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CHARACTERIZATION OF AN AGGREGATION PHER0M0NE AND ITS SITE OF PRODUCTION IN TRIBOLIUM CASTANEUM (HERBST) (COLEOPTERA: TENEBRIONIDAE) WITH COMPARATIVE NOTES ON ANALOGOUS STRUCTURES IN OTHER COLEOPTERA FAMILIES.
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University M icrd fiim s intemanonai Characterization of an Aggregation Pheromone and its
Site of Production in Tvibol-ium oastaneum (Herbst)
(Coleoptera: Tenebrionidae) with Comparative Notes on
Analogous Structures in Other Coleoptera Families.
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
D. L. Faustini, A.A., B.A., M.S.
* * *
The Ohio State University
1980
Reading Committee: Approved By
C. A. Triplehorn
M. E. Clay Adviser D. L. Denlinger Department of Entomology DEDICATION
I wish to dedicate this work in honor of ray parents John Mario and Evelyn Elizabeth Faustini and to my sisters, Alyce Dorner, Madonna Battaglia and Elayne Miller.
ii ACKNOWLEDGEMENTS
I wish to thank my major professor, Charles A. Triplehorn, for his assistance and support in this work, and for his warm
friendship. I would also like to thank Dr. James E. Sargent
who generously offered valuable advice and assistance with
problems relating to this study. I am grateful to Dr. A. C.
Waldron whose laboratory most of this work was performed.
Special thanks are due to David W. Stutes and Dr. Thomas
N. Taylor, Scanning Electron Microscopy Laboratory, Columbus,
Ohio for facilities and technical assistance which allowed for the SEM micrographs in this study.
Biological assays with the flour beetle were made possible by a travel grant from the Ohio State University
Graduate School and courtesy of the Stored Products and
Household Insects Laboratory, USDA, Department of Entomology,
University of Wisconsin, Madison. I particularly wish to thank Dr. Wendell E. Burkholder, professor of Entomology at the above laboratory, for helping to arrange the visit, and whose laboratory the work was done.
Finally, I am most grateful to my loving family, my assistants, Erik and Heidi and dearest wife Vicki for their many personal sacrifices during this study.
iii VITA
February 22, 1950 . . Born - Cleveland, Ohio
1973...... A.A., Mount San Antonio College, Walnut, California
197^...... B.A., California State College, San Bernardino, California
197^-1977 ...... Research Assistant, Stored Grain Laboratory, California State College, San Bernardino, California
1976...... M.Sc., California State College, San Bernardino, California
1979...... Teaching Associate, Department of Entomology, The Ohio State Univer sity, Columbus, Ohio
1979-1980 ...... Research Assistant, Insect Physiology Laboratory, The Ohio State University, Columbus, Ohio
PUBLICATIONS
Faustini, D. L. (1976). The effect of sex and irradiation on crossing-over in Tribolium castaneum (Herbst). Tribolium Inform. Bulletin 19:88-89.
Sokoloff, A., D. L. Faustini, M. A. Sokoloff and E. A. Sokoloff (1977). Observations of a natural population of Tribolium brevicornis LeConte. Tribolium Inform. Bulletin 20:135-138.
Faustini, D. L. (1980). The ultrastructure of a male sexual dimorphic character in Tribolium castaneum (Herbst). Tribolium Inform. Bulletin {in -press).
iv PROFESSIONAL PAPERS
The effect of sex and irradiation on crossing-over in Tribolium castaneum. Biological Sciences Undergraduate Research Conference, Santa Clara, California. 1977-
The morphology of a sexual dimorphic character in Tribolium oastaneum (Herbst) (Coleoptera: Tenebrionidae). Annual Meeting of the Entomological Society of America, Denver, Colorado. 1979.
AWARDS
Graduate Student Alumni Research Award, The Ohio State University, 1979-
FIELDS OF STUDY
Major Field: Functional Morphology and Behavior of Stored Grain Insects
Studies in morphology: Professor Charles A. Triplehorn
Studies in pheromone behavior: Professor Wendell E. Burkholder.
v TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
VITA iv
LIST OF TABLES viii
LIST OF FIGURES x
LIST OF PLATES xi
INTRODUCTION ...... 1
GENERAL LITERATURE REVIEW...... 5
Tribolium spp...... 5 Pheromones...... 8
GENERAL EXPERIMENTAL PROCEDURES...... 12
Insect Rearing...... 12 Handling Pupae...... 15 Handling Adults ...... 16
IDENTIFICATION OF THE EXOCRINE GLAND ...... 17
Introduction ...... 17 Morphology of the Male Setiferous Puncture . . . 23 Scanning Electron Microscopy ...... 23 Solubility Properties of the Globular Secretion ...... 23 Ultrastructure of the Setiferous Puncture . 26 Secretion Accumulation with Beetle Age . . 28 Histology...... 28 Discussion...... 42
LABORATORY BIOASSAY AND CHEMICAL ANALYSES ...... 47
Introduction...... 47 Materials and Methods ...... 49
vi TABLE OF CONTENTS (CONTINUED)
Page
Handling of Insects...... 49 Olfactometer and Laboratory Bioassay Conditions...... 50 Multiple-Choice Olfactometer ...... 50 Isolation of the Male Setiferous Secretion...... 52 Chemical Analyses...... 52 Gas Chromatography - packed column. . 5 3 Gas Chromatography - glass capillary column...... 53 Experimental Results...... 5^ Female and Male Behavioral Response .... 5^ Multiple-Choice Olfactometer Bioassay . . . 5^ Female Response ...... 5^ Male Respo n s e ...... 59 Analysis of the Secretion...... 76 Discussion...... 8l
ANALOGOUS SEXUAL DIMORPHIC MALE SETIFEROUS STRUCTURES 86
Introduction...... 86 Materials and Methods ...... 87 Scanning Electron Microscopy ...... 87 I n s e c t s ...... 88 Experimental Results...... 88 Tenebrionoidea ...... 88 Staphylinoidea...... 101 Bostrichoidea...... 101 Dermestoidea ...... 108 Cleroidea...... 108 Curculionoidea ...... 108 Scarabaeoidea...... 119 Discussion...... 128
SUMMARY...... 131
REFERENCES...... 13^
APPENDICES ...... 147
Light Microscopy Solutions...... 147
vii LIST OF TABLES
Table Page
1 Solubility properties of the male prothoracie leg'secretion ...... 27
2 Light microscopy processing procedure ...... 33
3 Multiple-choice olfactometer female response to whole-wheat flour...... 55
4 Multiple-choice olfactometer 12-hour post- eclosion female response to male globules . . 57
5 Multiple-choice olfactometer 30-day-old female response to 50 male globules...... 58
6 Multiple-choice olfactometer 12-hour post- eclosion female response to 150-day-old males with large globules...... 60
7 Multiple-choice olfactometer 1-day-old female response to 150-day-old males with large globules...... 6l
8 Multiple-choice olfactometer 30-day-old female response to 150-day-old males with large globules...... 62
9 Multiple-choice olfactometer 30-day-old female response to 90-day-old males without globule s*. ••••••■•••»•••••• 63
10 Multiple-choice olfactometer 30-day-old female response to 150-day-old and 90-day- old m a l e s ...... 64
11 Multiple-choice olfactometer male response to whose-wheat flour...... 65
12 Multiple-choice olfactometer 12-hour post- eclosion male response to 50 male globules• • 87
viii LIST OP TABLES (CONTINUED)
Table Page
13 Multiple-choice olfactometer 30-day-old male response to 50 male globules ...... 68
14 Multiple-choice olfactometer 12-hour post- eclosion male response to. 150-day-old males with large g l o b u l e s ...... 69
15 Multiple-choice olfactometer 1-day-old male response to 150-day-old males with large globules...... 70
16 Multiple-choice olfactometer 30-day-old male response to 150-day-old males with large globules...... 71
17 Multiple-choice olfactometer 30-day-old male response to 90-day-old males without globules...... 72
18 Multiple-choice olfactometer 20-day-old male response to 150-day-old and 90-day- old m a l e s ...... 73
19 Sexual dimorphic setiferous puncture characters in the Coleoptera...... 124
20 Light microscope solutions...... 1^7
ix LIST OF FIGURES
Figure Page
1 Diagrammatic interpretation of the prothoracic femur setiferous puncture on Tribolium castaneum males ...... 39
2 Histogram of x beetle response to odors from the male setiferous secretion...... 75
3 Packed column gas chromatogram of male pro- thoracic setiferous puncture secretion. .. . 78
4 Glass capillary column gas chromatogram of the male prothoracic setiferous puncture secretion...... 78
5 Glass capillary column gas chromatogram of the male secretion with high attenuation. . . 80
x LIST OP PLATES
Plate Page
I Stereoscope Micrographs of Tribolium castaneum ...... 20
II Stereoscope of Male T. castaneum. . . . 22
III Scanning Electron Micrographs of Male Prothoracic Leg Setiferous Puncture Secretion...... 25
IV SEM Micrographs of the Male Setiferous Puncture Ultrastructure ...... 30
V SEM Micrographs of the Male Setiferous Puncture in Beetles of Various Ages . . 32
VI Light Microscope Micrographs Showing Histological Sections of the Prothoracic Femora Setiferous Puncture of Tribolium castaneum Males ...... 36
VII LM Micrograph Showing Histological Section of T. castaneum Male Setiferous Puncture ...... 41
VIII SEM Micrographs of the Male Femoral Setiferous Punctures in T. confusum . 90
IX SEM Micrographs of Setiferous Sexual Dimorphic Characters in Tenebrionidae 93
X SEM Micrographs of Sexual Dimorphic Setiferous Characters in Tenebrionidae 95
XI SEM Micrographs of Sexual Dimorphic Setiferous Characters in Tenebrionidae 97
XII SEM Micrographs of Female Bolitotherus cornutus Lacking Setiferous Punctures 100
xi Plate Page
XIII SEM Micrographs of Sexual Dimorphic Setiferous Characters in Staphylinidae. . . 103
XIV SEM Micrographs of Sexual Dimorphic Setiferous Characters in Staphylinidae. . . 105
XV SEM Micrographs of Sexual Dimorphic Setiferous Characters in Ptinidae and Leiodidae...... 107
XVI SEM Micrographs of Sexual Dimorphic Setiferous Characters in Dermestidae. . . . 110
XVII SEM Micrographs of Sexual Dimorphic Setiferous Characters in Cleridae ...... 112
XVIII SEM Micrographs of Sexual Dimorphic Setiferous Characters in Cleridae...... 114
XIX SEM Micrographs of Sexual Dimorphic Setiferous Characters in Cleridae ..... 116
XX SEM Micrographs of Sexual Dimorphic Setiferous Characters in Anthribidae. . . . 118
XXI SEM Micrographs of Sexual Dimorphic Setiferous Characters in Anthribidae. . , . 121
XXII SEM Micrographs of Prothoracic Leg Setiferous Structures in Lamellicorns . . , 123
xii INTRODUCTION
Frequently, within a single population or between con- specific populations morphological differences occur between individuals (Mayr, 1976). Where only one sex exhibits a unique characteristic, the individual is considered sexually dimorphic (Darwin, 1871). Primary sexual characteristics are associated with copulatory organs, whereas, secondary sexual characteristics are traits not directly related to reproduction. These characters possessed by one sex (e.g.3 horns on the male Lamellicorns) play a prominent role in the sexual selective process to maximize the propagation of the species through more efficient reproduction. Generally, in animals where there is no parental care of offspring, a secondary sexual character plays a significant part in mating behavior (Ward and Humphries, 1977; Otte, 1979).
