Title: 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
Physical Description: xiv, 157 leaves : ill. ; 28 cm.
Language: English
Creator: Phillis, William Avery, 1942-
Copyright Date: 1975
 Subjects
Subject: Ticks   ( lcsh )
Screwworm   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
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Statement of Responsibility: by William Avery Phillis III.
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 144-155.
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00098154
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000408110
oclc - 02163948
notis - ACF4523

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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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