 1972). An electron mi orographic study of the cone cells in several species of eucone insects and the embryol ogical development of the pseudocone and Semper cells in dipteran eyes will be needed to determine positively if the pseudocone eye is an advanced form of eucone eye.

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119 Irradiated Cochliomyla hominovorax Irradiated flies received from the rearing facility in Mission, Texas, were abnormal in a number of respects. The most common abnormality was a large increase in the number and size of vacuoles in the retinular cell cytoplasm. The cytoplasm was highly vacuolate and appeared poorly fixed but the fixation is not believed responsible for the abnormalities encountered. Eyes of Anastrepha suspensa (Tephr i tidae ) were fixed concurrently with the irradiated screwworm fly eyes. The eyes of Anastrepha were well fixed and exhibited none of the abnormalities in the irradiated screwworm flies. A large increase in the number of tracheoles near and between the retinular cells was also observed inthe irradiated eyes (Plate XXII B, Plate XXI B, C). Abnormally shaped rhabdomeres, rhabdomeric microvilli, and central cell transitions (Plates XXII, XXIII, and XXIV) were not consistently observed but were present in many of the individuals examined. The irradiated flies were highly variable on an individual and weekly basis. Eichenbaum and Goldsmith (1968) published an electron micrograph of an abnormal central cell transition zone in Musca eyes transplanted as imaginal discs into the abdomen of a larva. This abnormal transition zone contains 8 rhabdomeres and is similar in some respects to Plate XXIV C although no

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120 bridge is present between R7 and R8. Eichenbaum and Goldsmith (1968) misnumbered the retinular cells (their No. 1 is actually No. 8) and stated that their el ectronmi crograph shows the "typical arrangement of photoreceptor cells" which is unfortunately incorrect. The abnormalities observed in the flies that were irradiated as pupae are of an unknown origin. Unirradiated flies reared in Fargo, North Dakota, and irradiated flies from Mission, Texas, were reared on an artificial diet, "nutria" (Hoffman, personal communication; Holt, personal communication). The observed abnormalities in the flies reared in Mission, Texas, may be due to irradiation damage. The possibility of dietary insufficiencies inducing the abnormalities cannot be dismissed. Moore (1974; believes that a true wild type-fly has not been produced even on the best available meat and blood diets used. He feels the accumulation of waste products from the flies may cause many quality problems in a massrearing program. Insects reared on artificial diets over many generations often deviate markedly from their wild counterpart. Agee (in press) has demonstrated considerable differences in the eye of Anastrepha suspensa due to the quality of their diet. Ana strepha reared on a superior diet were as much as 10 times more responsive to visual stimulation than those reared on other artificial diets.

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CHAPTER III THE SPECTRAL SENSITIVITY OF THE COMPOUND EYE OF Cochl i omyia homi novorax The importance of the screwworm fly has already been described in the previous chapter. The following research was done in part to substantiate the microanatomical findings and determine if there were any neurophys i ol og i ca 1 abnormalities associated with the fly. L iterature Review The spectral sensitivity of the compound eyes of only a small number of insects has been determined using electrophysiological methods. Spectral sensitivities for insects representing only 7 orders have been determined. Spectra for the following have been published: Lepidoptera, Heliothis zea (Boddie), hL virescens (F.) (Agee, 1973) and Manduca s exta (L.) (Hoglund & Struve, 1970); Coleoptera, Pi n e u t e s c i 1 i a t u s (Forsberg) (Bennet, 1967); Hymenoptera, Apis mel 1 ifera L. (Goldsmith, 1960, 1961); Odonata, Libe l Tula 1 uc tuosa Burimerster (Ruck, 1965) and Li bel 1 u 1 a needhami Westfall (Horridge, 1969) and Hemiptera, Notonecta s p p . (Bennett & Ruck, 1970) . 121

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122 The entire subject of insect vision has been covered in a book by Mazokl i n-Porshnyakov (1969). Burkhardt (1964) and Goldsmith (1964) have written review articles treating electrophysiological studies on the insect compound eye and insect vision. The most widely studied eye in terms of spectral sensitivity is that of Ca 1 1 i p h o r a e rythrocepha 1 a M e i g . It has been the subject of many detailed electrophysiological and behavioral vision studies along with 2 mutant eye types, the "chalky" eye and the "white-apricot" eye. These 2 mutant forms have been used to determine the effect of screening pigments on the spectral sensitivity, particularly the presence of a red receptor in the flies. Early studies of the spectral sensitivity of C_. ery throcephal a reported 3 peaks (Autrum & Stumpf, 1953; Walther & Dodt, 1959; Mazokl i n-Porshnya kov , 1960). The most prominent peak was in the green, around 490 nm, a second peak occurred around 350 nm in the ultraviolet, and a third in the red region, about 620 nm. Subsequent studies on the 2 mutant strains of C a 1 1 i p h o r a lacking eye pigments, the "chalky" mutant and the "white-apricot" mutant, failed to produce a peak in the red near 620 nm. The first study by Autrum (1955) on the "white-apricot" mutant, lacking brown-red pigments, produced only 2 peaks, 350 nm and 490 nm. A second study (Hoffman & Langer, 1961) on the "chalky" mutant, lacking all eye pigments,