For years, taxonomists and geneticists have used sexual dimorphic characteristics as tools for distinguishing sexes.
Little emphasis was placed upon the function of these morphological structures. Halstead (1963) in a review of the secondary sexual characteristics of stored product beetles, gives the abbreviation of elytral striae and a femoral setiferous puncture as differences for distinguishin between male and female red flour beetles, Tribolium castaneum (Herbst). The sub-basal setiferous puncture is an obscure structure on the male prothoracic femora. This structure, described by Hinton (19^2), has been beneficial to aid in distinguishing the sex of the flour beetle.
Sokoloff (1972) observed that the femoral pit in males is encrusted with flour in old beetles and suggested that this accretion could result from a beetle secretion.
Presently in the literature, no studies have been recorded on sexual dimorphic pheromone production sites.
In the past decade, studies of insect pheromones have dis closed that these natural chemical products play a signifi cant role in the life cycle of many insects. Complex be havioral patterns observed in the presence cf trace amounts of pheromone demonstrate the potency of these compounds.
In recent years, the concept of biological control through sophisticated microanalytical methods has led to a current interest in insect pheromones as tools for manipulating populations (Burkholder, 1976).
The role of sex pheromones in mating behavior has been reported for hundreds of insect species. Pheromones may be produced by either females or males and typically release a sterotyped sequence of behaviors in the receiving sex. Because sex and aggregation pheromones often appear to play an essential function in mating activities and population recruitants, numerous studies on the chemical
identity and biological function have been conducted with the goal of expediting development of safe and effective population survey and control programs (Burkholder, 1970).
Aggregation pheromones function in the drawing of
individuals within a population together. A pheromone may be secreted by either sex and serve as an attractant for both males and females. Coupled with sex pheromones, these two distinct chemical communicators function as a complex system uniting the ecological, environmental, and physio logical factors of the insect.
Little information is presently available concerning the morphology of pheromone glands among the Coleoptera.
However, glands have been observed within the abdominal tissues' in a number of species: the June beetle, Phyllophaga lanoeolata (Say) (Travis, 1939); the banded cucumber beetle,
Ddabvot-iea ba.ltea.ta LeConte (Cuthbert and Reid, 1964); the
Douglas-fir beetle, Dendroetonus pseudotsugae Hopkins
(Zether-Moller and Rudinsky, 1967); Trypodendron lineatum
(Olivier) (Schneider and Rudinsky, 1969); the Khapra beetle,
Trogoderma granarium Everts (Stanic et al., 1970); Trogoderma sp. (Hammack et al.3 1976); and the cigarette beetle,
Lasioderma serrieorne (P.)'(Coffelt, 1971)* The lack of specific information on pheromone glands is partially attri buted to the whole body, and/or mixed sex extraction methods conducted by biochemists during the pheromone isolation process. Generally, large numbers of insects are homogenized and extracted with an organic solvent to remove the biolog ically active compounds with little concern for the localiza tion of the pheromone glands.
The purpose of this study was: (1) to describe the ultrastructure of the sub-basal setiferous puncture on the male prothoracic femora in Tribolium castaneum (Herbst);
(2) to investigate the function of this obscure structure, with particular emphasis on the relationship to pheromone production; and (3) to compare analogous male sexually dimorphic setiferous characters in several families of the
Coleoptera. GENERAL LITERATURE REVIEW
Tribolium spp.
The red-rust flour beetle, Tribolium castaneum (Herbst), is a well-known cereal and stored product pest of cosmopoli tan distribution. This insect can survive on raw materials of both animal and plant origin, and the ease with which it can be cultured in the laboratory makes it the most thoroughly studied of all stored-product insect pests. The earliest evidence of the human-flour beetle association is from a specimen contained within an urn in a pharaonic tomb of the sixth Dynasty {ca. 2500 B.C.) (Andres, 1931).
Presently, the most comprehensive treatise of this genus was compiled in Sokoloff's monograph series, The
Biology of the Tribolium (Sokoloff, 1972, 197^, 1977). This review of Tribolium biology includes topics of evolution, morphology, behavior, ecology, geographical distribution, and genetics. Larval, pupal, and adult systematic keys are also presented.
Several species of this genus have become synanthropic, two of these, T. castaneum and T. confusum DuVal are the primary species found in the United States. Good (1936) observed that T. castaneum is generally an insect of warm climates, rarely found north of the fortieth parallel (in the eastern U.S.) except in heated buildings. T. confusum is more frequently found in the northern states. Both species thrive when in close association with stored commodities, flour mills, or milling machinery. Their presence in stored products and cereals results in sub stantial economic losses due to a decrease in nutrition
(Eden, 1967). Because of this insects' economic importance numerous research programs have been designed to generate more suitable control measures. The measures include the use of carbon dioxide (Ali Niazee, 1971), low oxygen atmospheres (Storey, 1977), synthetic growth regulators
(Loschiavo, 1975, 1976*, Williams and Amox, 197^; Kramer and McGregor, 1977), and grain protectants (LaHue, 1975;
LaHue and Dicke, 1976; Watters, 1977; Salameh et al.3 (1977) as potential control agents.
Hinton (19^8) listed six species of Tribolium for
North America, three of which are considered indigenous.
T. audax (often incorrectly cited as T. madens (Charp.),
T. parallelum (Casey), T. brevicornis Le Conte are indi genous species, while T. castaneum3 T. confusum and T. madens were thought to be introduced by commerce. T. confusum3
T. castaneum and T. madens originated in Africa, India and
Europe, respectively (Hinton, 19^8). It should be noted that in Linsley's 19^4 paper, T. audax was referred to as T. madens, the latter recognized as a new closely related species, T. audax (Halstead, 1969). Triplehorn (1978) recently described T. setosum Triplehorn, a new species indigenous to North America. Additional information and references regarding the distribution of Tribolium species within North America were given by Chittenden (1895, 1911),
Pall (1901), Blatchley (1910), Essig (1926), Sinha (1963b,
1965b), Sokoloff (197*1), Okumura and Strong (1965) and
Strong (1970). Most of these papers deal with infestations occurring in stored commodities, particularly grains and cereals. Additionally, T. castaneum has been reported in association with several other stored products including whole grains (Birch, 19*0), dried fruits, spices, nuts, milk and chocolate (USDA Manual 500), forest products {i.e., ash, pine, slippery elm), and preserved museum specimens, both insect and herbaria (Good, 1936; Sokoloff, 197*+) -
Although many species of Tribolium are synanthropic, considerable information has been documented on the natural habitats of this insect. T. castaneum has frequently been found under the bark of trees throughout the world: Great
Britian (Stephans, 1839j Fowler, 1891), France (Lepesme,
19*1*0, Algiers (Lucas, 18*19), United States (Fall, 1901),
India (Blair, 1930), Orient and Indo-Malaysian areas (Good,
1936; Lepesme, 19*1*1 \ Linsley, 19*1*1).
