I I
71-7537
PITMAN, W.J., 1924- NEUROSECRETORY SITES IN THREE SPECIES OF COPEPODS: DIAPTOMUS STAGNALIS, DIAPTOMUS SANGUINEUS AND CALANUS FINMARCHICUS.
The Ohio State University, Ph.D., 1970 Zoology
University Microfilms, A XEROX Company, Ann Arbor, Michigan
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED NEUROSECRETORY SITES IN THREE SPECIES OF COPEPODS:
DIAPTOMUS STAGNALIS, DIAPTOMUS SANGUINEUS
AND CALANUS FINMARCHICUS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University
by
W. J. Pitman, B.S., M.S. ft*ftftftft
The Ohio State University 19 70
Approved by
Aciviser r Department of /Zoology ACKNOWLEDGMENT
Grateful acknowledgment is made to Dr. Willard Myser, under whose direction this dissertation was completed.
Dr. Myser faced a particularly difficult task in assuming the role of adviser after the departure from the University of former advisers, Dr. Carter Broad, and Dr. Alan Brockway. His cooperation and constructive criticism made possible the completion of this work.
Very special thanks is extended to Dr. Jerry Hubschman who provided guidance and direction throughout the entire research program.
I especially wish to express my appreciation to
Dr. Loren Putnam for his continuing guidance, genuine interest, and constant encouragement throughout my graduate career. f Acknowledgment is made to Dr. Charles Leone, resident in the Department of Ophthalmology, for his assistance in the photocoagulation experiments.
I express gratitude to Mrs. Judy H. Wilson for her faithful assistance in the writing and typing of this manuscript. TABLE OF CONTENTS
Page
ACKNOWLEDGMENT...... ii
LIST OF FIGURES...... iv
INTRODUCTION...... 1
METHODS AND MATERIALS...... 8
Collections...... 8 Histological methods...... 11 Extract methods...... 13 Photocoagulation methods...... 14
RESULTS...... 20
Histological observations...... 20 Extract experiments...... 57 Photocoagulation experiments...... 6 8
DISCUSSION...... 75
CONCLUSIONS...... 82
LITERATURE CITED...... 85
AUTOBIOGRAPHY...... 92 LIST OF FIGURES
Figure Page
< 1 Chromatophore stages of Hogben and Slome (1931)...... 15
2 Diagram of light coagulator...... 19
3 Frontal section of D. stagnalis...... 21
4 Composite drawing of D. stagnalis in frontal section...... 22
5 Frontal organ and eye in frontal section.,... 26
6 Sagittal section of frontal organ...... 2 7
7 Sagittal section of frontal organ...... 2 8
8 Sagittal section of eye and frontal organ.... 28
9 Schemmatic drawing of sagittal section...... 29
10 Sagittal section of D. stagnalis...... 31
11 Eye in sagittal section ...... 33
12 Sagittal view of dorsal ocellus ...... 35
13 Dorsal ocellus in sagittal section...... 35
14 Eye and optic nerve of D. stagnalis...... 37
15 Three ocelli and brain of copepod...... 38
16 Schemmatic diagram of the eye and its nerves ...... 39
17 Semi-diagrammatic representation of the eye.. 39
18 Pigmented membranes and secretory droplets of eye...... 41
19 Dorsal ocellus in frontal section ...... 41
20 Central cells and dorsal ocelli ...... 43
21 Secretory droplets in central cells...... 44
22 Relative position of the three ocelli...... 46
iv LIST OF FIGURES (Continued)
Figure Page
2 3 Three ocelli of nauplius eye...... 4 8
24 Ventral ocellus and frontal organ...... 49
25 Ventral ocellus and frontal organ...... 50
s 26 Frontal section of optic nerves...... 5 3
2 7 Diagrammatic representation of innervation of eye...... 54
2 8 Neurosecretory cells in brain...... 55
29 Neurosecretory cell in brain ...... 56
30 Neurosecretory cells in the brain...... 56
31 Sagittal section of fifth naupliar stage of Calanus finmarchicus...... 58
32 Sagittal section of C. finmarchicus, fifth stage...... 59
33 Fifth stage nauplius eye in sagittal section ...... 59
34 Cross section of fifth stage nauplius eye...... 61
35 Transverse section of C. finmarchicus 62
36 Third stage C. finmarchicus...... 6 3
37 Secretory droplets in C. finmarchicus, third stage...... 7...... 64
38 Eye of C. finmarchicus...... 64
39 Blanched Uca pugilator...... 66
40 U. pugilator in state of maximum *” dispersion...... 66
v LIST OF FIGURES (Continued)
Figure Page
41 Relationship between chromatophorotrophic response of Uca and copepod extracts.... 67
42 D. stagnalis following photocoagulation.... 70
43 D. stagnalis following photocoagulation.... 71
44 D. stagnalis following laser treatment 71
45 Sagittal section of photocoagulated eye.... 72
46 Section through one photocoagulated ocellus...... 72
47 Section through two photocoagulated ocelli...... 72
48 Relationship between chromatophorotrophic response of Uca and extracts from photocoagulated copepods...... 7 3 INTRODUCTION
Throughout the Animal Kingdom histological and physio
logical evidence shows that there are groups of neurons which function as endocrine glands. The most characteristic
feature of neurosecretory cells is their ability to snythe-
size and release specific chemical agents (hormones) which
act upon various tissues some distance from the point of
release. A neurosecretory cell can be recognized by its
appearance and by the active substances which it can be
shown to contain (Carlisle and Knowles, 1959). Nerve cells
containing appreciable cytological signs of secretion in the
cytoplasm, in the form of granules, globules, or droplets, may be considered possible neurosecretory cells. The
granules may aggregate into inclusions of various sizes which then become stainable and visible with the light microscope (Bern and Hagadorn, 1965).
E. and B. Scharrer (1954) consider the neurosecretory
cell to be an elongate gland cell which secretes apically.
This type of nerve cell produces neurosecretory material which is transported along the axon to bulb-like terminals and released into the circulatory system (Turner, 1966), distinguishing them from neurohumors such as ajre located at the synapse and myoneural junction, whose secretion
1 functions only for a short distance. The neurosecretory system of crustaceans may have more than one center of release. However, the secretory cells are located in specific sites in the nervous system and their products are usually composed of discrete aggregates (Carlisle and
Knowles, 1959).
In vertebrates, neurosecretion has been amply demon strated. The neurohypophysis consists of non-medullated nerve fibers, neuroglea, and blood vessels. The nerve fibers have their origin in the hypothalamus from which the cell bodies elaborate neurosecretory granules. These granules migrate along the nerve axon to the posterior lobe of the pituitary where they are stored for release at a later time.
There are also non-neurosecretory structures in arthro pods, namely, the thoracic or prothoracic glands of insects, and the Y-organ of Malacostracans (Echalier, 1954 and 1955).
The Y-organ, as first hypothesized by Gabe, is the molting gland of the Malacostracans. It is a paired, compact epithelial structure located in the head region in the antennule or maxillary segments. The hormone produced by
' - i this structure is presumed to be the same as ecdysone pro duced by insects, since both hormones exhibit similar bio logical activity (Passano, 1960).
In invertebrates, neurosecretory cells play a decisive role in the operations of the endocrine system (Gorbman and
Bern, 1962). For example, located in the supraesophageal ganglion of annelid worms is a very large number of nerve cells which have the morphologic characteristics of gland cells and which exhibit endocrine functions (Lee and Knowles
1965). The roundworm, Ascaris sp., also shows neurosecre tory cells in a ganglion of its cephalic nerve ring
(Gersch, 195 7). However, most of the endocrine studies in the invertebrates have been concerned with the arthropods
(Gorbman and Bern, 1962). Growth, differentiation, meta morphosis, molting, diapause, color change, control of chitin formation, decomposition of lipids, and rate of oxygen consumption have been well established as being under the control of the endocrine system (Gorbman and Bern,
1962 and Carlisle and Knowles, 1959).
The first modern study of crustacean endocrinology was the work of Koller (192 7) and Perkins (192 8) in which they observed that color changes in crustaceans were controlled by chemical agents in the circulatory system. However, prior to this Kroyer (1842) had observed color change in the prawn Hippolyte sp. He and other early investigators believed that color change was mediated by nerves (Fingerman
196 3). Perkins’ (192 8) work on the prawn Palaemonetes sp, and Koller’s (1925, 192 7, 19 30) work on the shrimp Crangon vulgaris demonstrated that the color changes observed were under hormonal rather than nervous control. Also Pouchet
(1876) demonstrated many years ago from his work on
Palaemon and Crangon that the eyestalk influences some
metabolic and behavioral phenomena in Crustacea.