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123 also produced 2 peaks, 1 at 350 nm and another at 500 nm. Intracellular recordings of single receptor cells produced evidence indicating that the red peak was an artifact (Burkhardt, 1962, 1964). Burkhardt (1964) postulated that the 620 nm red pseudopeak is the result of differential absorption by the screening pigment. He postulates that the screening pigment does not absorb at 620 nm allowing extra light to pass and stimulate the receptor cells. This could account for a mass response in a spectral region where screening pigment does not absorb causing a false peak to appear. The spectral sensitivity of single photoreceptor cells of wildtype C a 1 1 i p h o r a agree with the spectral sen si tivity of the "chalky" mutant (Burkhardt, 1964). All the photoreceptor cells of the wildtype flies investigated had 2 peaks (Burkhardt, 1962, 1974). One peak is approximately 350 nm in the ultraviolet. The second peak can be in 3 different positions, 490 nm, 470 nm , or 521 nm. Approximately 80% of the receptor cells, the so-called green type, have a peak at 490 nm. Two other cell types, the yellow-green and the blue type, account for approximately 20% of the receptor cells. The yellow-green type has a peak at 470 nm and the blue type has a peak at approximately 521 nm. The mean peak of these 3 cell types

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124 will fall at about 496 nm when large numbers of photoreceptor cells are plotted (Burkhardt, 1964). An ommatidium illuminated by scattered light or reduced light exhibits an interesting spectral shift. The ultraviolet peak at 350 nm remains but the 490 nm peak shifts to approximately 515 nm and a 620 nm peak appears (Burkhardt, 1962, 1964; Autrum, 1955). As the intensity of light is increased the 620 nm peak disappears and the visible peak returns to 490 nm. In Mu sea domes ti ca L. 2 sensitivity peaks are present at 350 nm and 490 nm (Goldsmith & Hernandez, 1968). Studies on a white-eyed mutant of M . domes tica failed to produce evidence of a red receptor (Goldsmith, 1965). An alternate approach to the determination of spectral sensitivity is the use of optomotor responses. An optomotor response is a behavioral response initiated by an insect to a patterned stimulus. A rotating striped pattern was presented Phormi a r e g i n a Meig. in spectral colors. The yawing force or torque developed by the fly in fixed flight was used as a measure of the optomotor reaction to various spectral colors and the spectral sensitivity was calculated. The sensitivity curve had 2 peaks, 1 in the ultraviolet at 353 nm and the other in the green at 490 nm (Kaiser, 1968; Kaiser & Liske, 1972) .

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125 Spectral sensitivity experiments either electrophysiological or optomotor have shown 3 peaks of sensitivity in flies closely related to C. hominovorax , an ultraviolet peak near 350 nm, a green peak at approximately 490 nm, and a red "pseudopeak" at 625 nm. Methods and Materials Screwworm flies, Cochl i omyia hominovorax , were shipped to Gainesville, Florida, by air mail from H. C. Hoffmann, USDA, APHIS, Mission, Texas, as irradiated pupae (ca 7 Krad). These flies were mass reared for release in the screwworm fly eradication program and were received in the release container. Approximately 200 pupae were placed in a holding cage (15 x 30 x 60 cm) consisting of tube gauze stretched over an aluminum frame. A mixture of honey and water was provided the newly emerged flies as a source of sugar and water. The flies were held in a dark cabinet to minimize damage to the wings caused by the flies striking against the cage when flies were held on a normal light-dark regimen. Flies 8 days of age were used for spectral sensitivity experiments and 3-day-old flies were used for the weekly experiments to measure the variation in visual sensitivity. Equal numbers of male and female flies were selected at random from the cage for testing. Approximately

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126 60-70% of the flies set up for testing from some weekly shipments had very low visual sensitivities, some as low as 50 uV. Only flies that required a neutral density attenuation of .6 or higher at a wavelength of 490 nm for a criterion response were selected for testing over the complete wavelength range, 350 nm to 650 nm. The flies used for experiments ti on were immobilized on a wax block with 3 "U"-shaped wire clips. One clip was placed behind the head, another over the thorax and wings, and the third held the abdomen. Electrical contact with the photoreceptor cells was made by positioning' an electrosharpened stainless steel electrode (Agee, 1971) into each eye. The tip diameter of the recording electrode was approximately 1.7 microns and was placed into the right eye. The indifferent electrode was placed into the noni 1 1 umi nated left eye. Both electrodes were placed in the dorsal hemisphere of the eye behind the vertical midline. The electrodes generally penetrated between the corneal facets and were placed approximately 50-100 microns below the surface of the eye. Following placement of the electrodes the eyes were allowed to dark adapt for 20 minutes prior to testing. Preliminary tests showed that visual sensitivity did not increase with additional time. Cochl i omyi a hominovorax eyes were found to fully dark adapt in about 15 minutes and the extra 5 minutes were added to insure complete dark adaptation.