In more unusual natural conditions, Stebbing (191*1) reported T. castaneum as a semi-predator of the bostrichid, Dinodevus minutus. Trehan and Rajarao (1945) observed
T. castaneum feeding upon eggs of the moth Covcyra eei
lonioa in the field. Bird nests (Ansari, 1951) and mammal
nests have served as reservoirs for T. castaneum. Kosolapova
(1965) cited both the Siberian marmot and domestic mouse
as harborors of this insect.
Aside from the general features shared by most insects,
the success of T. castaneum as a stored grain pest has been
characterized by several factors: the ability to sustain
adverse environmental conditions (extreme moisture fluctu
ations), variety of food commodities, lack of diapause,
high resistance to starvation, small body size and two to
three year adult survivorship (Linsley, 1944).
Pheromones
Karlson and Butenandt (1959) offered the term pheromone
to describe externally released chemical substances that
initiate communicative signals between members of the same
species. Releaser pheromones stimulate immediate behavioral responses, while primer pheromones, somewhat slower in response, cause alterations in the physiological processes
in receiving individuals (Wilson, 1963). Both aggregation and sex pheromones are classified as releaser pheromones in that either a spontaneous attraction of individuals to the releaser ( e . g . odor source) or a sexual behavioral response is generated. The presence of a sex pheromone
can trigger a variety of sexual behavior characteristic" in
the receiver. Tschinkel et at. (1967) observed male
Tenebrio molitor L. attempting to copulate with an inanimate
object treated with a sex pheromone.
In recent years, the subject of insect pheromones has
opened a new field of chemical ecology focusing on the
biological function of these natural substances. Part of
this interest lies in the potential use of these natural
products to manipulate and regulate pestiferous insect
populations (Beroza, 1970; Jacobson, 1972; Roelofs, 1975,
1978; Shorey, 1976, Kondratev, 1978). The use of phero
mones as lures to control or eradicate insect pests has
met with considerable success. Roelofs (1975) was able
to control leafroller moths in a field-trapping study.
Shorey (1976), through the use of a synthetic sex attrac-
tant, was able to control the pink bollworm, Peatinophora
gossypiella (Saunders), in cotton and cabbage fields.
Similarly, the oriental fruit fly, Daous dorsalis Hendel,
was eradicated during a field-trapping program (Jacobson,
1972). Vick et at. (1978) reported the successful
disruption of mating in the Angoumois grain moth, Sitotroga
aerealella (Olivier), through the use of a synthetic sex
attractant.
There are two primary factors that are essential in using pheromones for pest control: (1) mass culturing of 10
Insect populations under laboratory conditions and (2)
sophisticated development of microanalytical equipment re
enable the extraction, isolation, characterization, and
synthesis of trace amounts {e.g., nanogram levels) of
active chemical compounds in insects (Capella and Zorzut,
1968; Beroza, 1975; Beroza and Bierl, 1967, 1976; Byrn et
al.3 1975, Hendry et al.3 1975; Tumlinson and Heath, 1976).
Although much of our pheromone knowledge is based on
studies with Lepidoptera, the Coleoptera are becoming an
increasingly important source of information on chemical
communication. Inscoe and Beroza (1976) listed twenty-four
beetle species in four families where the active pheromone
properties have been characterized. More recently, Kuwahara
et al. (1978) identified the sex pheromone of the drugstore
beetle, Stegobium panieeum (L.). Greenblatt et at. (1976)
reported a candidate sex pheromone for Trogoderma glabrum
(Herbst) as (E)-l4-methyl-8-hexadecen-l-ol. The sex
pheromone of another Dermestid, Attagenus elongatus (Casey)
was identified as Z,Z-3,5-tetradecadienoic acid (Pukui
et al. 3 1977).
Keville and Kannowski (1975) identified 1-pentadecene, n-hexadecane, 1-heptadecene, and heptadecadiene, as phero mones secreted by both sexes of T. confusum that are attractive only to male Tribolium confusum Duval. Later,
Ryan and 0 ’Ceallachain (1976, 1977) found: (1) a male secreted aggregation pheromone attractive to both sexes; 11 and (2) a female secreted sex pheromone attractive to male
T. oonfusum. Suzuki et al. (1975) while working with
T. oonfusum and T. oastaneum identified seven unsaturated hydrocarbons: 1-pentadecene; 1-heptadecene; 1,8-heptadeca- diene; 1-tetradecene; 1-hexadecene; 1,6-pentadecadiene; and heptadecatriene . These substances have repellent qualities which act as alarm pheromones. Suzuki and
Sugawara (1979) isolated a structurally unknown aggregation pheromone from mixed species of both sexes.
Jacobson (1965) compiled an extensive review on the subject of insect pheromones. To date, numerous articles have appeared in the literature which focus upon the topic of insect attractants Wilson (1963), Wilson and Bossert
(1963)j Jacobson and Beroza (1964, 1970), -Jacobson (1965,
1966, 1972, 1974), Butler (1967), Shorey and Gaston (1967),
Shorey et al, (1968), Regnier and Law (1968), Blum (1969),
Wood et al, (1970), Law and Regnier (1971), Birch (1974),
Roelofs (1975, 1978)3 Burkholder (1976), Hendry (1976),
Shorey (1976), Shorey and McKelvey (1977), Matthews and
Matthews (1978) and Payne (1979). GENERAL EXPERIMENTAL PROCEDURES
Insect Rearing
Tv-Lbol-Lum species used for this study were obtained from stock cultures maintained at the Tribolium Stock Center,
California State College, San Bernardino, California. The beetles were reared in %-pint glass jars containing approx imately 20 g of a mixture of whole-wheat flour (Shapphire brand) and brewers' yeast powder in a 19:1 ratio. Before introducing the insects, medium ingredients were sifted through 30 mesh screen (U.S. Standard sieve series) to facilitate later insect removal. Insects were maintained at 28 ± 2°C and 60 ± 5% relative humidity with a 16:8 light:dark photocycle regime; lights-on occurred at 6:00
A.M. EST.
Initial stock insects were selected and handled by one of three procedures:
1. For the experiments designed to generate
the waxy pheromone secretion on the prothoracic
femur in T. oastaneum, cultures were started with
approximately 50 newly emerged adult males. These
were placed directly on medium and left undisturbed
for approximately 150 days.
12 2. For subsequent experiments conducted with
T. castaneum} test insects were obtained from cultures
started with eggs. Groups of 75, 5- to 10-day old virgin females were placed with 75, 5- to 10-day old virgin males. The jars were sealed with Parafilm to prevent escape of the insects. A maximum of three days was allowed for mating and oviposition, and the parent stock was removed after that time and trans ferred to fresh flour. This was performed primarily to limit egg and larval cannibalism by the adults
(Chapman, 1928; Park, Mertz, Grodzinski, and Prus,
1965). The sifted medium containing eggs was trans ferred to preconditioned medium in a %-pint container.
Larval development was completed in approximately
25 days. At the onset of pupation, the contents of a jar were sifted through a series of three screens.
A l6-mesh screen on the top retained most of the pupae while allowing passage of most larvae and rear ing medium. A 20-mesh screen in the middle retained the largest larvae. The smallest larvae were collected on a 30-mesh screen at the bottom. Frass and medium particles passed through all three screens and were collected in a pan below the 30-mesh screen.
Extraneous material (large wheat particles, exuviae, and frass) was separated from the insects, and the papae were sexed using the genital lobe characteristics described by Ho (1969). Although this procedure proved highly reliable, sex was later veri fied in teneral or 1- to 2-day old adults using the sexual dimorphic setiferous puncture on the male pro- thoracic femora (Hinton, 19^2). If an error was sub sequently detected, female pupae and all adults held with the mis-sexed individual were discarded. The practice of segregating the sexes during the pupal stadium not only minimized the possibility of females becoming contaminated with male pheromone, but, also assured the accuracy of age groups for experiments utilizing virgin adults. Each culture yielded approximately 500 pupae. After removal of pupae from the culture jars, the remaining larvae were returned to their respective containers with pre-conditioned medium and placed back into the environmental rearing incubator. After five to seven days, the above pro cedure was repeated for each jar and a second group of pupae was obtained. This routine was repeated, as needed, for each jar until most of the individuals had been collected. When the majority of the insects had been removed, the culture was either destroyed by autoclaving or the remaining insects were used as breeding stock.