Hanstrom (19 33 , 19 34, 19 39) determined that there are
two structures in the eyestalks of crustaceans, the sinus
gland and the X-organ, which have endocrine activity. He
suggested that the secretory nature of the X-organ was
demonstrated by the occurrence of acidophil droplets and
other larger, unidentified accumulations of secretory pro ducts. Knowles and Carlisle (1956) proposed that the X-organ
described by Hanstrom be termed the sensory pore X-organ or sensory papilla X-organ. In regard to color changes,
Carlson (19 36) noted that the removal of the eyestalks from
the fiddler crabs Uca pugilator and U. pugnax caused a con
centration of the pigment within the chromatophores result ing in blanching of the animals. Subsequent injection of
the eyestalk extract led to darkening of the animals within
two hours due to the dispersion of the pigment within the
chromatophores. Brown and Cunningham (19 39), Abramowitz and
Abramowitz (1939, 1940), and Kleinholz and Bourquin (1941), in further studies, found that eyestalk removal in crusta ceans altered the rate of molting, in addition to bringing about color changes. A variety of other neuroendocrine functions, such as control of metabolism, water metabolism, blood sugar level, growth, and development, have been 5 associated with the crustacean eyestalk.
Novak (1966) and others established that hormones are not species specific in their effects. Mammalian hormones have been shown to produce their effect in birds, amphibians, and other vertebrates. A hormone from the pituitary gland of pigs, and the same hormone from the pituitary of oxen, both have similar effects on amphibian chromatophores. Hor mones from the thyroid and pituitary will accelerate meta morphosis in some amphibians but not in others. Frog tad poles change into adult frogs if injected with extracts from the thyroids of sheep (Lee and Knowles, 1965). Skin from mammals and mammalian malignant tissue has a chromato= phorotrophic effect on the fiddler crab U. pugilator (W. J.
Pitman, unpublished data). Insect hormones have been shown to be effective in other arthropod groups.
Crustacean chromatophores are known to be under hor monal control; therefore, any significant pigment movement in crustaceans is considered indicative of the presence of hormonal material. Crustaceans, such as fiddler crabs, crayfish, and shrimp (Palaemonetes) , have been used exten sively in bioassay experiments involving hormonal and sus pected hormonal material.
Few observations have been made on the sites of hormonal secretion in copepods. Most of the work on crustacean neurosecretory systems has been concerned with adult Mala- costraca (Hanstrom, 1931; Enami, 1951a, 1951b; Bliss and t
6
Welsh, 1952 ; Carlisle, 19 59 ; and Matsumoto, 195 8).
Hubschman (1963), in more recent studies, gave experimental and histological data concerning neurosecretory sites in the eyestalk of Palaemonetes in the larval forms. Prelimi nary investigations by Carlisle and Pitman (1961) of Calanus finmarchicus and C. helgolandicus revealed cells on the anterior part of the brain which were thought to produce neurosecretory material. Fahrenbach (1962) briefly described neurosecretory cells in the adult stage of Diarthrodes cystoecus, a harpacticoid copepod, using Gabe's paraldehyde fuchsin stain. The cells found in the lateral and dorsal region of the protocerebral lobes, and in the median region of the antennular ganglia, showed intensely purple stained granules in their cytoplasm. Naupliar and copepodid brains, however, showed an apparent absence of secretory products when stained with the Gabe's paraldehyde fuchsin stain.
Novak (1966) describes diapause (obligatory) as a temporary interruption of development occurring without regard to environmental conditions. This type of diapause is broken by a period of adverse conditions, then develop ment of the organism is resumed. He described a second type of diapause, facultative diapause, which occurs in response to the environmental conditions. Diapause is broken when the environmental conditions change and development resumes.
Cole (195 3) suggested that some cyclopod and harpacticoid copepod resting stages are analogous to diapause as found in insects. For example, Elgmork (1959) observed that the sub species Cyclops strennus strennus (Fisher) possesses a weak facultative diapause in the copepodid IV stage. Also,
Brewer (1964), using field observations correlated with laboratory investigations, indicated that there is a lag in the development of the egg of Diaptomus stagnalis. The 10- month "resting egg stage" in D. stagnalis takes place during the summer when the eggs are exposed to high temperatures and followed by low temperatures of the winter. This resting phase is considered by Brewer to be a period of diapause which he considers in principle to be identical to the phenomenon of diapause found in insects.
As has been cited, much is known concerning crustacean neurosecretion. However, documentation is scarce on the subject of neurosecretion in the lower crustaceans. The purpose of this investigation is to determine and describe the sites of neurosecretion in the copepod species Diaptomus stagnalis, D. sanguineus and Calanus finmarchicus. METHODS AND MATERIALS
Collections
Diaptomus stagnalis is a monocyclic inhabitant of ponds and swamps in the North Central and Eastern United
States, and south to the Gulf of Mexico. Copepodids and adults, for this investigation, were collected from 1963 to 196 7 in Calanus Swamp, Pickaway County, Ohio, approxi mately 30 miles south of Columbus. This is a poorly drained area, varying in size from approximately 100 square yards in the summer months (dry period) to 4 acres in the fall and winter months. During the fall and throughout the spring months, the depth may vary from 6 inches to 4 feet.
Collections were made with standard Number 12 (12 5 meshes to the inch) and Number 20 (175 meshes to the inch) plankton towing nets. The nets were thrown out into the lake, approximately 20-30 feet, and pulled toward shore.
One group of copepods was kept in refrigerator incubators att a temperature of 13°C. a second group was kept out of doors in an aquarium.
In central Ohio where D. stagnalis collections were made, the eggs hatch in early to late March, developing into adults in about a month after passing through six nauplius stages and five copepodid stages. This approximates the
8 9 time of hatching and development observed by Brewer (1964).
In the 1966 collections, red nauplius stages were first found in great abundance on 15 March in 8.5°C water of pH
7.1. Four days later, at a temperature of 4.5°C and pH 7.1, the red nauplius stages were still present. The first cope podid stages were noted on 24 March when the temperature was
15°C and pH 7.0. The copepodid stages molted every 48 hours in the laboratory incubator at a temperature of 13°C.
This compares favorably with field observations' made by
Brewer and personal observations in the field made from
17 March through 12 April, 1965,
The adult forms of D. stagnalis began to appear in the
4 April collections. As the breeding cycle progresses, females usually outnumber the males in natural populations.
This may be explained in part by the shorter life span of the male, and by the fact that the maximum number of males occur earlier in the breeding cycle (Marshall and Orr, 1955).
In a collection made on 16 April, of 225 animals counted, there were 112 males and 113 females, while in the 21 April collections, 12 7 males and 146 females were counted.
Diaptomus sanguineus, like D. stagnalis, is an in habitant of ponds and swamps. It breeds for short periods in the spring. The complete life cycle from stage I thru VI
(adult)requires 2 to 3 weeks, each stage lasting from 2 to 3 days. Diaptomus sanguineus was collected near Golden Pond,
Kentucky, 10 miles east of Hurray State University, from
February through April during 1966 and 196 7. The pond is small, covering less than one acre, and generally becomes almost dry during the summer months. Methods used in collections and maintenance were the same as described for
D. stagnalis.
Calanus finmarchicus is a species widely distributed over most of the oceans of the world. It is abundant in the north where most studies have been made, the northernmost record being 79°N (Marshall and Orr, 1955). During the winter months, development is arrested and over-wintering takes place in the last larval instar, the fifth copepodid stage, in locations such as the English Channel, or in the third or fourth copepodid stages in colder locations such as East Greenland (Russell, 192 8). There may well be three pauses occuring in the normal development of C. finmarchicus
16-cell stage, Nauplius III, and copepodid V stages
(Lebour, 1916, and Marshall and Orr, 1955).
The time required to complete the cycle from egg to adult depends upon the temperature, taking 2 months in the
English Channel and the Clyde Sea allowing 30 days for egg maturation and 30 days for development. In colder areas such as East Greenland, the cycle may take a full year
(Marshall and Orr, 1955).. There are six nauplius stages and 11 six copepodid stages; the sixth copepodid stage is the adult.
Calanus finmarchicus was collected by boat in the
English Channel, March through September of 1960. Speci mens fixed in Bouin's were received from Plymouth, England during the summer and winter months of 1965-66-6 7.