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127 The electrodes were connected push-pull to a Grass Model P-6 preamplifier with an amplification of 500X. The electrophysiological response was displayed on a Tektronix Model 565 dual trace oscilloscope. The upper beam was connected to a photocell that monitored the flash duration and the lower beam displayed the summed electrical response of the illuminated photoreceptor of the eye. Optical Stimulation The stimulating light source was a Bausch and Lomb No. 33-86-39-01 tungsten (quartz iodide) coil filament lamp. This lamp provided a continuous spectrum from the near ultraviolet to the infrared (300 nm to 700 nm). The lamp was operated at a voltage of 120 V AC and an amperage of 400 ma monitored with meters. Monochromatic light was obtained with a Bausch and Lomb grating monochrometer Cat. No. 33-86-07 that provides a continuous spectrum from the ultraviolet to the infrared wavelengths. The purity of the light emitted from the exist slit is a product of the dispersion of the system and the width of the entrance and exit slits utilized. A slit width combination that gave a 10 nm bandpass was used. The entrance slit was 2.3 mm and the exit slit was set at 1.35 mm (Agee, 1973) . The light was chopped with a mechanical vane attached to a solenoid controlled by a Grass S-4 stimulator. Light

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128 flashes of 235 milliseconds duration at a rate of 1 per 2.5 seconds were used. Filters were utilized to control the quality and quantity of light delivered to the illuminated eye. Selected Corning filters were used to block second and third order wavelengths characteristically produced by grating systems. The higher orders are produced at integral multiples of the first order wavelengths. The blocking filters used were Corning No. 1-69 utilized in the wavelength range 350-700 nm , No, 5-59 at wavelengths of 365-480 nm and No. 4-65 used from 500-700 nm. Appendix A illustrates the percent transmittance of the various filters. The equal response method for determining spectral sensitivity was used. The intensity of light stimuli delivered to the insect was regulated at each wavelength with a neutral density wedge. Light was attenuated to provide a criterion response defined for the purpose of this study as a 1 cm negative deflection ("on" response) on the oscilloscope screen. A response of 250 pV was neces sary to provide the criterion response. The neutral density wedge is a circular quartz iconel filter calibrated continuously from neutral density (ND) to a ND of 2. Due to the low visual quality of the irradiated flies a ND of 1.5 was the highest value used compared with a 2.5 ND of the considerably smaller eye of Anastrepha suspensa

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129 (*Agee, unpublished). The wedge delivered a 90% attenuation of light at a ND of 1.0 and a 50% attenuation at a ND of 0.3. Light radiation was conducted from the monochrometer to a light-proof box through a tube painted internally with a flat black paint that absorbed the incident light (Agee, 1972). Flies were tested in an electrically shielded light-proof Faraday cage. .The light-proof box contained a pair of micromanipulators and a dissecting microscope to aid in positioning the indifferent and recording electrodes. The optical system was calibrated with a 12 junction bismuth-silver thermopile (Eppley) and a mi 1 1 imi crovol t meter. Most ERG studies have dealt with undefined relative units that are of limited value due to the problem encountered when attempting to relate two different studies 2 or insects. Absolute units, watts per cm , were calculated at full light intensity at each wavelength tested. Measurement of absolute units is attainable by calibration with a thermopile (comparable to National Bureau of Standards 1 amps ) . The intensity of light measured with the thermopile and microvolt meter at 530 nm used in the weekly variance tests was calculated to be 2.63 p watts/cm 2 at full intensity. The value from the calibration for the intensity 2 of light at a particular wavelength in watts/cm was

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130 multiplied by the percent attenuation of the ND wedge necessary to produce the criterion response. The reciprocal of this electrical response was graphically presented to show the spectral sensitivity of the screwworm fly eye. Resul ts The average spectral sensitivity of 9 C_. hominovora x in the spectral range of 350 nm to 6 50 nm is presented in Figure 5 . Three peaks of sensitivity are present. There is a peak in the ultraviolet region presumably at 350 nm. Only a portion of this peak is present since it falls on the first wavelength tested. The second and largest peak is present in the green region of the spectrum at 490 nm. A third smaller peak is present near 625 nm in the red region of the spectrum. The visual sensitivity of 6 weekly shipments of irradiated screwworm flies as measured by the ERG at 530 nm is shown in Figure 6. Discussion The spectral sensitivity curve for irradiated C. hominovorax as determined by e 1 ec trophs i ol ogi ca 1 techniques is very similar to those previously reported for C a 1 1 i p h o r a (Walther & Dodt, 1959; Autrum & Burkhardt, 1961; Burkhardt, 1962, 1964; Mazokl i n-Porshnya kov , 1960), Musca domestica

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135 (Mazokl i n-Porshnyakov , 1960; Goldsmith & Fernandez, 1968), and Phormia reqina (Kaiser, 1968). Three peaks are present in all 4 species, a UV peak at approximately 350 nm, a larger peak in the green range near 490 nm, and a third peak at 625 nm. The third peak in the red region is probably a "pseudopeak" resulting from the lack of absorption by screening pigments around 625 nm. (Gol dsmi th , 1965; Burkhardt, 1962, 1964; Autrum, 1955; Hoffman & Langer, 1961 ). An unpublished electrophysiological study of the spectral sensitivity of 4 strains of reared irradiated C_. homi novorax showed 2 peaks, 1 at 550 nm (blue-green) and 1 at 630 nm (red) (Holt, unpublished report). These results are not consistent with prior work on related genera or the results of this study. No ultraviolet peak (350 nm) was reported since the spectra tested, 490 nm to 710 nm, did not include the UV portion of the spectra. The 630 nm red peak is probably a "pseudopeak" as discussed above. The primary sensitivity peak at 550 nm in all 4 strains is unexpected and unexplained. Scattered or reduced light has been reported to shift the green peak of sensitivity in flies from 490 nm to about 515 nm (Burkhardt, 1962). The criterion response used by Holt was 300 y volts which was higher than the 250 uvolt criterion utilized in this study.