3. For general colony maintenance, surplus
10-15 day old adults were placed in pint containers in groups of 100 each, males and females. Live adults
were removed after 90 days. The dead were remove:: by
aspiration and progeny were deposited into separate
containers. For all procedures it was often necessary
to anesthetize the adults in a funnel etherizer to
facilitate handling (Sokoloff, I960). This not only
limited movement but allowed accurate identification
of the sexual dimorphic characteristic on the pro-
thoracic leg.
Handling of Pupae
Isolated groups of male or female pupae, usually in groups of 25-30, were placed in clean 3 dram vials. A
12.7-mm filter paper disc was attached to the top of the vial and secured in place with Parafilm® to prevent the insects from escaping. Small holes in the top of the filter paper provided adequate ventilation. When holes were not provided the flour turned M pink" due to ethyl- quinone contamination (Lloyd, 1961).
Female pupae were returned to a separate rearing incubator and males were transferred to a separate room and placed in a female-free incubator. Rearing conditions for both sexes were identical. 16
Handling of Adults
Newly-emerged T. aastaneum male adults were collected within 24 hrs of emergence and transferred to vials in groups of 25-30 and held in the incubator until testing.
Unless otherwise stated, males used in all series of tests in this study were from < 12 hours, 1-day, 30-days, 90-days, or 150-days old at the time of testing.
Females were also collected at daily intervals and transferred to new vials. Groups of 45-50 were maintained in the incubator and, unless otherwise noted, used for test ing after they were < 12 hours, 1-day, or 30-days old. IDENTIFICATION OF THE EXOCRINE GLAND
Introduction
The majority of studies on pheromone gland ultrastruc ture have focused on Lepidoptera (Roelofs and Feng, 1968;
Percy et al. s 1971; Percy and Weatherston, 1971s 1974 ;
Lalanne-Cassou et al. s 1977; Percy, 1974, 1979). Among the species examined, ductless glands are located in the intersegmental membranes between the eighth and ninth abdominal segments (Percy and Weatherston, 197*0* By contrast, the site of pheromone production has been local ized in a few Coleoptera and found to occur within the abdominal tissue (Travis, 1939; Soo Hoo and Roberts, 1965;
Cuthbert and Reid, 1964; Coffelt, 1971j Hammack et al.3
1973; and Peschke, 1978).
In a review of the secondary sex characters of the confused flour beetle, Tribolium oonfusum Duval, and the red flour beetle, T. oastaneum (Herbst), Park (1934) noted that the genital lobes of the female are more pronounced than'those of the male. Likewise, Hinton (1942) differ entiated the sexes of both species by the presence of a sub-basal setiferous puncture on the ventral side of the
17 male femur, a structure which is absent in the female (Plate
I). Hinton observed that in T. castaneum the setiferous
puncture is restricted to the prothoracic legs, whereas in
T. oonfusum, the punctures are much reduced in size but
occur on all three pairs of legs. He also noted that this
structure is more reduced in the latter species. A decade
later, Hope (1953) discovered a sex difference in the
elytral striae and El-Kifl (1953) reported both a sclerc-
tized medial projection on the seventh abdominal tergite
that is unique to the male and internally, a medial anterior
apodeme in the eighth abdominal sternite present only in
the female. In 1967} Lange found the pregenital setae to
be different in the two sexes of T. castaneum. Sokoloff
(1972) observed the femoral pit of males to be encrusted
with flour in old beetles (Plate II) and suggested that
this accumulation could be resulting from a beetle secretory
product. Furthermore, Sokoloff (1972) presented scanning
electron micrographs of male sex spots, however, ultra-
structural detail was lacking due to poor resolution. At
this time, a function for this somewhat obscure sexual
dimorphic structure has not been proposed.
This investigation was undertaken to: (1) describe
the ultrastructure and relationships of certain main
components of the sexual dimorphic setiferous puncture,
including sensilla; and (2) to determine whether there were morphological modifications in this structure that PLATE I
Explanation of Figures
A Teneral female and male Tvibolium castaneum.
B Small pigmented setiferous puncture (SP) on the male prothoracic femora (circle).
C Sub-basal setiferous puncture secretion (S) on the prothoracic leg (circle).
D Secretion is removed with an insect minuten pin.
19 «ss
PLATE i
Sfii: *• PLATE II
Explanation of Figures
A Comparison of male beetle to globular secretion on insect pin.
B Male prothoracic leg secretion weighing oa. 1.2 ng.
21 PLATE II 23 could be related to the production of a subcuticular secretion.
Morphology of the Male Setiferous Puncture
Scanning Electron Microscopy
Male beetles were mounted on Cambridge stubs using a fast drying silver colloidal paint. Preparations were coated with ca. 200 X of a 60:40 goldtpalladium mixture applied by sublimation under vacuum, using a Hummer I sputter coater and viewed on a Hitachi S-500 scanning electron microscope with an accelerating voltage of 20 kV.
Scanning electron microscopy (SEM) observations dis closed, in non-solvent treated specimens, the existence of a globular secretion covering the sub-basal setiferous puncture on the prothoracic leg in 150-day old males
(Plate III A and B). The amorphic texture of the globule suggest that small flour particles are trapped within the secretion (Plate III-B; Plate V-D).
Solubility Properties of the Globular Secretion
Following the same scanning electron microscopy pre parations, 150-day old beetles were placed into a three dram vial containing two mis solvent for a 35 min period.
Several organic solvents with different bonding polarities were tested to examine the solubility properties of the globular secretion (Table 1). SEM observations revealed PLATE III
Explanation of Figures
A Ventral view of the male prothoracic femoral puncture covered by a secretion (S).
B Enlargement of prothoracic femur (PF) and globular secretion (S).
C Ventrolateral view of setiferous puncture (SP) in newly emerged male showing sensilla extend ing above cuticle.
D Ventral view of setiferous puncture with fluted sensillae (FS) and secondary reservoirs (SR) interdispersed between the sensillae. Area marked with circle is shown in Plate IV-A.
24 PLATE III 26
increased solubility of the secretion as the solvent
polarity decreased. As seen from Table 1, the latter four
non-polar solvents, carbon tetrachloride, diethyl ether,
toluene and hexane readily dissolved the prothoracic femoral
secretion. This suggests that the secretion was lipid in
nature.
Ultrastruoture of the Setiferous Punoture
To dissolve the waxy secretion for ultrastructure
studies, the entire insect was bathed in hexane bath for
oa. 35 min. Removal of the secretion revealed the setifer
ous puncture contained 51 ± 4.6 fluted sensillae (Callahan,
1975) (Plate III-C). The sensillae appear smooth, short and ovate. These structures clearly arise from within the pit and continue above the plane of the cuticle. The
sensillae are particularly sparse within the center of the
setiferous puncture and appear dense in arrangement at the periphery of the puncture. The outer margin of the set- tiferous puncture is devoid of sensillae where the femoral cuticular surface forms a sculptured texture.
Numerous pores (secondary reservoirs) were observed to be interdispersed randomly between the fluted sensillae along the cuticular basement of the setiferous puncture
(Plates III-D; IV-A). These secondary reservoirs measure
3.3 ± .2198y by 4.3 + .l68ly (N = 25 from 25 individuals).
Upon close examination of the secondary reservoirs, minute, TABLE 1. Male prothoracic femoral setiferous puncture secretion solubility properties. (N = 10 for each solvent)
Solvent Solubility Results
water — >3 -P acetic acid (RCOOH) - •H !h - cti ethanol (ROH) 1—1 o acetone (RCOR) ± a (U bO ethyl acetate (RCOOR) + ±b G •H carbon tetrachloride (RX) + + CQ ctf CL> diethyl ether (ROR) + + + G O toluene (ArH) + + + 1) Q ' + hexane (RH) + + + a The outside border of the setiferous puncture showed some indication of secretion solubility in a few specimens.
The setiferous puncture secretion in several specimens were not soluble in ethyl acetate. 28 cuticular ductlike openings (tertiary reservoirs) with a diameter of 1.3 ± .2544y (N = 25) were observed within these structures (Plate IV-A and B). Each secondary reservoir contained 5.2 ± 2.1 tertiary reservoirs (N = 25).
Secretion Accumulation with Beetle Age
Scanning electron microscope observations suggests the secretion accumulates as the beetles become older (Plate
V). In 10-day old post-eclosion males, the secretion has migrated out of the setiferous puncture onto the outlying cuticle (Plate V-A)j but the fluted sensillae and secondary reservoirs can be clearly observed within the puncture.
In a 30-day old male, secretion has further accumulated in the central region of the puncture (Plate V-B). In 60-day old beetles, the secretion has increased in volume, covering all of the sensillae within the puncture and exuding over the margin of the pit (Plate V-C). By 150 days, the secretion extends beyond the margin of the pit and continues over the femoral cuticle (Plate V-D). In the latter case, the secretion has amassed such a large volume that it extends well above the plane of the prothoracic femora cuticle.
Histology
For light microscope thick sectioning (4 ym), femora were excised from teneral adult male beetles within 24 hrs of eclosion. Older beetles are less suitable for sectioning since the cuticle is much harder. PLATE IV
Explanation of Figures
A Enlargement of the secondary reservoirs (SR) within the setiferous puncture. Note the tertiary reservoirs (TR) or ducts within each secondary reservoir.