Histological methods
Freshwater organisms to be used for histological pur poses were collected in the early morning and fixed immediate ly in Smith's Alcoholic Bouin's according to Guyer's method
(1953), Zenker's fluid, 5 per cent formalin, formalin- alcohol-acetic acid (F.A.A. or A.F.A.), and ethyl alcohol.
In order to insure good penetration, the cuticle of each specimen was punctured and placed in the various fixatives for a period of 8-24 hours, except for Bouin's which re quired 24 hours or longer (Humason, 1962). Smith's Alco holic Bouin’s reagent and formalin were found to produce the most satisfactory results.
Prior to embedding, the specimens were dehydrated in alcohol and one animal in each group was treated with mush room extract, using Carlisle's method (1960), to soften the chitin. This procedure gave better results histologically by lessening the tendency of the chitin to tear when sectioned, and no discernible difference in intercellular structure was noted in treated as compared to the untreated tissues. 12
Each animal was individually oriented and embedded in polyester wax (melting point, 37°C), Fisher's Tissuemat
(melting point, 60-62°C), or Bioloid paraffin compound
(melting point, 50-52°C). Serial sections were cut 6-8 microns thick at room temperature (Tissuemat and paraffin) and mounted with Mayer's albumin adhesive on glass slides
(Guyer, 195 3). Tissue embedded in polyester wax was sectioned in a room held at a constant temperature of 10°C.
The most satisfactory staining procedure was a modi fication of azocarmine-aniline blue, orange-G process of
Gomori (19 39-1946), modified by Hubschman (1962). Gomori's
(1950) trichrome stain, which has been used extensively in vertebrate and invertebrate cytological studies of skin, muscle, connective tissue, endocrine and neurosecretory
cells, was also used in this study. For the nuclear staining, Delafield's hematoxylin stain (Guyer, 19 34) was found to give better results than Ehrlich's hematoxylin.
Sections for general histological studies were stained with hematoxylin and eosin and Mallory's triple stain. Paralde- hyde-fuchsin used by Gabe (1953) and Gomori's (1941)
chromium-hematoxylin-phloxine were also preferred as they have been widely used in crustacean tissue.
The histological sections were photographed through an
A. 0. Spencer (Microstar) compound microscope "With Ortho illuminator and 35 mm camera attachment. 13
Extract methods
Extracts of various copepod tissues were assayed for chromactivating activity in the fiddler crab Uca pugilator.
The three specific extracts tested on the chromatophores of eyestalkless U. pugilator were taken from (1) the anterior rostral area; (2) the region of the eye, and (3) tissue posterior to the eye. A fourth extract was also prepared from the eyestalks of U. pugilator.
In four experiments 20 fiddler crabs were used. The eyestalks were removed from 20 adult fiddler crabs by clipping them as close to their base as possible. They were then frozen at -18°C until ready for use in preparing the eyestalk extract. The eyestalkless animals were placed in a refrigerator for a period of one hour after which they were removed and placed in an incubator (19°C) for 24 hours.
By this time they had thoroughly blanched and were ready
for use. Injections of the extracts were then made in four
groups of crabs. A fifth group was injected with Neptune salt water as a control. Three or four eyestalkless Uca were used in each of the 5 groups.
Extracts were prepared following the method of Welsh
and Smith (1960). Copepod tissue was thoroughly ground in
a mortar with 10 ml of Neptune salt water (hydrometric
reading 1.20). Eyestalks from 10 Uca were prepared in a
similar manner to be used as a control tissue. Each extract was then placed in a test tube and the test tubes were 14 placed in a 100°C water bath for 5 minutes. This was done to denature proteins and to free smaller molecules. After the extracts had cooled, the supernatant was decanted and the remaining fluid was expressed from the tissue. Approxi mately .1-.3 ml of the extract was injected into the body cavity at the bases of the first legs of the respective crabs as noted above, with the amount injected determined by the size of the animal.
The groups of bioassay animals were observed for a period of at least one hour, and the stage of chromatophore activity reached was recorded. The method of staging chro matophores followed that of Hogben and Slome (1931)
(Figure 1). In this research only the degree of dispersion of the black pigment was recorded.
Photoco'agulation methods
The use of the laser has been described as a tool for the study of embryology and cytology (Lang, Barnes, Daniel and Maisel, 1964), with the advantage that particular cells in a monolayer culture may be coagulated and killed with no visible effects on surrounding cells. The helium-neon laser permits selective destruction of tissues. Witt, Reed, and
Tittle (1964) used direct-radiation of a laser beam and pro duced lesions in the nervous system of the spider which in turn brought about changes in the behavior of the organism.
The laser beam apparently coagulated the spider tissue in 15
* J :vf *\ • •/ <
Figure 1. Chromatophore stages of Hogben and Slome (1931) showing degrees of melanophore pigment dispersion. 1, punctate; 2, punctate-
stellate; 3» stellate; U, stellate-reticulate, and 5, reticulate. 16 such a way that leakage of body fluid did not occur.
Havener (1960) describes the use of the photocoagulator in the creation of chorio-retinal adhesions in the retina and in the destruction of unwanted tissue.
The Zeiss photocoagulator produces an intense con trolled source of light from the visible portion of the spectrum. Xenon high pressure is used as the source of radiation, and the density of the light can be regulated and focused on organs or tissues of a given animal (Meyer-
Schwickerath, 1960). Darker pigmented tissues absorb more energy from the beam, produce more heat, and are more readily coagulated (Meyer-Schwiekerath, 1960). The eye of the copepod seemed ideally suited for laser coagulation as it is pigmented with a lens-like mass located anteriorly and dors ally.
The animals to be treated were placed in single de pression slides with a small amount of water, and in order to slow their movement the slide was placed on an ice cubicle prior to treatment with the laser. Another method used to restrict the movement of the copepods was to place a wide mesh gauze over them during the treatment. To local ize the bums, the coagulation temperature was raised in as short a time as possible (Meyer-Schwickerath, 1960). The photocoagulator was placed on its highest setting: overload or 6500° (Figure 2). The iris diaphragm (6) was set to full 17 open (maximum power), and the variable diaphragm (8) was opened to .5° and 1.5° which produced burn areas of between
.1 mm and .5 mm in diameter (Havener, 196H). Each animal was exposed to the beam for 1 to 3 seconds and over a period of four weeks several groups of animals were treated.
To compare the differences between tissue from normal and photocoagulated animals extracts were prepared from the three areas of the animals as previously described.
Tissues fron the frontal region, the target site, and the remainder of the animal were assayed for chromatophoro trophic effect on the crab U. pugilator. After each in jection observations were made, the information recorded for a period of 3 hours following the experiment. 18
Legend for figure 2. 1, concave mirror; 2, high pressure lamp;
3, filament; U, shutter; $, condenser; 6, iris diaphragm;
7, filter; 8, field diaphragm; 9, objective; 10, observer;
11, smaller aperture; 12, ophthalmoscope mirror; 13, object to be coagulated. 11 10
- 1 -
Figure 2. Diagrammatic presentation of the optical system of the light coagulator.
(After H. Littmann, Heidelberg Berichte, 1957.)
CD RESULTS
Histological observations
The observation of stainable granule in a nerve cell presumably indicate secretory function (Bern and Hagadorn,
1965). By the use of Gabe's paraldehyde-fuchsin, Gomori, and Hubschman's stains granules were revealed in sections of the frontal organ, the eye, and the brain of the copepods D. stagnalis and D. sanguineus (Figures 3 and 4).
Extensive investigation has been done on the frontal organ of both Malacostracans and Entomostracans. Dahl
(1953, 1957, 1959) found groups of sensory cells in the anterior part of the cephalothorax and in the rostrum which he considered to be the frontal organ in the copepods
Harpacticus sp. He believed the frontal organ to be in nervated by nerves in close association with the ocular nerves.
Fahrenbach (1962) , in a study of harpacticoid cope pods, briefly described a mass of ganglion and gland cells which he considered to be the frontal organ. The nerves to this organ were seen to pass from the lateral surface
of the protocerebrum, anteriorly to the rostrum where the organ was located. Fahrenbach made no attempt to discuss the function of this structure, however.