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136 The visual sensitivity measured by the ERG at 530 nm exhibited considerable weekly variation. The highest weekly mean sensitivity was approximately 10 times more sensitive than the lowest weekly sensitivity. Visual quality has been demonstrated as a means of assessing the quality of mass-reared insects (Agee & Park, 1975). The wide variance of weekly values indicates considerable difference in the visual quality of flies released. Visual quality in Cochl j omyi a hominovorax may therefore serve as a means of determining the quality of the flies being reared and released. This area of screwworm fly biology deserves more attention to determine if visual quality can be used successfully as a quality control measure in the screwworm release program. The estimated production of the new screwworm plant near Tuxtla Guiterrez, Chiapas, Mexico, and the Mission, Texas, screwworm facility is estimated to be approximately one-half billion flies per week (Bushland, 1974a, b). The success of a rearing program of this magnitude will require a means of assessing the quality of the flies produced. Visual quality could be useful as a tool in the screwworm program and more research along these lines should be undertaken.

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CHAPTER IV CONCLUSIONS The research performed in this study has led to the following conclusions on the three areas investigated: A. The microanatomy of the eyes of the "lonestar tick," Amblyomma americanum . 1 . Amblyomma americanum L. has a functional eye. The photoreceptor neurons possess all the microanatomica 1 features common to other rhabdomeric photoreceptors. 2. The eye of A. americanum is primitive and not secondarily reduced. 3. The eye of A. ameri canum is the first arhabdomate eye in the Arthropoda. 4. The photoreceptor neurons show striking affinities with the turbellarian PI a ty helm inthes (flat worms) and pulmonate Mollusca (snails). All three have arhabdomate eyes, terminal microvilli lacking microvillar interaction, and very similar organelles and cellular organization. 137

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138 5. The affinities of the eyes of flatworms, snails, and ticks place the arthropods in the main line of rhabdomeric photoreceptor evolution. Evolutionary dendrograms previously had placed the flatworms and snails outside the mainstream of evolution. The microanatomy of the eye of the "screwworm fly," Cochliomyia hominovorax (Coquerel). 1. The microanatomy of the peripheral retina and lamina of unirradiated screwworm flies is very similar to the other genera of cyclorraphan Diptera studied. 2. The Semper cells maintain the distal trapezoidal pattern of the open rhabdom as do the rhabdomere caps, desmosomal and gap junctions of the Semper cells and the retinular cells. The significance of this configuration is structural continuity and permits according to many scientists visual data processing. 3. A 7-armed extension of the ommatidial cavity extends into the Semper cells. The ommatidial cavity is fluid filled and contains an unknown electron dense material. 4. "Pigment granules" of previous authors are actually vacuoles filled with crystals of pi gment .

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139 5. Retinular cells have no apparent metabolic functions and appear to be basically light transducers , 6. Morphological evidence indicates two types of polarized light reception. 7. The cone cells of eucone eyes and the Semper cells of pseudocone eyes appear to be homologous. The pseudocone eye is believed to be an advanced type of eucone eye. 8. Irradiated flies showed a number of abnormalities. a. Obconical rhabdomeres b. highly vacuolate cytoplasm c. increased number of trachea at all levels in the peripheral retina d. abnormally shaped microvilli e. decrease in number of microvilli f. abnormal central cell transition zone. 9. Although both irradiated and unirradiated flies were reared on artificial "nutria" diet, nutritionally induced abnormalities cannot be discounted. Spectral Sensitivity Studies. 1. Irradiated flies all demonstrated poor visual quality. Often 70% or more of a test group failed to give an adequate criterion reponse.

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140 2. Considerable weekly variance in visual sensitivity was determined. 3. The spectral sensitivity curve of C. hominovorax was 3 peaks. The largest peak is at 490 nm. A second peak of sensitivity is present in the ultraviolet region at 350 nm and a third "pseudopea-k" is present at 625 nm The large 490 nm peak would indicate a strong response to the color green. Confirmation of this research should be done with the optomotor technique.

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LITERATURE CITED Agee, H.R. 1971. Flicker fusion frequency of the compound eye of Hel iothi s zea . Ann. Tentomol . Soc. Amer. 64:942-945. . 1972. Sensory response of the compound eye of adult H el iothi s zea and H. vi rescens to ultraviolet stimuli. Ann. Entomol . Soc. 65:701-705. . 1973. Spectral sensitivity of the compound eyes of field-collected bollworms and tobacco budworms. Ann. Entomol. Soc. 66:613-615. . 1975. Article in preparation on the structure of the peripheral retina of Anastrepha suspensa . and M. L. Park. 1975. Use of the el ectroreti nogram to measure the quality of vision of the frui fly. Environmental Letters (in press). Autrum, H. 1955. Die spektrale empf i ndl i chkei t der augenmutation white-apricot von Cal 1 i phora erythrocephala . Biol. Zbl. 74:515-524. and D. Burkhardt. 1961. Spectral sensitivity of single visual cells. Nature 190:639. and H. Stumpf. 1953. El ektrophys i ol og i sche untersuchungen Liber das farbensehen von Ca 1 1 i phora . Z. Vergl. Physiol. 35:71-104. iaccetti, B. and C. Bedini. 1964. Research on the structure and physiology of the eyes of a lycosid spider. Arch. Ital. Biol. 102:97-122. Seadle, D. J. 1972. Fine structure of the integument of the ticks, B o o p h i 1 u s de col oratus Koch and B. m i c r o p 1 u s (Canastrini") (Acari na : Ixodid-ae). Int. J. Insect Morphol . and Embryol . 3:1-12. Sennett, R.R. 1967. Spectral sensitivity studies on the whirligig beetle, Dineutes c iliatus . J. Insect Physiol. 13:621-633. 144