B Secondary reservoir containing oa. 5-7 tertiary reservoirs.
29 PLATE IV PLATE V
Explanation of Figures
A Ventral view of the setiferous puncture on a 10-day-old male. Secretion (S) is deposited on the margin of the puncture.
B Ventral view of the setiferous puncture on a 30-day-old male. Secretion (S) is observed increasing in mass in the central area of the puncture.
C Ventral view of the setiferous puncture in a 60-day-old male. Secretion (S) has completely coated the setiferous structure and sensillae.
D Ventro-lateral view of male prothoracic femora. The setiferous puncture secretion has extended beyond the margin of the pit and continues over the cuticle.
31 PLATE V 32 33
TABLE 2. Light Microscopy Processing
Material Time k% Glutaraldehyde in 0.1 M Sorensen's phosphate buffer, pH 7-2, 4°C 2 hr Three washes with osmotically adjusted SPB, 4°C 10 min 1$ Osmic acid in SPB, 4°C 1 hr 35$ ethanol 30 min 50$ ethanol 30 min 70$ ethanol 30 min 90$ ethanol 30 min 95$ ethanol 30 min 100$ ethanol, four rinses 30 min 1:1 ratio, 100$ ethanol:propylene oxide 30 min 3:1 ratio, propylene oxide:Spurr standard mixture 30 min 1:1 ratio, propylene oxide:Spurr standard mixture 30 min 1:3 ratio, propylene oxide Spurr standard mixture 30 min Pure Spurr standard mixture, 20 lbs vacuum, 35°C overnight Pinal Spurr standard mixture, 60°C 12 hr Prior to femur excision, the male beetles were lightly anesthetized (Sokoloff, I960) using anhydrous ether. Excised femora were immediately placed into glass capillary tissue processing units with screen tops (Pelco, Inc., Tustin, Ca.).
These tubes confined the specimens within a limited area and allowed the tissue to be permeated with various pro cessing solutions (Table 2). The specimens were embedded under vacuum (25 psi) in Spurr low viscosity resin (Poly sciences) (Spurr, 1969).
Sections were cut at 4 pm with a glass knife on a
Sorvall Porter-Blum MT-2 ultramicrotome (Sorvall, Inc.,
Norwalk, Conn.). Glass knives were made using a LKB 78OO B knifemaker and only the left half knife was used for section ing. Every tenth section through the femur was retained.
Sections were stained with 2% toluidine blue, 2% sodium borate, 1% pironine B (see Appendix A), examined, and photographed with an Olympus compound light microscope.
The exterior surface of the setiferous puncture contains fluted sensillae that extend beyond the margin of the cuticle (Plate VI-A). Each sensilla arises from a subcuticular trichogen cell. The cuticle overlying the secretory region consists of a dense layer with minute gaps representing cuticular pores.
Clusters of secretory cells of modified epidermal origin are located internally within the setiferous puncture.
The secretory cells are large and homogeneous in appearance. Abbreviations c Cuticle N Nucleus EL Epidermal layer SCL Secretory cell layer FS Fluted sensilla SD Secretory ducts LSR Lipid secretory reservoir SP Setiferous puncture M Muscle V Vacuole
PLATE VI
Explanation of Figures
A Photomicrograph from slide mounted Thick Spurr cross section from a T. oastaneum male prothoracic femora through the region of the setiferous punc ture showing secretory tissue on the inner surface of the cuticle.
B Cross section from a T. oastaneum male prothoracic .femur through the center of the setiferous puncture showing subcuticular secretory ducts (tertiary ducts) that lead to basement of the pit.
35 PLATE vi 36 This homogeneity suggests that the cells serve a similar,'
perhaps a limited function (i.e.j the production of a
specific product). Each cell contains a nucleus and
extends externally via a secretory duct (tertiary reservoir
duct) which is lined with a cuticular intima. These ducts
extend to the basement of the setiferous puncture and
appear as invaginations of the integument. The long cuticle
lined duct joins the secretory reservoir and terminates
at the surface of the setiferous puncture. This suggests
that the associated lipid secretion is carried from the
secretory reservoir through the cuticular duct, then to
the surface of the puncture. This is summarized in Figure
1 .
Internally, large lipid secretory reservoirs are found
to be a component of these modified epidermal cell mass.
The reservoirs probably function in the storage and trans
port of the setiferous secretion. Numerous muscle bundles
are found directly beneath these secretory reservoirs
(Plates VI; VII). The secretory cell layer observed beneath the setiferous puncture is bordered laterally by
unmodified, cuboidal epidermal cells (Plates VI-A). Except
for the region directly below the setiferous puncture, the epidermal cells maintain a consistant, unmodified border beneath the femoral cuticle. FIGURE 1
Diagrammatic interpretation of the Tribolium aastaneum male prothoracic femoral setiferous puncture showing prominent subcuticular features. The lipid secretion follows a probable pathway of transport from the lipid secretory cells through the cuticular ducts and passes through the cuticle where it is liberated on the surface of the seti ferous puncture.
38 FLUTED SENSILLA
— PRIMARY RESERVOIR
CUTICLE SECONDARY RESERVOIR
SECRETORY DUCT NUCLEUS
LIPID SECRETORY Abbreviations
N Nucleus SD Secretory ducts LSR Lipid secretory reservoir SP Setiferous puncture
PLATE VII
Explanation of Figures
A Thick Spurr cross section from a T. eastaneum male prothoracic femur through the center of the setiferous puncture showing secretory tissue below the basement of the pit.
40 P L A T E vii 41 Discussion
The ultrastructure of the femoral puncture, combined with the presence of a specialized secretory epidermal layer on the inner surface of this structure, implies that • the male has a unique exocrine gland that synthesizes and releases a lipid to the surface of the body. The substance exudes out from minute cuticular ducts onto the cuticular basement of the setiferous puncture. As the beetle ages, the secretion enters into the primary reservoir covering the basement of the cuticle. Prom here, possibly due to capillary action or simple diffusion, the secretion travels along the length of the fluted sensillae, subsequently coating these sensory structures.
The fluted sensillae associated with the male setiferous puncture structure of T. castaneum appears to be analogous in morphology to that of the termite. The sensillae in
T. oastaneum probably act as sites of increased evaporation.
The trail marking pheromone gland of the termite, Zooter- mopsis spp.j localized in the region of the fourth abdominal sternite, is equipped with a distinct pattern of campani- form sensilla which function as proprioceptors (Stuart,
1964; Stuart and Satir, 1968). These authors postulate that these sense organs function as part of a feedback control system from substrata stimuli that determines the quantity of trail pheromone deposited. 4 3
Epidermal pheromone glands have been described ir. ::
variety of insects, occurring in epithelial linings of
different organs or in the digestive tract. In the moth
Rhyacionia frustana (Comstock), the sex pheromone gland is
a columnar epithelial sac located in the abdomen (Baer
et al., 1976). Pheromone is released by hydrostatic
pressure following gland extension. The sex pheromone gland
in the cockroach Bvyostria fumigata (Guerin) (Moore and
Barth, 1976) is localized in the genital atrium. The
setiferous puncture of T. aastaneum does resemble sex
pheromone glands in other arthropods. The sex pheromone
gland in six species of Trogoderma, including the Khapra
beetle, is formed from enlarged columnar epithelial lining
of the inner surface of the 7th abdominal sternite, and is
associated with minute cuticular pores (Hammack et al,3
1973). The foveal gland in ixodid ticks, Bermaoentor spp.,
consists of lobular clusters of secretory cells which
release sex pheromone through minute pores in the fovea
dorsales (Sonenshine et al., 1977). The unique chemical
feature of the flour beetle setiferous puncture is the
accumulation of the lipid secretion (Faustini, 1980).
Because the male femoral punctures are morphologically similar among twelve Tribolium species (Table 19), it is probable that the tissues produce a secretion as in
T. aastaneum. In many Lepidoptera, the sex pheromone gland cells are ductless, hypertrophied epidermal cells (Percy and
Weatherston, 1974; Percy, 1979). However, pheromonal duct systems associated with epidermal secretory cells have been described in the desert locust (Strong, 1971) where the epidermal glandular tissue was formed by two cell types: one type for cuticular deposition and the other type forming ducts from secretory cell vacuoles to the cuticular surface. The sex pheromone glands in the
Mecoptera are composed of two major cell types: secretory and duct transporting epidermal cells. Each cuticularized duct drains a deep, membrane-bound invagination of the secretory cell, termed an end apparatus. Hodosh et al.3
(1979) report a canaliculi-duct system which transports the male sex pheromone through the anus of Drosophila grimshawi Oldenburg. Comparable secretory units specialized for synthesis and deposition of noxious defense compounds which are toxic to the secretory cell have been reviewed in numerous articles (Roth, 1943; Loconti and Roth, 1953;
Beams and Anderson, 1961; Happ, 1968; Happ and Happ, 1970;
Kendall, 1972; Tschinkel, 1975a, 1975c; Sehildknecht et al.3
(1976); Klinger and Maschwitz, 1977). In T. aastaneum the cuticular ducts appear to provide increased secretory surface area and an individual excretory ciuct for each secretory cell. Furthermore., the duct appears to reduce the pathway involved in the transport of the secretion from the exocrine cells.