20 21
Figure 3» Frontal section of the copepod Diaptomus stagnalis. 22
Figure U. Composite drawing of frontal section of Diaptomus
stagnalis♦
w
Legend for figure 3 and U. A, frontal organ; B, eye;
C, brain; D, esophagus; E, gut; F, longitudinal muscles. 23
Park (1966) gave a detailed description of frontal organs in the calanoid copepod Epilabiodocera amphitrites.
He found a pair of frontal organs, cylindrical in shape, lying close to the anterior exoskeleton of the rostrum, the frontal nerve being formed by three tapered nerve cells
emanating from the frontal organ. Park was unable to
determine the function of the organ.
Elofsson (1966a, b, c) made a series of comparative
studies of the frontal organs in several species of cope pods . In Pareuchaeta norvegica he found the organ in the
anterior part of the head. Two nerves ran from the antero-
dorsal margin of the brain to the anterior end of the
animal, just above the rostrum. For most of the course,
they ran parallel but divided distally and turned to the
lateral surface of the head. Elofsson's description of the
frontal organ was the same for Calanus finmarchicus. He
concluded nothing as to the possible secretory activity of
the organ.
Recently, the so-called frontal organ has been found
to be a homologue to the X-organ of other crustaceans
(Elofsson, 1966a and Dahl, 1963). However, no evidence has
been available on possible neurosecretory functions of the
structure, although some authors report that the frontal
organs are glandular (Dahl, 1953 and Fahrenbach, 1962). 24
In D. stagnalis the frontal organ is found in the region of the anterior cephalothorax just beneath the exoskeleton (Figure 5). It is located dorsal to the rostral filament in the frontal sinus, and measures approximately 95 p in length. In some histological sections it appears to be intimately connected with the cephalothorax at the anterior, medial border.
Stained sections show that the frontal organ of the adult copepod consists of two types of tissue which are intimately connected to one another (Figures 6, 7, and 8).
The dorsal part of the structure is composed of two large, elongated cells. The dorsal of the two cells is 64 in length and has a maximum width of 19 ji. The ventral cell is less elongated and has a maximum width of 14 jj. and length of 16 ji. The two cells lie end to end, giving the dorsal portion of the structure a total length of 80 ji.
Both cells contain deeply staining granules in the cyto plasm, and have a large nucleus occupying almost the entire cell.
A neck composed of 2 or 3 elongated, granular cells extends from the dorsal-most cell, and is connected to a nerve which passes dorsally over the eye joining the frontal organ with the brain. None of the histological sections revealed the complete nerve fiber running the entire length from the structure to the brain, but portions of it could be seen in different sections. A composite drawing of its connection to the brain is shown in Figure 9. Figure 5. Frontal section of anterior region of
Diaptomus stagnalis showing the frontal organ.
fO
1R
Legend for figure 5. BR, brain; E, eye; FO, frontal organ. Figure 6. Sagittal section of the frontal organ showing the two types of tissue present.
, DFO
Legend for figure 6. DFO, dorsal portion of frontal
organ; E, eye; VFO, ventral portion of frontal organ. Figure 7* Sagittal section of the frontal organ, showing its relationship to the eye.
Figure 8. Sagittal section through the eye
and frontal organ. Figure 9. Schemmatic sagittal section of Diaptomus stagnalis. BR, brain; DFO, dorsal portion of frontal organ; E, eye; NFO, nerve to frontal organ; ON, optic nerve; VFO, ventral portion of frontal organ. 30
The ventral portion of the structure is composed of at least four cells, having a total length of 51 p. The maximum width is 16 p and the minimum width is 8 p. Each cell contains a large granular nucleus, and also has posi tive staining granules in the cytoplasm. There is a "sac" surrounding the 4 cells which is also granular. The ventral part of the frontal organ is more granular than the dorsal part.
The eyes of D. stagnalis and D. sanguineus were found to be essentially alike; therefore, only D. stagnalis will be shown and described. Examinations were made in both sagittal (Figures 10 thru 14) and frontal sections (Figure
15); Figures 16 and 17 show composite drawings of the eye in D. stagnalis. The eye is composed of three ocelli; two dorsal ocelli, and an unpaired, ventral ocellus. The two dorsal ocelli are of approximately the same size and shape, and are located in a position which is anterior and dorsal to the protocerebrum. They are roughly oval in shape, and
are situated in the dorsal, mid-region of the head. They
occupy about one-fourth the area of the frontal sinus of
the head. Each ocellus is 35 p wide and 50 p long (Figures
18 and 19).
Flattened cells form the anterior surfaces of the
dorsal ocelli. The posterior surface is formed by a group
of "tapetal cells" (Fahrenbach, 1964) which form a reflecting
surface. A dark staining membrane separates the two ocelli Figure 10. Sagittal section of Diaptomus stagnalis.
ON DQ' yo
FA
Legend for figure 10. BR, brain; DO, dorsal ocellus;
ESO, esophagus; FA, first antennae; G, gut; VO, ventral
ocellus. Figure 11. Sagittal section through the anterior aspect of Diaptomus stagnalis showing the dorsal ocellus of the nauplius eye.
Legend for figure 11. BR, brain; ON, optic nerve;
FM, pigmented membrane; RC, retinular cells; SD, secretory droplets. Legend for figure 11.
PM SD
ON >s
PC
IP 34
Figure 12. Sagittal view of dorsal ocellus.
Figure 13. Sagittal section of the dorsal ocellus and optic nerve. „ 35 Figure 12.
Figure 13. 36
Figure lU. Posterior aspect of the dorsal ocellus including
the optic nerve and the anterior aspect of the brain.
Secretory granules are shown in both the protocerebrum and
the ocellus.
Legend for figure lU. BR, brain; DO, dorsal ocellus;
ON, optic nerve; SG, secretory granules. 37 Figure lb*
Legend for figure lb. 38
Figure V->. Frontal section through the anterior half
of Dlaptomus stagnalis. Shown are the three ocelli of
the eye, and their relationship to the brain. VO
LDO v / RDO
ON
Figure 16. A schemmatic diagram of the three ocelli and their nerve fibers. LDO, left dorsal ocellus; ON, optic nerves; RDO, right dorsal ocellus; VO, ventral ocellus.
Figure 17. Semi-diagrammatic representation of the three ocelli of the eye of Diaptomus stagnalis in frontal section.
LDO, left dorsal ocellus; RDO, right dorsal ocellus;
VO, ventral ocellus. Figure 18. Frontal section at a high dorsal level through
the right and left dorsal ocelli. A pigmented membrane
separates the two ocelli, and secretory droplets occur
anterior and posterior to the ocelli.
Figure 19. The dorsal ocelli as seen in frontal section.
Granules are present posterior to the two lateral ocelli. 41 o Figure 18
Figure 19 42
(Figure 18) , and continues posteriorly to form their posterior margins. A dark staining membrane also separates each cell of each ocellus (Figure 11).
The greatest volume of the eye is composed of retinu- lar cells as described by Elofsson (1963s 1965, 1966a, b, c), Park (1966), Fahrenbach (1964), and Vaissiere (1961).
These cells, in D. stagnalis, are of a variety of shapes.
The anterior ones are triangular, the posterior ones are spherical, and the lateral ones are oblong with their long axis in the lateral plane. The membranes separating the retinular cells are filamentous and tortuous (Figure 11).
There are two granulated "central cells" adjacent to one another, located in the ventral posterior region between the two dorsal ocelli (Figures 20 and 21). The cells are large, measuring 24 p in length and 16 p in width. A large, round nucleus is located in the mid-pos terior region of each cell. The cells give a positive reaction when stained with the paraldehyde-fuchsin stain of
Gabe (195 3) and Gomori's chromium-hematoxylin-phloxine stain (1941). The cells are pyramidal in shape and have apical fibers which extend anteriorly between the two dorsal ocelli to form an anterior, membrane-bounded vesicle in which granules are found. These granules are located in the anterior-most part of the eye complex (Figure 18).
Granules also surround the central cells in the area be tween the ocelli. Figure 20. Frontal section through the two central cells
and the dorsal ocelli.