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145 lennett, R.R. and P. Ruck. 1970. Spectral sensitivities of dark and light adapted Notonecta compound eyes. J. Insect Physiol. 16:83-88. , J. Tunstall, and Horridge. 1967. Spectral sensisitivity of single ret inula cells of the locust. Z. Vergl. Physiol. 55:195-206. Sernard, G.D. and W. H. Miller. 1968 ters in the cornea of Diptera ophthalmology 7:416-434. Interference f i 1 Invertigative 1972. Optical Vol .VII. In Sernhardt, C.G., G. Gemne, and G. Seitz. properties of the compound eyes. M.G.F. Fuortes (Ed.), Physiology of photoreceptor organs. Berlin: Springer-Verlag. Sinnington, K.C. 1972. The distribution and morphology of probable photoreceptors in eight species of ticks (Ixodidea). Z fur Paras i ten ki nde 40:321-332. iishopp, F.C. and H.L. Trembley. 1945. Distribution and hosts of certain North American ticks. J. Paras i tol. 31:1-54. lonnet, A. 1907. Recherches sur 1'anatomie compare" et le devel oppement des Ixodides. Ann. Univ. Lyon 20: 1-180. loschek, C.B. 1971. On the fine structure of the periph' eral retina and lamina ganglionaris of the fly Musca domestica. Z. Zellforsch 118:369-409. . 1972. Synaptomol ogy of the lamina ganglionaris in the fly. In R. Wehner (Ed.), Information processing the visual systems of arthropods. New York: Springer-Verlag. Braitenberg, V. 1967. Patterns of projection in the visual system of the fly. I. Retina-lamina projections. Exp.. Brain Res. 3:271-298. . 1972. Periodic structures and structural gradients in the visual ganglia of the fly. In R. Wehner (Ed.), Information processing in the visual systems of arthropods. New York: Springer-Verlag. Brammer, J.D. 1970. The ul trastructure of the compound eye of a mosquito Aedes aegypti L. J. Exp. Zool . 175:181-196. "" '"

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146 Burkhardt, D. 1962. Spectral sensitivity and other response characteristics of single visual cells in the arthropod eye. Symposia Soc. Exp. Biol. 16:86-109. . 1964. Colour discrimination in insects. Adv. Insect Physiol . 2:131-173. Burton, P.R. and K.A. Stockhammer. 1969. Electron microscopic studies of the compound eye of the toad bug, Gel astrocori s ocuscol atu s . J. Morph. 127: 233-257. Bushland, R.C. 1974a. Screwworm eradication program. Science 184:1010-101 1 . . 1974b. Founders' memorial lecture presented at the Entomological Society of America annual meeting, Minneapolis, Minn., December 2, 1974. Butler, L., R. Roppel , and J. Zeigler. 1970. Post emergence maturation of the black carpet bettle, Attagenus megatoma (Fab.): An electron microscope study. J. Morph. 130:103-128. Campos-Ortega, J. A. and N.J. Strausfeld. 1972. The columnar organization of the second synaptic region of the visual system of Musca domesti ca L . I . Receptor terminals in the medulla. Z. Zellforsch 124: 561-585. Cooley, R.A. and G. M. Kohls. 1944. The genus Amblyomm a (Ixodidae) in the United States. J. Paras i t o 1 . 30:77-111 . Cromroy, H.L. 1971. Successful uses of radiation in agriculture. In: Conference innovative applications of radiation. Southern Interstate Nuclear Board, 29p. Curtis, D.J. 1970. -Comparative aspects of the fine structure of the eyes of Phalangida (Arachnida) and certain correlations with habitat. J. Zool. London 160:231-265. Dannel, R. 1957. Uber den feinbau der ret inula bei D rosophila mel anogaster . Z. Naturforsch. 12B: 580-583.