Crossley and V/aterhouse (1969) while working with
Mecoptera sex pheromone glands, suggested that muscular
contraction increased haemolymph pressure leading to
glandular eversion and volume reduction of the secretory
cell end apparatus. As a result of this compression, the
secretion was transported through the duct and onto the
surface cuticle. Hodosh et al.3 (1979) offered a compar
able explanation for sex pheromone release in the intra-
anal lobe sex pheromone gland of Drosophila gvimshawi.
They suggested that the secretory product was passed out the anus through simple turgor pressure or muscle contrac tion. Based upon histological evidence of the setiferous puncture in T . aastaneum, muscle bundles located beneath the secretory cell layer probably are the active force or aid in the release of the lipid secretion.
The external morphology of the male femoral puncture
suggests that after release from the exocrine cells, the
secretion flows along the surface of the cuticle within the puncture and coats the sensilla. Furthermore, the
secretion accumulates in older beetles.
The cells of the setiferous puncture appear to be active secretory cells. They differ from unmodified epithelial cells in having cuticular secretory ducts,
larger nuclei, and lipid secretory reservoirs. The overall morphology of the puncture resembles those of pheromon
producing glands in other arthropods {e.g., Sonenshlne
1977; Hammack et al., 1973; Hodosh et al., 1979). LABORATORY BIOASSAY AND CHEMICAL ANALYSES
Introduction
In Tribolium, Keville and Kannowski (1975), Suzuki et
al. (1975), Ryan and 0' Ceallachain (1976, 1977), and Suzuki
and Sugawara (1979), demonstrated the existence of sex,
alarm, aggregation pheromones in the species T. eonfusum
and T. castaneum. In each of these studies, beetle response
to the attractant was demonstrated within a reliable bio
assay arena.
In recent years, the process of insect pheromone isola tion, characterization and synthesis has flourished due to advances in analytical chemistry (Beroza, 19751 Beroza and
Bierl, 1967, 1976; Tumlinson and Heath, 1976). It is well established, however, that behavioral variations can occur in the insect, depending on structural differences of various pheromone isomers. Prior to micromethodology leading to pheromone identification, a reliable bioassay must be devised to monitor insect behavior. The design of the system must provide continuous sensitive response during isolation cleanup procedures of the potentially biologically active components (Beroza, 1975). Lack of consideration for the
47 48 physiological or environmental variables may produce non-
reproducible data (Shorey, 1970).
Of further consideration are insect behavioral character
istics selected as criteria for positive response in the presence of a chemical odor. Englemann (1970) demonstrated
insect responses to be sequential and observed that higher
concentrations may be required to produce more sophisticated
behavioral traits. In previous studies, the primary behav
ioral characteristics have been established as: (1) antennal
flagging, observed in the yellow mealworm, Tenebrio molitor
L. (Valentine, 1931); (2) wing fluttering, demonstrated in
the greater was moth, Galleria mellonella L. (Roller et al.,
1963)j pink bollworm, Peotinophora gassypiella Saunders
(Bergen et al., 1964) and the cabbage looper, Trichoplusia ni Hubner (Gaston and Shorey, 1964); and (3) locomotion towards the odor source, as reported in Trogoderma glab rum
(Herbst) (Burkholder and Dicke, 1966) and Dermestes maculatus
(DeGeer) (Abdel-Kader and Barak, 1979). Regardless of the trait selected, it should establish an accurate reflection of a specific pheromone response signal.
Some sensitive and generally applicable methods for the determination of insect pheromones and their analogs have been developed in which gas liquid chromatography and gas chromatography and mass spectrometry are employed. In previous gas chromatographic studies on volatile extracts from T. aastaneum and T. confusum relatively simple H9 chromatogram profiles were presented using packed columns
(Keville and Kannowski, 1975 ); Suzuki et al. 3 1975 ; Baker et al. , 1978 ; and Markarian et al. 3 1978). Many of the major component fractions represented hydrocarbons (Keville and Kannowski, 1975; Suzuki et al. 3 1975; Baker et al. 3
1978; and Markarian et al. 3 1978), quinones (Markarian et al.3 1978) and bisolvents (Markarian et al.3 1978).
The purpose of the present study was to: (1) examine the biological activity of the male prothoracic femur secretion in T. aastaneum; (2) to establish a reliable, quantitative laboratory bioassay for use in subsequent studies; (3) to correlate the perception of pheromone response with sexual maturity in both sexes; and (4) to describe a more sensitive technique for pheromone research in glass capillary column gas chromatography.
Materials and Methods
Handling of Insects
The beetles were from the Vera Cruz wild-type strain of
Tvibolium aastaneum obtained from Professor A. Sokoloff,
California State College, San Bernardino. They were main tained in ^ oz. culture jars, containing 20 g of medium, consisting of whole-wheat flour. The beetles were sexed as pupae by examination of genital lobes (Ho, 1969) and isolated in separate culture containers, with medium, to 50 develop into adults. The sexed beetles were provided with separate environmental chambers maintained on a 15:8 LD photoperiod regime, with lights on at 6:00 AM EDT.
Olfactometer and Laboratory Bioassay Conditions
The multiple-choice was selected for use in the study of the potential pheromonal properties of the male setiferous secretion of T. aastaneum. Bioassays were performed in a room separate from that used for culturing and handling of the stock insects. The bioassay room was controlled at
27 ± 2°C and 55 ± 8% relative humidity, under illumination by cool white fluorescent lighting. To reduce assay con tamination the room was provided with ventilation. Tested insects were used for only one observation or replicate.
Multiple-Choice Olfactometer
The fan-shaped multiple-choice olfactometer bioassay chamber has been described in detail (Burkholder, 1970).
Briefly, it consists of an attractant choice available to the test insects at one or more of five alternatives equidistant from the point of release.
. Twenty-five test beetles were released into the chamber floor and their distribution in the olfactometer was recorded every 1 min for 16 min. Air flow rate for each choice was controlled at 2.0 liters/min using Gelman flow meters. The total number of beetles within each air plume was counted 51 at each observation. Pour replicates were conducted for each test. Each replicate lasted 16 min.
Controls consisted of five vessels containing 'whole wheat flour. Treatments consisted of a central vessel
(vessel #3) containing either 50 globules that had been removed from the male or globules still remaining on the male prothoracic leg. The remaining four vessels contained whole-wheat flour. Both male and female responses were tested against these treatments.
Experimental groups consisted of males or females < 1,
1, or 30 days old. These insects were used to monitor the activity of the male globular secretion or the globular secretion still intact on the prothroacic leg in 150-day males. Response for each group was also examined for activity to 90-day-old males without the presence of a prothoracic femoral globule.
Following each replicate the olfactometer vessels and associated glassware were washed with detergent, rinses of distilled water and acetone, and dried in an oven. This was performed to remove any beetle contaminates within the enclosed system.
After each experiment, the total number of beetles responding to each choice was calculated and expressed as a mean (x) value for each choice/min. Data were subjected to analysis by Wilcoxon’s Rank Sum non-parametric statisti- 52
cal test (Hollander and Wolfe, 1973), differences at the -
5% level or lower only were accepted as statistically
significant.
Isolation of the Male Setiferous Secretion
Under light ether anesthesia, experimental males with large globular secretions on the prothoracic femora were held in place with jewelers’ forceps. Samples were collected by lifting off the secretion with an insect minuten pin mounted on a wood applicator stick (Plate II). The globules were placed on the bottom of an empty vessel and mounted in the center choice of the multiple-choice olfactometer.
A colony of 100-day old males were the source of the
setiferous secretion. Globules isolated from this particular age class weigh approximately 1.2 ng/globule (n = 25).
Weights were obtained using a Cahn Gram Electrobalance®
(Vernon Instruments Corp., Vernon, Ca.).
Chemical Analyses
Material for chemical analysis was usually obtained by removing 50 whole male globules (see Isolation section) and placing each into a 2 dram vial containing double distilled hexane. 53
Gas Chromatography - Packed Column
Aliquots of beetle secretion were injected into a
Hewlett-Parkard, model 5710A, gas chromatograph equipped with flame ionization detector (FID). Nitrogen was used as the carrier gas at a flow rate of 5^ ml/min through a
5-5 m x 6.0 mm OD glass column packed with 10% SE-30® stationary phase. Temperature was programmed from 150° to 310°C at 8°C/min. The detector temperature was 350°C, and the injector port was 350°C.
GC-Glass Capillary
The male secretion was concentrated under a stream of nitrogen and analyzed on a Varian, model 3700, gas chromatograph equipped with flame ionization detector.