SD
SD
Legend for figure 20. AF, apical fibers of central cells
N, nucleus of central cell; SD, secretory droplets. Figure 21. Frontal section at a high dorsal level through the right and left dorsal ocelli. Secretory droplets are present in the central cells. 45
The ventral ocellus is roughly oval and is approxi mately 35 ju wide and 45 p long. The ocellus points anteriorly with the long axis lying along the longitudinal axis of the animal. It lies directly beneath the dorsal ocelli s although in some preparations appears to lie posterior to the ocelli and only slightly ventral to them
(Figures 22 and 23). In one preparation the anterior margin of the third ocellus is continuous with the frontal organ by a membrane which encloses the whole eye complex
(Figures 24 and 25). There is an area between the ventral ocellus and the frontal organ which is enclosed by this membrane, forming a type of vesicle 24 p wide and 11 p long. This anterior vesicle contains large granules which give a positive reaction with all three stains. In the same preparation, the frontal organ is in continuation with the cells of the anterior portion of the ventral ocellus. In every preparation observed, the ventral portion of the frontal organ and the whole eye complex stained identically, suggesting that they contain the same type of tissue.
In some animals, granules are located posterior to the ventral ocellus (Figure 18). In several preparations granules were found in the brain, and continued along the optic nerve (Figure 14), indicating that some of the granules may originate in the brain. Figure 22. Frontal section through the brain and three
ocelli. The ventral portion of the two dorsal ocelli and
the dorsal portion of the median ocellus seem to appear on
the same plane.
Legend for figure 22. BR, brainj DO, dorsal ocelli;
VO, ventral ocellus or median ocellus. 47
Figure 23. Frontal section through the brain and eye.
This section is at a low dorsal level; the most ventral aspect of the two dorsal ocelli and the mid-region of the median ocellus are in view.
Legend for figure 23. BR, brain; DO, dorsal ocellus;
VO, ventral ocellus. 48 Figure 23.
Legend for
figure 23. Figure 2U. Frontal section through the ventral ocellus
and frontal organ.
AV I
VO
Legend for figure 2U. AV, anterior vesicle;
BR, brain; FO, frontal organ; VO, ventral ocellus. 50
Figure 2£. Oil immersion view of ventral ocellus in
continuation with the frontal organ. 51
Granules associated with the eye. exhibit a wide range
of deminsions. The large ones appear to be aggregates of
smaller granules, indicating that areas where they are
present may function as blood sinuses.
There are three optic nerves. The two nerves to the
dorsal ocelli leave the anterior area of the brain and
penetrate the ocelli at their posterior lateral border.
The paired nerves appear to be symmetrical, and are con
tinuations of the neurons of the protocerebrum. The large
nerves branch just before entering the posterior margin of
the ocelli, appearing to innervate the different cells of
the ocelli (Figure 26).
The third optic nerve leaves the ventral, anterior
surface of the medial region of the brain and extends
anteriorly to the ventral ocellus. It enters the ocellus
posteriorly and medially as shown in Figures 26 and 27.
Hubschman’s stain revealed a pair of cells, apparently
^ secretory in nature, in the brain. These large cells are
located in the posterior, ventral aspect of the brain just
beneath the level of the ventral ocellus of the eye. They
are approximately equal in size, 21 p long and 10 )i wide.
Each cell has a very large, oval nucleus, measuring approxi
mately 14 ji in length and 9 ^ in width. Positively stain
ing granules appear in both the cytoplasm and the nucleus,
as well as in the nerves extending anteriorly from each cell.
The path of the nerves could not be traced with certainty, Figure 26. Frontal section of the optic nerves, brain, and two dorsal ocelli.
Legend for figure 26. BR, brain; DO, dorsal ocellus;
LOF, lateral optic fibers; MOF, median optic fibers. 53 Figure 26.
Legend for
figure 26. DO DO
» LOF
LOF MOF BR 54
I
9
Figure 27. A diagrammatic, dorsal view of the innervation of the eye. BR, brain; DO, dorsal ocelli; LOF, lateral optic fibers; MOF, median optic fibers. Figure 28. Protocerebrum from the dorsal side showing
the two neurosecretory cells.
NNSC— 4k* % — NNSC
Qeift*«r«*»/sSL....
Legend for figure 28. FO, frontal organj NNSC, nerve
from neurosecretory cell; NSC, neurosecretory cell. Figure 29. A section through one neurosecretory cell.
Figure 30. Part of a frontal section through the brain, including the two neurosecretory cells. 57 however, they appear to extend anteriorly just ventral to
the margin of the brain, perhaps ending in the eye (Figures
28, 29, and 30).
The three neurosecretory structures were found in all
stages of development in D. stagnalis, D. sanguineus, and
C. finmarchicus with one exception. Granules were found in
the eye of the latter species in the early copepodid
stages, but were absent in the fifth stage which is the
"resting stage." (See Figures 31, 32, 33, 34, and 35 to
Figures 36, 37, and 38.)
Endocrine experiments
The test universally employed for determining the
chromactivity of hormone-like extracts is the response of
eyestalkless animals to injections of an extract from
suspected tissue (Knowles, 1956). In this series of
experiments, eyestalkless Uca was used as the bioassay
animal. Pigment dispersion of and among chromotophores
was taken as a criterion for the presence of hormones.
The degree of dispersion, staged according to Hogben and
Slome (1931) , was indicative of the concentration or
activity of the hormone. Hogben and Slome's method de
scribes the degree of pigment dispersion numerically, as
shown in Figure 1. Stage 1, punctate, indicates maximum
pigment concentration. Stage 5, reticulate, is the stage
of maximum pigment dispersion. Stages 2, 3 , 4 (punctate- Figure 31. Sagittal section through nauplius eye of Calanus finmarchicus. The four cells in the lateral ocellus contain no secretory droplets.
Legend for figure 31. BR, brainj E, eye; FO, frontal organ;
G, gut; RF, rostral filament. Figure 32. Sagittal section of Calanus finmarchicus in
fifth stage.
Figure 33. Fifth stage nauplius eye as seen in sagittal section. Note apparent absence of secretory droplets. 60
Figure 3k. Cross section through nauplius eye of Calanus finmarchicus. Note the absence of secretory droplets between the three ocelli.
Legend for figure 3U. DO, dorsal ocelli; FA, first antennae;
VO, ventral or median ocellus. | Figure 3UC1
figure 3k Figure 35. Transverse section of the head of
Calanus finmarchicus in fifth stage at the level
of the nauplius eye. Figure 36. Calanus finmarchicus at third stage seen in sagittal section. Note secretory droplets present in the eye.
SD
VO
Legend for figure 36. BR, brain; DO, dorsal ocellus}
SD, secretory droplets; VO, ventral ocellus. Figure 37. A lateral view of the anterior aspect of Calanus finmarchicus, third copepodid stage. Note the presence of large secretory droplets between the ocelli of the eye.
Figure 38. The eye of Calanus finmarchicus, third stage,
in detail. 65 stellate, stellate, stellate-reticulate, respectively) are intermediate stages of dispersion. The absence of chro- mactivating substances in the blood of the bioassay animals, prior to injection of extracts, was indicated by their blanched condition (Figure 39).
Fresh extract from the rostral area of the test copepod, which contains the frontal organ with the two morphologically different types of granular tissue, resulted in migration of pigment within the chromatophores from punctate (stage 1) to punctate-stellate (stage 2) when injected into Uca. The eye plus a small amount of the rostral area extract when injected into Uca caused the pigment pattern to change from punctate to stellate- reticulate or reticulate, which is the maximum response that the organism can exhibit (Figure 40). The extract from the remainder of the animal, the area posterior to the eye which includes the brain, caused dispersion of the pigment in Uca to stage 2 (punctate-stellate). In only one animal observed was there migration past stage 2.
This one showed dispersion to early stage 3 (stellate).
Pigment dispersion progressed to stage 5 (reticulate) when eyestalkless Uca was injected with Uca eyestalk extract. Time of response and degree of dispersion paral leled that of the extract of the copepod eye (Figure 41).
The control injection, sea water, effected no movement; Figure 39. Fiddler crab, Uca pugilator, in blanched
condition.
Figure UO. Uca pugilator following injection of eye extract from Diaptomus stagnalis. Condition of the chromatophores is maximum dispersion. 67
§ D3
I I H
CM
rH
0 10 20 30 uo 50 60 MINUTES
Figure Ul. Relationships between responses of melanophores of Uca pugilator to extracts of Uca eyestalk and tissues from the copepod
Diaptomus stagnalis. Uca eyestalk, •; eye, o; frontal organ, e; brain and abdominal tissue, ej salt water control, «. 68 that is, the chromatophorcs reiiui i tied punctate. Due to handling and environmental changes, two of the animals showed a small amount of migration to stage ? (punctate- stel1 ate) .