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147 Dorrscheidt-Kaf er , M. 1972. Die empf i nd 1 ichkei t eizelner photorezeptoren im komplexauge von C a 1 1 i p h o r a ery thro cephala . J. Comp. Physiol. 81:309-340. Douglas, J.R. 1943. The internal anatomy of Dermacentor andersoni Stiles. Univ. of Calif. Pub. in Entomol. 7:207-272. Eakin, R.M. 1965. Evolution of photoreceptors. Cold Spr. Harb. Symp. on Quant. Biol. 30:363-370. 1968. Evolution of photoreceptors. Vol. 2. In T. Dobzhanski (Ed.), Evolutionary biology. New York: Appl eton-Century . and J.L. Brandenberger . 1971. Fine structure of the eyes of jumping spiders. J. Ultrastr. Res. 37: 618-663. Eguchi, E. and T.H. Waterman. 1966. Fine structure patterns in crustacean rhabdoms. In C.G. Bernhard (Ed.), The functional organization of the compound eye. London: Pergamon Press. . 1968. Cellular basis for polarized light perception in the spider crab, L i b i n i a . Z. Zellforsch. 84:87. Eichenbaum, D.M. and T.H. Goldsmith. 1968. Properties of intact photoreceptor cells lacking synapsis. J. Exp, Zool. 169:15-32. Fahrenbach, W.F. 1968. The morphology of the eyes of Limul u s . I. Cornea and epidermis of the compound eye. Z. Zellforsch. 87:278-291. . 1969. The morphology of the eyes of Limu 1 us . II. Ommatidia of the compound eye. Z. Zellforsch. 93:451-483. 1970. The morphology of the LimuTus visual system, III. The lateral rudimentary eye. Z. Zellforsch 105:303-316 _. 1971. The morphology of the L i m u 1 u s visual system, IV. The lateral optic nerve. Z. Zellforsch. 114: 532-545. Fischer, A. and G. Horstmann. 1971. Der feinbau des auges der mehlmotte, E p h e s t i a kuehni el 1 a Zeller (Lepidoptera: Pyra 1 i d i dae~]7 Z. Zellforsch. 116:275-304.

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148 Fuge, H. 1967. Die p igmentbi 1 dung i m auge von Dorsophila mel anogaster und ihre beei nf 1 ussung durch ben white locus. Z. Zellforsch. 83:468-507. Goldman, L.J. 1971. The el ectroreti nogram and spectral sensitivity of the compound eye of Aedes aegyp ti . Ph.D. dissertation, University of Florida. 7 5~p~p. Goldsmith, T.H. 1960. The nature of the retinal action potential and the spectral sensitivities of ultraviolet and green receptor systems of the compound eye of the worker honeybee. J. Gen. Physiol. 43:7757 9 9. . _. 1961. The physiological basis of wavelength discrimination in the eye of the honeybee. In W.H. Roseblith (Ed.), Sensory communication. Cambridge, Mass.: M.I.T. Press. _. 1964. The visual system of insects. In M. Rockstein (Ed.), The physiology of insecta. New York: Academic Press. _. 1965. Physiol . Do flies have 49:265-287. a red receptor? J. Gen. _ and H.R. Fernandez. 1968. The sensitivity of housefly photoreceptors in the mid-ultraviolet and the limits of the visible spectrum. J. Exp. Biol. 49:669-677. Gossel, P. 1935. Beitrage zur kenntnis der hautsi nnesorgane und haustorusen der cheliceraten und der augen der Ixodiden. Z. morph. und Okologie (Berlin) 30:177205. Hasselmann, E.M. 1962. Uber die relative spektrale emf i ndichkei t von kaferund schmetter lingsaugen bei verschiedenen helligkeiten. Zool. Jb. Physiol. 69:537-576. Hoffman, C. and H. Langer. 1961. Die spektrale auger eupf i ndl ichkei t der mutante "chalky" von Cal 1 i phora erythrocephal a . Natur. wi ssenschaf ten 48:605. Hoglund, G. and G. Struwe. 1970. Pigment migration and spectral sensitivity in the compound eye of moths. Z. Vergl. Physiol. 67:229-237.

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149 Holt, G.G. 1973. Vision studies of four strains of the screwwornt fly. Unpublished report submitted to ARS, APHIS Screwworm Laboratory, Mission, Texas. Hoogstral, H. 1970. Bibliography of ticks and tickborne diseases: From Homer (about 800 b.c.) to 31 December 1969. Vol. II Special Publication U.S. Naval Medical Research Unit Number Three (NAMRU-3), Cairo, Egypt, U.A.R. 495 pp. Horridge, G.A. 1965. Arthropod receptors for light and the optic lobe. Vol. II. In T. Bullock and G.A. Horridge (Eds.), Structure and function of the nervous system of invertebrates. San Francisco: W.H. Freeman. 1969. Unit studies on the retina of dragonflies Vergl. Physiol. 62:1-37. Jones, B.M. 1950. The sensory physiology of the harvest mite, Trombicul a autumnal is Shaw. J. Exp. Biol. 27:461^49^ ~ Kaiser, W. 1968. Zur frage des unterscheidungsvermogens fur spectralfarben: eine utersuchung der optomotrik der koniglichen glanzfliege Phormia regina Meig. Z. Vergl. Physiol. 61:71-102. and E. Liske. 1972. A preliminary report on the analysis of the optomotor system of the bee-behavioral studies with spectral lights. In R. Wehner (Ed.), Information processing in the visual systems of arthropods. New York: Springer-Verlag. Karnovsky, M.J. 1965. A forma 1 dehyde-gl utera 1 dehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol . 27: 137A. Kirschfeld, K. 1967. Die projektion der optischen umweld auf das raster der rhabdomere im komplexauge von Musca Exp. Brain Res .. 3 : 248-270 . _. 1972. The visual system of Musca : studies in optics, structure and function. In R. Wehner (Ed.), Information processing in the visual systems of arthropods. Berlin: Springer-Verlag. _ and W. Francheschini. 1968. Optisch eigenschafter der ommatidien im complexauge von Musca . Kybernetik 5:47-52.