The glass capillary columns (20 m x .03 mm) were prepared and statically coated with SE-52® as stationary phase conditioned at 300°C. for 16 hrs. The carrier gas flow was 8.0 ml/min of hydrogen. The temperature of the GC oven was raised by programming it from 60° to 200°C at 70°C/min. The final temperature was 200° to 330°C at 4°C/min. The injection port and the detector were maintained at 320°C and 350°C3 respectively. The amount injected was 1.5 yl in the splitless mode. Gas chromatographic retention values and peak heights from the male setiferous secretion were compared by relating the position of the peaks of the gas chromatogram to that of the closest saturated, straight 54 chain hydrocarbons (from 5-30 carbons long) available reference compounds. An uncontaminated whole-wheat flour sample, extracted in hexane, was also used as a reference.
Experimental Results
Female and Male Behavioral Response
The characteristic responses by either sex to the source of the male setiferous globules and males with large globules on the prothoracic leg were demonstrated by the following traits'. (1) extension of the prothoracic legs, often accompanied with bobbing up and down movements sub sequently followed by locomotion; (2) antennal protraction; and (3) locomotory movements in a zig-zag (orthokinesis) manner directly beneath the air plumes of the pheromone source. In each of the bioassays, the beetles favored the walls of the olfactometer, maintaining a high degree of wall-surface contact (this was reduced in conditions where a pheromone source was tested).
Multiple-Choice Olfactometer Bioassay
Female response
There was consistency between assays, using females and males to ascertain the effect of age on pheromone perception.
Table 3 summarizes the results of females responding in the multiple-choice olfactometer to vessels containing 55
TABLE 3
Distribution of Tribolium aastaneum females in a multiple- choice olfactometer to which no male secretion was presented (control).
Total number of females counted Total number of Replicatea in indicated choice in 16 minb insects counted 2 3 4 5 per replicate1
I 93 20 16 30 94 253
II 83 25 25 49 84 266
III 73 64 27 41 76 281
IV 81 52 34 31 76 274
Total0 330 161 102 151 330 1074 a 25 untested females/replication.
sum of sixteen consecutive one min readings. c significant deviation from random distribution between choice slots at the 5% level as determined by Wilcoxon's Rank Sum Test. whole-wheat flour (controls). Following the introduction of
females into the olfactometer, random movement in seve: 1
directions was observed. Since the insects favored the
outer walls of the system, their distribution was not equal
in choice. After 16 min the vast majority of the insects
were recorded in slots 1 and 5. Some variation occurred
between replicates for beetles counted within each choice
slot. This was attributed to variation in time of day in
which the assays were conducted versus peak biological
rhythms.
When a source of 50 male globules (25 male equivalent) were provided in the central vessel (#3), 12-hr post-
eclosion females responded significantly to the source of
odor (P < 0.05) (Table 4). Locomotor response consisted of antennal protraction accompanied by orthokinetic movements to the odor. This differed significantly from the results obtained where only flour was used in vessel 3 (Table 3).
Females moved in a zig-zag manner in and out of the odor
iferous air plume, pausing occasionally to extend their prothoracic legs in a bobbing manner. In many of the replicates, females still maintained contac-t with the walls of the olfactometer, however, movements continued within the system towards the source of attractant. Thirty-day post-eclosion virgin females showed a slightly higher response to 50 male globules (25 ME), Table 5- Response by the females 57
TABLE 4
Distribution of Tribolium aastaneum 12-hour post-eclosion virgin females in a multiple choice olfactometer containing male globular secretion.
Total number of females counted Total number of cl Replicate in indicated choice in 16 min*3 insects counted 1 2 3C 4 5 per replicate
I 63 6 14 7 12 102
II 41 22 91 11 54 219
III 69 12 38 22 65 206
IV 51 22 74 22 53 222
Total 224 62 217d 62 184 749 a 25 untested females/replication. 1^ sum of sixteen consecutive one min readings. (3 contained 50 male globules. d P < 0.05. 58
TABLE 5
Distribution of Tvibotium aastaneum 30-day post-eciosion females in a multiple-choice olfactometer containing male globular secretion.
Total number of females counted Total number of Replicatea in indicated choice in 16 min*3 insects counted 1 2 3C 4 5 per replicate
I 86 14 64 23 56 243
II 70 16 91 27 61 265
III 41 22 91 11 54 219
IV 69 12 46 22 65 214
Total 266 64 292d 83 236 941 a 25 untested females/replication. r,. sum of sixteen consecutive one min readings. 0 contained 50 male globules. d P < 0.05. 59
appears to have only increased gradually to the male
globules when compared to 12-hr post-eclosion female
response.
Twelve-hour post-eclosion female response was signifi
cant (P < 0.05) to odors from 150-day-old males with large
prothoracic femoral globules (Table 6). However, female
response was slightly lower when compared to 12-hr post-
eclosion female response to 50 crude globules (Table 4).
One-day and 30-day-old post-eclosion female responses were
greater than 12-hr post-eclosion females and controls
(P <0.05). Male odors clearly excited older females
(Tables 7 and 8).
When 30-day-old females were provided with 90-day-old
males without prothoracic femoral globules (Table 9), a
non-significant response was recorded (P > 0.05). Further
more, where two sources of male odors were provided (Table
10), females showed a strong response to 150-day-old males
with prothoracic leg globules (vessel #2) and a non-signifi
cant response to 90-day-old males without globules (vessel
#4).
Male Response
Males responding to whole-wheat flour in the multiple- choice olfactometer was similar to female control response
(Table 11). Males exhibited similar behavioral characteristics
{i.e., antennal protraction, leg extension, zig-zag locomotion). 60
TABLE 6
Distribution of Tvibolium aastaneum 12-hour post-eclosion virgin females in a multiple-choice olfactometer containing 150-day-old males with large globules on the prothoracic femur.
Total number of females counted Total number of Replicatea In indicated choice in 16 min'3 insects counted 3C 4 5 per replicate12
I 44 26 38 29 40 177
II 67 30 30 30 67 224
III 71 32 35 36 70 244
IV 69 40 37 37 77 260
Total 251 128 l40d 132 254 905 a 25 untested females/replication. d sum of sixteen consecutive one min readings.
contained 25 males 450 globule equivalent) d P < 0.05. 61
TABLE 7
Distribution of Tvibolium oastaneum 1-day post-eclosion virgin females in a multiple-choice olfactometer containing 150-day-old males with large globules on the prothoracic femur.
Total number of fe males counted Total number of Replicatea in indicated choic e in 16 min13 insects counted 1 2 3C 4 5 per replicate
I 40 13 154 19 43 269
II 28 5 57 12 20 122
III 39 17 97 30 35 218
IV 48 18 99 27 50 242
Total 155 53 407d 88 148 851 a 25 untested females/replication.
sum of sixteen consecutive one min readings,
contained 25 males 450 globule equivalent) d P < 0.05. 62
TABLE 8
Distribution of Tribolium oastaneum 30-day post-eclosion virgin females in a multiple-choice olfactometer containing 150-day-old males with large globules on the prothoracic femur.
Total number of females counted Total number of Replicate in indicated choice in 16 min13 insects counted 1 2 3C 4 5 per replicate
I 94 63 109 46 43 355
II 45 16 131 18 46 256
III 44 25 111 34 58 338
IV 49 45 67 17 65 243
Total 232 149 484d 115 212 1192 a 25 untested females/replication, k sum of sixteen consecutive one min readings. fi contained 25 males 450 globule equivalent). d P < 0.05. 63
TABLE 9
Distribution of Tribolium oastaneum 30-day post-eclosion virgin females in a multiple-choice olfactometer containing 90-day-old males without globular secretion.
Total number of females counted Total number of Replicate3- in indicated choice in 16 min13 insects counted 1 2 3C 4 5 per replicate
I 51 23 16 32 53 143 ’
II 42 27 19 35 33 156
III 61 31 16 27 57 192
IV 54 32 12 30 59 187
Total 208 113 63d 124 202 678 a 25 untested females/replication.
sum of sixteen consecutive one min readings. c contained 25 males. d P > 0.05 (non-significant). 64
TABLE 10
Distribution of Tribolium aastaneum 30-day post-eclosion virgin females in a multiple-choice olfactometer containing 150-day-old males with large globules^ and 90-day-old males without globulese .
Total number of females counted Total number of Replicate3" in indicated choice in 16 min^ insects counted 1 2c 3 4c 5 per replicate
I 76 35 22 70 247
II 49 130 22 67 56 324
III 92 54 11 31 70 258
IV . 73 78 46 37 63 297
Total 290 306d 114 157e 259 1126
cL 25 untested females/replication,
sum of sixteen consecutive one min readings. c contained 25 males. d P < 0.05. e P > 0.05 (non-significant). 65
TABLE 11
Distribution of Tribolium oastaneum males in a multiple- choice olfactometer to which no male secretion was presented (control).