P h o t o e o ag u1 a t i o n c xp c r i men t s
Identical extracts Were made of the animals which had been subjected to photocoagulation experiments. Those extracts were of the rostral area, the area of the eye, and the abdominal, posterior region of the animal. Figures
U2 thru 5 7 show the copepod after photocoagulation. None of the extracts from the photocoagulated copepod produced any pigment dispersion in the melaiiophores of Uca past stage 7 or stage 3 (punctate-stellate or stellate, res pectively). This small amount of dispersion may have been due to the presence of a minute amount of hormonal material, remaining in the animal (Figure M8).
In both the normal extract and photocoagulated extract experiments, the chrornatopharotrophic response of the Uca to the injected material was observed at intervals of 10 minutes for a period of one hour. In each case, the move ment of the dark pigment occurred within a 10 minute period,
The two positive responses, the Uca eyestalk extract and the untreated copepod eye extract, initiated pigment migra tion in the chromatophores within 5 minutes, and complete dispersion of the pigment within 40 minutes (Figures Ml and 69
Figure U2. Anterior region of Diaptomus stagnalis following
photocoagulation.
Legend for figure U2. BR, brain; DO, dorsal ocellus;
ON, optic nerves; VO, ventral ocellus. Legend for
ON DO
yo Figure 1*3 • Photocoagulated ocelli of Diaptomua
stagnalis.
Figure 1*1*. Photocoagulated ocelli in detail. • 9
Figure ii!?. Sagittal section through the eye showing
destruction by photocoagulation.
Figure U6. Section through one photo Figure U7« Section through two photo
coagulated ocellus. coagulated ocelli. 73
SI o
CVl
0 10 20 30 50 60 MINUTES
Figure U8. Relationships between responses of melanophores of Uca pugilator to extracts of Uca eyestalks and tissues from the copepod
Diaptomus stagnalis. Uca eyestalk, •; normal eye, o; eye from photo coagulated copepod, ©; frontal organ from photocoagulated copepod, e; brain and abdominal tissue from photocoagulated copepod, ej salt water control, o. 48). The pigment also remained in the dispersed state from 2 to 3 hours. The copepod extracts did not appear to be toxic since there were no deaths of the experimental animals, and a second response could be effected by using the same animals the next day for re-experimentation.
The responses to the copepod extracts were typical physio logical responses, complete over the entire organism and exactly parallel to the responses to the Uca eyestalk extract itself. Hence, the results conform to the cri teria outlined by Knowles (1956) in that the responses were immediate, prolonged, typical, and non-toxic. DISCUSSION
The major objective of this investigation was to identify possible neurosecretory structures in the fresh water copepods Diaptomus stagnalis. During the course of the study critical comparison was made with another fresh water organism Piaptomus sanguineus and a marine copepod
Calanus finmarchicus.
The gross histology was similar in the two fresh water forms. However in Calanus finmarchicus the location of the eye was more posterior and devoid of granules in the 5th copepodid stage.
Three specific staining techniques were used: Gomori's chromium-hematoxylin-phloxine (CHP), Gabe's paraldehyde- fuchsin (PAG) and a modification of azan stain by
Hubschman. These stains render neurosecretory cells very prominent in histological preparations (Novak, 1966).
Gomori's CHP is known to selectively stain the contents of neurosecretory cells in both invertebrates and vertebrates
(Welsh, 1961). With the use of these stains the neurose cretory granules could be identified in the perikaryon, which was its site of production, as well as along the axon fibers.
Three distinctive areas are evident in D. stagnalis and D. sanguineus which, by their histological character istics and/or physiological activity, are recognized as
75 76 neurosecretory cells. Areas containing appreciable amounts of secretory material were found in the regions of the eye, brain, and frontal organ.
A light sensitive nauplius eye is present in almost all Crustacea, displaying a wide variety of types in
Copepoda (Horridge, 1965). The nauplius eye is tripartite in the species under consideration, as in most other cope- pods, developing from epidermal cells (Dahl, 1959), The three ocelli are dorsal and antero-dorsal to the brain in the calanoid copepod. The eye assumes a similar position in the harpacticoid copepod according to Fahrenbach’s (1962) description.
The eye is composed primarily of retinular cells, each surrounded by a membrane (Figure 12). These cells constitute the greatest volume of the eye of most copepods, a fact described in comparative studies by Fahrenbach (1964),
Vaissiere (1961), Elofsson (1966a, b, and c), and Park
(1966). Reflecting surfaces of the eye are formed by tapetal cells (Fahrenbach, 1964). In a study of the ultra structure of the nauplius eye of the harpacticoid copepod
Macrocyclops albidus , Fahrenbach (1964) reported four pig mented "central cells" located between the rounded surfaces of the three ocelli. Elofssen (1966a, b, and c), in a comparative study of non-Malacostraca, never found more than three of these pigmented cells. In the present study of 77
D. stagnalis and D. sanguineus, only two granulated central cells were found between and anterior to the three ocelli of the nauplius eye (Figure 20).
The cytoplasm of all central cells investigated seemed to be completely filled with some type of inclusion.
The granular filled central cells in D. stagnalis stained positively with Gomori's, Gabe's, and Hubschman's modified azan stain. Granules which stain positively with the
Gomori stain consist of nonbasic protein and contain tyrosine and relatively large amounts of cystine. This has been demonstrated in decapod crustaceans, the posterior pituitary of vertebrates, and the corpora cardacia of insects (Bern and Hagadorn, 1965). Also, Fahrenbach (1964) reported that some similar inclusions in M. albidus were of a lipid nature. He further mentioned the presence of small, spherical masses of granules which he could not identify. The granules in D. stagnalis, however, were not lipids as this material would have dissolved in the xylene and alcohol during the fixation and staining process, and would have given a positive reaction when stained with oil red and nile blue stain. It seems apparent, from the histological study, that this is an endocrine, gland which persists as an active structure throughout the life of
D. stagnalis. During larval development, the entire eye complex containing this gland is in close contact with the 78
frontal organ. In the adult, however, the whole complex has moved posteriorly, leaving a space between the frontal
organ and the eye. Similar glands, such as the X-organ,
the sensory pore X-organ, and the sinus gland, have been
found in other crustaceans.
A second organ, the frontal gland, contains character
istic stainable granules and is located in the most
anterior part of the cephalothorax. Elofsson (1966a) makes
reference to an unpaired heap of cells in the upper part of
the head of the copepod Pareuchaeta norvegica. Results of
his study suggest that these may be similar structures, but
nothing was concluded as to secretory activity in P.
norvegica.
In D. stagnalis, the median frontal organ is an un
paired group of six cells divided into two cytologically
distinct regions (Figures 6 and 7). The most ventral
portion, composed of four cells, stained more densely and
appeared to contain a greater number of granules than the
two cells of the dorsal portion (Figures 7 and 8). Inner
vation is by a nerve which passes dorsally over the eye,
and enters the frontal organ from the dorsal aspect of the
two-cell complex (Figure 9). The secretory material was a
constant feature seen in all sections of these cells.
In addition to the optic gland and the median frontal
organ, a third possible endocrine secretory site, located in 79 the brain, gave a positive reaction when stained with
Gomori's, Gate's, and Hubschman's stains. Two cells in the mid-region of the brain contained appreciable quantities of secretory granules. These were large, nucleated cells, very densely stained (Figures 28, 29 and 30). The termi nation of their distal connections was not clear in any of the preparations.
Neurosecretion has been found in nearly all groups of
Crustacea in the central nervous system (Bern and Hagadorn,
1965). Neurosecretory cells usually occur in groups, with cytological signs of neurosecretion in the form of droplets, globules, or granules, containing secretion bearing axons.
The cells in the brain are paired, located in the mid- protocerebrum (Figures 28 and 30). These cells are strik ingly different from the other neurons in the brain, being twice the size of a characteristic neuron, and con taining densely staining granules which were absent in the cytoplasm of the other nerve cells (Figures 29 and 30).
In the crustacean decapods, the endocrine structures which control molting and metabolism also govern color changes. Color changes in crustaceans are due to chromato- phores which lie among the hypodermal cells, and the migration of pigment within these cells is controlled by hormones (Brown and Sandee, 19H8). 80 Carlson (19 35) showed that removal of both eyestalks from the fiddler crab Uca pugilator brought about blanching of the animal, due to the concentration of the melanin.
Subsequent injections of eyestalk extracts brought about melanin dispersion. Hanstrom (1933) presented evidence that the sinus gland and the X-organ in the eyestalk were endocrine structures. Carlson (19 36) reported that the major melanin-dispersion hormone was in the sinus gland of the eyestalk.