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150 Kirschfelf, K. and W. Francheschi ni . 1969. Ein mechanismus z u r steurerung des sichflusses in den rhabdomeren des komplexauges von Musca . Kybernetik 6:13-22. and W. Reichardt. 1970. Optomotor i sche versuche an M usca mi t inear polar isiert em licht. Z. Naturforsch. 25B : 228 . Land, M.F. 1969. Structure of the principal eyes of jumping spiders in relation to visual optics. J. Exp. Biol. 51:443-470. Lasansky, A. 1967. Cell junctions in ommatidia of Limul us . J. Cell Biol. 33:365-384. Locke, M. 1959. The cuticular pattern in an insect, Rhodni us pro! i xus . Stal. J. of Exp. Biol. 36: 459-477." 1961. Pore canals and related structure in insect cutible. J. Biophys. Biochem. Cytology 10: 589-618. 1969. The structure of an epidermal cell during the formation of the protein epicuticle and the uptake of moulting fluid in an insect. J. Morphology 127:7-39. Ludtke, H. 1953. Retinomotrik und adaptationsvorg'a'nge in auge des ruchenschwimmers ( Notonecta gl anca L.) S. Vergl. Physiol. 35:129-152. McEnroe, W.D. and M.A. McEnroe. 1973. Questing behavior in the adult American dog tick, D ermacentor v ariabilis Say. Acarologia 15:37-42. Machan, L. 1966. Studies on structure, el ec troret i nogram , and spectral sensitivity of the lateral and median eyes of the scorpion. Ph.D. dissertation, University of Wisconsin. 162 pp. MacRae, R.K. 1964. Observations on the fine structure of photoreceptor cells in the planarian D u g e s i a tigrin a. J. Ultrastr. Res. 10:334-349. Mazokl i n-Porschnyakov , G.A. 1960. Colormetric study of the properties of colour vision of insects as exemplified by the house fly. Biofizida 5:295-303. 1969. 306 pp. Insect vision !ew York PI enum Press

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151 Melamed, J. and 0. Tru j i 1 1 o-Cenoz . 1966. The fine structure of visual system of Ly cosa (Araneae: Lycosidae). Part I. Retina and optic nerve. Z. Zellforsch. 74:12-31 . and 1968. The fine structure of the central cells in the ommatidia of dipterans. J. Ultrastr. Res. 21:313-334. Meyer-Rochow, V.B. 1973. Fine structural changes in darklight adaptation in relation to unit studies of an insect compound eye with a crustacean-like rhabdom. J. Insect Physiol. 20:573-589. Miller, W.H. 1960. Visual photoreceptor structures. In J. Bracket and A.E. Mirsky (Eds.), The cell. New York: Academic Press. Mills, L.R. 1973. On the detailed morphology of the visual system, dorsal setae, and glands of the two-spotted spider-mite, Tetranychus urticae Koch, 1836. Ph.D. dissertation, Stanford University. 141 pp. Moody, M.F. and J.D. Robertson. 1960. The fine structure of some retinal photoreceptors. J. Biophys. Biochem. Cytol. 7:87-91. Moore, R.F. 1974. Report of nutritional studies on the screwworm conducted at the screwworm research laboratory. ARS, APHIS, Mission, Texas. Unpublished report. Mote, M.I. and T.H. Goldsmith. 1970. Spectral sensitivities of color receptors in the compound eye of the cockroach, Periplaneta . J. Exp. Zool . 173:137-146. Nathanson, M.E. 1967. Comparative fine structure of sclerotized and unscl eroti zed integument of the rabbit tick, Haema physal is leporispalustris (Acari: Ixodides: I sod i da e) . Ann. Entomol. Soc. 60:1125-1135. Nolte, J. and J.E. Brown. 1971. The anatomy of the median ocellus of Limulus . Z. Zellforsch. 118:297-309. Pedler, C. and H. Goodland. 1965. The compound eye and first optic ganglion of the fly. J. Roy. Micr. Soc. 84:161-179. Reynolds, E.S. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17:208-212.

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152 Rivers, T.M. and H.L. Horsfall, Jr. (Eds.). 1959. Viral and rickettsial infections of man. Philadelphia: J.B. Lippincott. 967 pp. Rohlich, P. and L.J. TbVdk. 1961. El ektronenmi kroskopi sche untersuchungen des auges von planarien. Z. Zellforsch. 54:362-381 . and 1963. Die feinstruktur des auges der wei nbergschnecke ( H e 1 i x pomati a L.). Z. Zellforsch 60:348-368. Ruck, P. 1965. The components of the visual system of a dragonfly. J. Gen. Physiol. 49:289-307. Satir, P. and N.B. Gilula. 1973. The fine structure of membranes and intercellular communication in insects. Ann. Rev. Entomol. 18:143-166. Schneider, L. and H. Langer. 1969. Die struktur des rhabdoms im doppelauge des wasserl auf ers G e r r i s 1 a c u s t r i s . Z. Zellforsch. 99:538-559. Seitz, G. 1968. Der strahlengang in appositonauge von Calliphora e rythrocephala (Meig.). Z. Vergl. Physiol. 59:205-231. Semtner, P.J., R.W. Barker, and J. A. Hair. 1971a. The ecology and behavior of the lone star tick (Acarina: Ixodidae) II. Activity and survival in different ecological habitats. J. Med. Ent. 8:719-725. , D.E. Howell, and J. A. Hair. 1971b. The ecology and behavior of the lone star tick (Acarina: Ixodidae) I. The relationship between vegetation habitat type and tick abundance and distribution in Cherokee Co., Oklahoma. J. Med. Ent. 8:329-335. _ and J. A. Hair. 1973a. The ecology and behavior of the lone star tick (Acarina: Ixodidae) IV. The daily and seasonal activity patterns of adults in different habitat types. J. Med. Ent. 10:337-345. _ and . 1973b. The ecology and behavior of the lone star tick (Acarina: Ixodidae) V. Abundance and seasonal distribution in different habitat types. J. Med. Ent. 10:618-6 28.

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153 Semtner, P.J., J.R. Sauer, and J. Hair. 1973. The ecology and behavior of the lone star tick (Acarina: Ixodidae) III. The effect of season on moulting time and postmoult behavior of engorged nymphs and adults. J. Med. Ent. 10:202-205. Smith, C.N. and M.M. Cole. 1941. Effect of langth of day on the activity and hibernation of the American dog tick, Dermacentor variabilis (Say). Ann. Entomol. Soc. 34:426-431. Smith, D.S. 1968. Insect cells: Their structure and function. Edinburgh, Scotland: Oliver & Boyd. 372 pp. Sonenshine, D.E., E.L. Atwood, and J.T. Lamb, Jr. 1966. The ecology of ticks transmitting Rocky Mountain spotted fever in a study area in Virginia. Ann. Entomol. Soc. 59:1234-1262. and G.F. Levy. 1971. The ecology of the lone star tick, Amblyomma amer i c anum (L) in two contrasting habitats in Virginia CAcarina: Isodidae). J. Med. Ent. 8:623-635. Spurr, A.R. 1969. A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastr. Res. 26:31-43. Strausfeld, N.J. and V. Brainenberg. 1970. The compound eye of the fly ( Musca domesti ca ) : Connections between the cartridges of the lamina ganglionar is. Z. Vergl. Physiol. 70:95-104. Tru j i 1 1 o-Cenoz , 0. 1965a. Some aspects of the structural organization of the arthropod eye. Cold Spr. Harb. Symp. Quant. Biol. 30:371-382. . 1965b. Some aspects of the structural organization of the intermediate retina of dipterans J. Ultrastr. Res. 13:1-33. _. 1969. Some aspects of the structural organization of the medulla in muscoid flies. J. Ultrastr. Res. 27: 533-553. _. 1972. The structural organization of the compound eye in insects. Vol. VII. In M.G.F. Fuortes (Ed.), Physiology of photoreceptor organs. Berlin: SpringerVerlag .

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155 Waterman, T.H. and K.W. Horch. 1966. Mechanism of polarized light perception. Science 154:467-475. Wehner, R. 1972. Information processing in the visual systems of arthropods. Berlin: Spr i nger-Verl ag . 334 pp. Wharton, G.W., W. Parrish, and D.E. Johnston. 1968. Observations on the fine structure of the cuticle of the spiny rat mite, Lael aps e c h i d n i n a (Acari: Mesosti gmata ) . Acarologia 10: 206-214. White R.H. 1967. The effect of li.ght and light deprivation upon the ultrastructure of the larval mosquito eye. II. The rhabdom. J. Exp. Zool . 166:405-425. Wolken, J.J. 1971. Invertebrate photoreceptors, comparative analysis. New York: Academic Press. 179 pp. Wright, J.E. 1969. Effect of photoperiod on patterns of o v i position of Anocentor ni tens Neumann (Acarina: Ixodidae). J. Med. Ent. 6:257-262. 1971. Response of ovipositing Amblyomma macul atum Koch (Acarina: Ixodidae) to photoperiod. J. Med. Ent. 8:529-531.

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BIOGRAPHICAL SKETCH William Avery Phi 11 is III was born in Evanston, Illinois, on March 1, 1942. He received his secondary education at Lutheran High School West, Detroit, Michigan, graduating in June, 1960. He attended Miami University, Oxford, Ohio, and received the Bachelor of Science degree in June of 1964 with a major in economics and finance. Following graduation, he worked for three years as a salesman of construction equipment for Brock Tool of Detroit, Inc. Mr. Phi 11 is attended Eastern Michigan University as a part-time graduate student in the Biology Department in 1966 and in 1967 as a full-time graduate student. He received the Master of Science degree in Biology in 1970. He taught Zoology and General Biology at Oakland County Community College, Farmington, Michigan, and attended the University of Michigan from September, 1970, until August, 1971 . In September, 1971, he enrolled in the Department of Entomology and Nematology of the University of Florida to work towards the Doctor of Philosophy degree in entomology. His research interests include the system at ics and natural history of parasitic mesosti gmatid mites and 156

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157 the anatomy, u 1 trastructu re , and function of arthropod photoreceptors . William Avery Phi 11 is III is the father of two children, Emily Love Phil lis and Colby Anne Phillis.

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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. * CI H a r v e y IZJ Crdmroy , C h a i r m a] n Professor of Entomology and Nematol ogy 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. m h Ak (P 4 JLJi-^ Associate Professor of Entomol ogy 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 degreee of Doctor of Philosophy. /V U>.Jd, H. C. Aid rich Associate Professor of Botany I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is f ul ly adequate , in scope and quality, as a dissertation for the degree of Doctor of Philosophy. I i, ,.
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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. &w F. Butler .ssociate Professor of Entomology This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. August, 1975 J / Dean, College of Agriculture Dean , Graduate School

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