Cell bodies in the brain and sub-thoracic ganglia have been shown to contain secretory material in decapods, and chromatophorotrophins have also been extracted from these sites. The next step, then, was an attempt to correlate the cytological evidence of neurosecretion in
D. stagnalis with chromatophorotrophic activity as physio logical evidence, even though D. stagnalis contains no chromatophores. Uca pugilator was used as the bioassay animal to determine if extracts from the copepod have a chromatophorotrophic effect.
The endocrine organs which were shown histologically to contain secretory material were extracted and injected into U. pugilator. Tissue homogenates consisting of parts of 20 copepods from each of the three aforementioned regions were used. The homogenates were injected into eyestalkless crabs at the base of the first cheleped, with 81 artificial sea water used as a control.
A definite correlation was found between the presence of the granules in the eye and the color change in the bioassay animal. Injections of eye tissue extracts into the eyestalkless Uca brought about a typical chromato phorotrophic response not unlike that of the hormones from the animal itself. The response consisted of a darkening of the Uca due to dispersion of pigment in the chromato- phores (Figure 40). Neither extracts of the frontal organ, nor the brain tissue homogenate containing the remainder of the central nervous system, brought about a significant change in the chromatophores of Uca when the animals were injected (Figure 41). Injections of homogenates from the area of the eye, after the eye tissue had been removed with an RC2 laser, brought about no melanin dispersing activity
(Figure 48). CONCLUSIONS
The endocrine structures in the copepod Diaptomus stagnalis have been made the subject of a histological and physiological study. Judgements on whether or not an area was to be considered endocrine in nature were based on the following two criteria: (a) the presence of stainable materials in neurons or neural material and
(b) the effect of injected extracts of the stainable material on bioassay animals.
The following conclusions were drawn from the histo logical evidence and/or chromatophorotrophic activity of extracts.
1. Sites of secretory material are similar or identical
in all three species observed; that is, in Diaptomus
stagnalis, Piaptomus sanguineus, and Calanus
finmarchicus.
2. The endocrine structures were found in all copepod
stages examined. One structure, the frontal organ,
varied slightly in position in some of the preparations.
3. Stainable granules are constant in all stages of
development examined, except in the overwintering fifth
stage of the marine species C. finmarchicus.
82 83
U. The presence of stainable granules indicated three
sites of neurosecretion: the eye, the frontal organ,
and the brain.
5. The neurosecretory site in the eye is localized in
two large "central cells" located between the three
ocelli of the nauplius eye.
6. The unpaired frontal organ is composed of two distinct
regions; the ventral portion being the more granular
of the two.
7. Secretory material in the brain is located in two
large neurosecretory cells, situated in the mid-dorsal
region of the protocerebrum.
8. Only the extract from the region of the eye elicited
chromatophorotrophic responses in U. pugilator. Lack
of concentration of the hormone in the other sites of
neurosecretion probably was responsible for the failure
of those extracts to elicit a response.
9. The extract of the eye effected no chromatophorotrophic
response after the region of the eye had been destroyed
by laser treatment.
In evaluating the significance of the responses of bioassay animals to injected material, the criteria recom mended by Knowles (1956) was applied. He suggested that
the response should occur within 5 minutes, that it should 84 last at least 30 minutes, that it should be a response typical of normal specimens under normal conditions, and that the injected substance should not be toxic.
The responses observed in this series of experiments met the requirements of Knowles, since the movement of chromatophores in U. pugilator began immediately after homogenate injection; the responses lasted longer than
30 minutes; they were identical to responses found under normal conditions, and they were not toxic.
\ LITERATURE CITED
Abramowitz, R. K. £ A. A. Abramowitz. 19 39. Moulting and viability after removal of the eyestalk in Uca pugilator. Woods Hole Biol. Bull., 77: 326-327.
Abramowitz, R. K. £ A. A. Abramowitz. 1940. Moulting, growth and survival after eyestalk removal in Uca pugilator. Biol. Bull. 78: 179-188.
Bern, H. A. £ I. R. Hagadorn. 1965. Neurosecretion. Structure and function intthe nervous systems of invertebrates. Vol. I. T. H. Bullock £ G. A. Horridge, eds. W. H. Freeman and Co., San Francisco.
Bliss, D. E. £ J. H. Welsh. 1952. The neurosecretory system of brachyuran crustacea. Biol. Bull. 103: 157-169.
Brewer, R. H. 196 4. The phenology of Diaptomus stagnalis (Copepoda: Calanoida): The development and the hatching of the egg stage. Ph.D. Thesis, University of Chicago. Published, Physiological Zoology, 37: 1-20.
Brown, F. A., Jr., and 0. Cuniingham. 1939. Influence of the sinus gland of crustacea on normal viability and ecdysis. Biol. Bull. 77: 104-114.
Brown, F. A. , Jr. 5 M. I. Sandeen. 1948. Responses of the chromatophores of the fiddler crab, Uca, to light and temperature. Physiol. Zool. 21: 361.
Carlisle, D. B. 19 59. On the sexual biology of Pandalus borealis (crustacea:decapoda). I. Histology of incretory elements. J. Mar. Biol. Assn. U. K., 38: 381-394.
Carlisle, D. G. 1960. Softening chitin for histology. Nature, 187: 1132-1133.
Carlisle, D. B. £ Francis G. W. Knowles. 1959. Endocrine control in crustacea. Cambridge Monographs in Exp. Biol. Cambridge Univ. Press.
85 86
Carlisle, D. B. S W. J. Pitman. 1961. Diapause, neuro secretion and hormones in copepoda. Nature, 190: 827-828.
Carlson, S. P. 19 35. The color changes in Uca Pugilator Proc. Nat. Acad. Sci., Wash., 21: 549.
Carlson, S. P. 19 36. Colour changes in Brachyura crus taceans, especially in Uca pugilator. Kungl. fysiogr. Sallsk. Lund Forhandl., 6: 6 3-80.
Cole, G. A. 1953. Notes on copepod encystment, Ecology, 34: 208-211.
Dahl, Erik. 1953. Frontal organs in free-living copepods. K. Fysiogr. Sallsk. Forh., 23: 1-7.
Dahl, Erik. 19 57. Embryology of X-organs in Crangon allmanni. Nature. 179: 482.
Dahl, Erik. 1959. The ontogeny and comparative anatomy of some protocerebral sense organs in nostracan phyllopods. Z. FI. microsc. Sci., 100: 445-462.
Dahl, Erik. 196 3. Main evolutionary lines among recent crustacea. In: Phylogeny and Evolution of Crustacea. Museum of Comparative Zool., Special Publ., H. B. Whittington 6 W. D. I. Rolfe, eds.
Echalier, G. 19 54. Recherches experimentales sur le role de *1’ organe Y' dans la mue de Carcinus moenas (L.). Crustace decapode. C. R. Acad~ Sci. , Paris, 238: 523-525.
Echalier, G. 1955. Role de 1'organe Y' dans la determinisme de la mue de Carcinides (Carcinus) moenas L. (Crustaces decapodes). Experiences d 1 implantation. C. R. Acad. Sci., Paris, 240: 1581-1583.
Elgmork, K. 1959. Seasonal occurrence of Cyclops strenuus strenuus. Folia Limnol. Scand. 11: 1-196.
Elofsson, R. 196 3. The nauplius eye and frontal organs in Decapoda (Crustacea). Sarsia, 12: 1-^68.
Elofsson, R. 1965. The nauplius eye and frontal organs in Malacostraca (Crustacea). Sarsia, 19: 1-54. 87
Elofsson, R. 1966a. The nauplius eye and frontal organs of the non-Malacostraca (Crustacea). Sarsia, 25: 1-128
Elofsson, R. 1966b. Notes on the development of the nauplius eye and frontal organs of decapod crusta ceans. Acta Univ. Lund. N. S. Nr, 27: 1-2 3.
Elofsson, R. 1966c. Some aspects of the fine structure of the nauplius eye of Pandalus borealis (Crustacea: Decapoda). Acta Univ. Lund! N .S. Nr, 28: 1-16.
Enami, Masashi. 1951a. The sources and activities of two chromatophorotropic hormones in crabs of the genus Sesarma. I. Experimental analysis. Biol. Bull., 100: 28-43.
Enami, Masashi. 19 51b. The sources and activities of two chromatophorotropic hormones in crabs of the genus Sesarma. II. Histology of the incretory elements. Biol. Bull., 101: 241-258.
Fahrenbach, W. H. 1962. The biology of a harpacticoid copepod. Ph.D. Thesis, Univ. of Washington. Published, Cellule, 62: 303-376.
Fahrenbach, W. H. 1964. The fine structure of a nauplius eye. Z. Zellforsch., 62: 182-197.
Fingerman, M. 196 3. The Control of Chromatophores. The Macmillan Co., New York.
Gabe, M. 195 3. Quelques acquisitions recentes sur les glandes endocrines des Arthropodes. Experientia, 9: 352-356.
Gersch, M. 19 57. Wesen und Wirkungsweise von Neuro- hormonen in Tierreich. Naturwissenschaften, 44: 525-532.
Gomori, G. 19 39. Studies on the cells of the pancreatic islets. Anat. Rec., 74: 439-460.
Gomori, G. 1941. Observations with differential stains on human islets of langerhans. Amer. J. Path., 17: 395-406.
Gomori, G. 1946. Staining of chromaffin tissue. Amer. J. Clin. Path. (Tech. Suppl.), 10: 115-117. 88
Gomori', G. 1950. Aldehyde fuchsin: a new stain for elastic tissue. Amer. J. Clin. Path., 20: 665-666.
Gorbman, A. S H. A. Bern. 1962. A textbook of compara tive endocrinology. John Wiley and Sons, Inc., New York.
Guyer, M. F. 19 34. Delafield's hematoxylin. Micrology III. U. Chicago Press, Chicago, 111.
Guyer, M. F. 195 3. Animal Micrology. U. Chicago Press, Chicago.
Hanstrom, B. 19 31. Neue Untersuchungen uber Sinnesorgane und Nervensystem der Crustaceen. I. Zeitschr. F. Morph, u. Okol. d. Tiere., 23.
Hanstrom, B. 19 33. Neue Untersuchungen uber Sinnesorgane und Nervensystem de Crustacean. II. Zool. Jb., Abt. Anat. Ontog. Tiere, 56: 387-520.
Hanstrom, B. 19 34. Neue Untersuchungen uber Sinnesorgane und Nervensystem de Crustacean. III. Zool. Jb., Abt. Anat. Ontog. Tiere, 58: 101-144.
Hanstrom, B. 19 39. Hormones in Invertebrates. Oxford.
Havener, W. H. 1960. Clinical experiences with the photocoagulator. Am. J. Ophth., 49: 1213.
Havener, W. H. 1964. Technical Aspects of Laser Coagu lation. Am. J. Ophth., 58: 38-41.
Hogben, L. T. S D. Slome. 19 31. The pigmentary effector system. VI. The dual character of endocrine coordination in amphibian color change. Proc. Roy. Soc. (London), Proc., B., 10 8: 10.
Horridge, G. A. 196 5. Arthropoda: Receptors for light .and optic lobe. In: Structure and Function in the Nervous Systems of Invertebrates. Vol. II. W. H. Freeman and Co., San Francisco.
Hubschman, Jerry H. 1962. A simplified azari process well suited for crustacean tissue. Stain Tech., 37: 379-380. 89
Hubschman, Jerry H. 196 3. The development and function of neurosecretory sites in the eyestalks of larval Palaemonetes (Decapoda:Natantia). Biol. Bull., 1251 96-113.
Humason, G. L. 1962. Animal Tissue Techniques. W. H. Freeman and Co., San Francisco.
Kleinholz, L. H. 8 E. Bourquin. 1941. Effects of eye stalk removal on decapod crustaceans. Proc. Nat. Acad. Sci. (Washington), 27: 145-149.
Knowles, F. G. W. 1956. Some problems in the study of colour change in crustaceans. Ann. Sci. Nat. (Zool.), 18: 315-324.
Knowles, F. G. W. 8 D. B. Carlisle. 19 56. Endocrine control in the crustacea. Biol. Reviews, 31: 396-473.
Koller, G. 1925. Farbwechsel bei Crangon vulgaris. Verh. dtsch. Zool. Ges., 30: 128.
Koller, G. 19 27. Uber chromatophorensystem, Farbensinn und Farbwechsel bei Crangon vulgaris. Z. vergl. Physiol. 5: 191.
Koller, G. 19 30. Weitere Untersuchungen uber Farbwechsel und Farbwechselhormone bei Crangon vulgaris. Z. vergl. Physiol. 12: 632-667.
Kroyer, H. 1842. Monographisk fremstilling af slaegten Hippolytes nordiske arter. K. danske vidensk. Selsk. Skr., 9: 209-361.
Lang, K. R., F. S. Barnes, J. C. Daniel, 8 J. C. Maisel. 1964. Lasers as tools for embryology and cytology. Nature, 201: 675-677.
Lebour, M. V. 1916. Stages in the life history of Calanus finmarchicus (Gunnerus), Experimentally reared by Mr. L. R. Grawshay in the Plymouth Laboratory. F. Mar. Biol. Ass. U. K . , 11: 1-17.
Lee, J. 8 F. G. W. Knowles. 196 5. Animal Hormones. H. M. Fox., ed., Hutchinson Univ. Library, London. 90
Lowes E. 1935. On the anatomy of a marine copepod, Calanus finmarchicus (Gunner). Trans. Roy. Soc. Edinburgh, 58: 561-603.
Marshall, S. M. 8 A. P. Orr. 19.55. The biology of a marine copepod Calanus finmarchicus (Gunnerus). Oliver 8 Boyd Ltd., Edinburgh.
Matsumoto, Kunio. 19 58. Morphological studies on the neurosecretion in crabs. Biol. J. of Okayama Univ. (Japan), 4: 103-176.
Meglitsch, P. A. 1967. Invertebrate Zoology. Oxford Univ. Press, New York.
Meyer-Schwickerath, G. 1960. The historical and experi mental background of radiation damage to the retina. C. V. Mosby Co., St. Louis.
Novak, V. J. A. 1966. Insect Hormones. Methuen and Co., Ltd., London.
Park, T. S. 1966. The biology of a calanoid copepod Epilabidocera amphitrites McMurrich. Cellule 66(2): 129-251.
Passano, L. M. 1960. Molting and its control. In: The Physiology of Crustacea. Vol. I. T. H. Waterman, ed., Academic Press, New York.
Pennak, R. W. 195 3. Freshwater Invertebrates of the •U.S., Ronald Press Co., New York.
Perkins, E. B. 1928. Colour changes in crustaceans, especially in Palaemonetes. J. Exp. Zool., 50: 71-195.
Pouchet, G. 1876. Les changement de coloration sous 1'influence des nerfs. J. d'Anat. et Physiol. Paris, 12: 1-90, 113-165.
Russell, F. S. 1928. The vertical distribution of marine macroplankton. VII: Observations on the behavior of Calanus finmarchicus. F. Mar. Biol. Ass. U. K. , 15: 429-454.
Scharrer, E. 8 B. Scharrer. 1954. Hormones produced by neurosecretory cells. Recent Progr. Hormone Res., 10: 183-240. • 9 1
Turner, C. D. 1966. General endocrinology. W. B. Saunders Co., Philadelphia.
Vaissiere, R. 1961. Morphologie et histologie comparees des yeux des crustaces copepodes. Arch. Zool. exp. et gen. 100: 1-125.
Welsh, J. H. 1961. Neurohumors and neurosecretion. The Physiology of Crustaceans, T. H. Waterman, ed., Vol. II., Acad. Press, New York.
Welsh, J. H. S R. I. Smith. 196 0. Laboratory exercises in invertebrate physiology. Burgess Publ. Co., Minneapolis.
Witt, P. N., C. F. Reed, S F. K. Tittel. 1964. Laser lesions and spider web construction. Nature, 201: 150-152. AUTOBIOGRAPHY
I, W. J. Pitman, was born in Calloway County,
Kentucky, August 16, 1924. I received my secondary school education in public schools of Murray, Kentucky, and my undergraduate training at Murray State College, which granted me the Bachelor of Science Degree in
1949 .
From the Ohio State University, I received the
Master of Science Degree in 1951. I taught at Mary
Washington College for 8 years, during seven summers.
I attended Mountain Lake Biology Station of the
University of Virginia and subsequently received a
National Science Foundation Science Faculty Fellowship
for 15 months. This grant enabled me to complete
language__course and resident requirements for the Doctor
of Philosophy Degree.
I have been an Assistant Professor of Biology at
Murray State University, Murray, Kentucky, since 1961.
92