Total number of males counted^ Total number of Replicatea in indicated choice in 16 min insects counted 1 2 3 4 5 per replicate
I 77 49 23 35 63 247
II 78 41 27 39 85 270
III 81 43 24 42 59 249
IV 75 38 25 43 88 269
Total0 311 171 99 159 295 1035 a 25 untested males/replication.
sum of sixteen consecutive one min readings. c significant deviation from random distribution between- choice slots at the 5% level as determined by Wilcoxon’s Rank Sum Test. 66
When 12-hour post-eclosion males were exposed to odors
from 50 globules (25 ME), the response was significant
(P < 0.05), Table 12. Thirty-day-old males showed a
slightly higher response than 12-hour post-eclosion males;
however, the latter case is still significant when compared
to the whole-wheat flour control (P < 0.05).
Twelve-hour post-eclosion males that were provided with
an odor source from 150-day-old males with large globules
on the prothoracic femora showed a significant response
when compared to the control (Table 14). Response dramati
cally increased for 1-day-old males to the older males
(Table 15), however, a decline in response was recorded
for 30-day-old males (Table 16), although the response was
was still significant (P < 0.05).
Like females, when 30-day-old males were provided with
90-day-old males without globules, a non-significant
response was obtained (Table 17)- Furthermore, when given
a dual choice of male odors, 30-day-old males selected odors
eminating from 150-day-old males with large globules
(Table 18), rather than 90-day-old males without globules.
Both female and male data are summarized in Figure 2.
Beetles of both sexes are clearly excited from odors
elicited from the male setiferous secretion. In this study,
for females it appears that 30-day-old individuals are the most responsive physiological age to the aggregation pheromone. 67
TABLE 12
Distribution of Tvibolium oastaneum 12-hour post-eclosion virgin males in a multiple-choice olfactometer containing male globular secretion.
Total number of males counted, Total number of Replicatea in indicated choice in 16 min insects counted 1 2 3C 4 0 per replication
I 28 19 124 11 60 242
II 47 17 26 34 67 191
III 62 20 44 26 57 209
IV 78 21 40 24 67 230
Total 215 77 234d 95 251 872
25 untested males/replication. b sum of sixteen consecutive one min readings. c contained 50 male globules. d P < 0.05. 68
TABLE 13
Distribution of Tribolium oastaneum 30-day post-eclosion virgin males in a multiple-choice olfactometer containing male globular secretion.
Total number of males counted^ Total number of Replicatea in indicated choice in 16 min insects counted 1 2 3C 4 5 per replicate
I 42 19 115 11 61 248
II 75 25 52 27 75 254
III 58 26 54 32 58 228
IV 80 39 55 21 60 255
Total 255 109 276d 91 254 978 a 25 untested males/replication.
13 sum of sixteen consecutive one min readings.
contained 50 male globules. d P < 0.05. 69
TABLE 14
Distribution of Tr-tbolium eastaneum 12-hour post-eclosion virgin males in a multiple-choice olfactometer containing 150-day-old males with large globules on the prothoracic femur.
o Total number of males counted, Total number of Replicate in indicated choice in 16 min insects counted 1 2 3C 4 5 per replicate
I 63 6 14 7 66 156
II 41 22 91 11 54 219
III 69 12 38 22 65 206
IV 62 24 49 28 67 230
Total 235 64 192d 68 252 811
25 untested males/replication.
■j ^ sum of sixteen consecutive one min readings.
c contained 25 males 4 globule equivalent).
d P < 0.05.
« 70
TABLE 15
Distribution of Tribolium oastaneum 1-day-old post-eclosion virgin males in a multiple-choice olfactometer containing 150-day-old males with large globules on the prothoracic femur.
Total number of mate s countedb Total number of Replicate3, in indicated choice in 16 min insects counted 1 2 3C 4 5 per replicate
I 92 12 71 10 76 261
II 75 14 119 12 86 306
III 111 9 97 8 87 312
IV 96 9 86 11 94 296
Total 374 44 373d 41 343 1175 a 25 untested males/replication. d sum of sixteen consecutive one min readings c contained 25 males 450 globule equivalent). d P < 0.05. 71
TABLE l'6
Distribution of Tviboli,jm oastaneum 30-day-old post-eclosion virgin males in a multiple-choice olfactometer containing 150-day-old males with large globules on the prothoracic femur.
Total number of males counted^ Total number of Replicate3" in indicated choice in 16 min insects counted 1 2 3C 4 5 per replicate
I 83 22 65 43 54 267
II '4 7 40 101 45 53 286
III 70 79 67 21 81 318
IV 86 40 60 29 38 253
Total 286 181 293d 138 226 1124 o 25 untested males/replication. d sum of sixteen consecutive one min readings.
contained 25 males 450 globule equivalent). d P < 0.05. 72
TABLE 17
Distribution of Tvibolium aastaneum 30-day post-elcosion virgin males in a multiple-choice olfactometer containing 90-day-old males without globular secretion.
Total number of males counted^ Total number of Replicate3- in indicated choice in 16 min insects counted 1 2 3C 4 5 per replicate
I 125 18 15 16 55 229
II 95 42 41 37 100 315
III 109 29 33 32 43 246
IV 96 23 11 35 114 279
Total 425 112 100d 120 312 1069 a 25 untested males/replication, k sum of 16 consecutive one min readings. c contained 25 males.
P > 0.05 (*non-significant). 73
TABLE 18
Distribution of Tribolium castaneum 30-day post-eclosion virgin males in a multiple-choice olfactometer containing 150-day-old males with large globules^ and 90-day-old males without globulese .
Total number of males counted Total number of ci Replicate in indicated choice in 16 min insects counted 1 2C 3 4C 5 per replicate
I 72 76 17 30 78 273
II 44 80 21 9 68 222
III 62 79 23 11 70 245
IV 65 87 16 13 72 253
Total 243 322d 77 63e 288 993 a 25 untested males/replication.
1^ sum of sixteen consecutive one min readings. c contained 25 males. d P < 0.05. e P > 0.05 (non-significant). FIGURE 2
Histogram of the mean (x) response to odors from the male setiferous secretion. Odors consist of two groups: (1) 50 globules (25 male equivalents); or (2) 25 males with large globules intact on the male prothoracic femora. The treatments consisted of females and males of various age groups, while controls were odors from whole-wheat flour.
74 X response of beetles to odors from tfsetiferous secretion 100 125 150 50 25 75 control <1 day 30 day 30 day <1 control 0 Globules 50 d* □ □ □ 9 iue 2 Figure 1a 1dy30 day 1day '1day By contrast, 1-day-old males show the maximum response from the male groups examined. As previously mentioned, globules of 150-day-old males weigh approximately 1.2 ng/globule (2.4 ng/beetle). Response to these globules was based upon either 50 crude globules (60 ng) or 25, 150-day-old males with large prothoracic femoral globules (ca. 60 ng). Therefore, a response to the male produced odors are within nanogram levels to elicit biological activity. Analysis of the Secretion Packed column GC analysis showed 2 major components in the beetles’ secretion (Pig. 3). Both peaks showed extreme shouldering indicating the presence of several components under these peaks. A gradual rise in the base line (10-35 min) indicates that there are many volatile components prior to the two major peaks. Glass capillary GC analysis revealed 9 major components in the beetle secretion (Fig. 4). Each of these peaks showed a shouldering edge, as those produced using packed columns. Several components consisted of fractions that were highly volatile (1-3) and fractions with long retention times (4-9), indicating a low volatility. The latter components may act as a carrier for the more volatile fractions. When the attenuation was increased (Fig. 5), well over 250 components in the male flour beetle secretion FIGURE 3 Gas Liquid chromatogram (packed column) components of fresh male Tribolium aastaneum prothoracic leg setiferous puncture secretion. Column: 5-5 m x 6.0 mm o.d. glass column, 10% SE-30©; Temperature programme - 150-310°C (programme rate, 8°C/min); Carrier gas - M2 flow rate, 56 ml/min; Detector - flame ionization sensitivity 16 x 101. (Numbered components have not yet been identified.) FIGURE 4 Glass capillary gas liquid chromatogram showing components of fresh male prothoracic femora setiferous puncture secretion. Column: 20 m x .03 mm i.d. glass column, SE-52 static coat; Temperature programme - 60-200°C (programme rate, 4°C/min); Carrier gas - N2 flow rate, 8.0 ml/min; Detector - flame ionization sensitivity 64 x 10” 12. (Numbered components have not yet been identi fied . ) 77 78 solvent peak 20 30 40 50 min Figure 3 Retention Time aolveiit.peak P 1 _L 8- ■mmm T 0 10 20 30 40 50 60 70 min Figure •! Retention Time FIGURE 5 Glass capillary gas liquid chromatogram showing ca. 250 components of fresh male prothoracic femora setiferous puncture secretion. Detector - flame ionization sensitivity 16 x 10“12. (All other conditions same as Fig. 4.) 79 co o % Detector Response Figure 5 Retention Tinie 81 were obtained. This latter chromotographic profile exempli fies the complexity of this secretion. The shouldering observed in the Fig. A peaks was a result of the multiple fractions observed in Figure 5- Based upon the complexity of fractions shown on the analytical chromatogram profiles, quantitative analyses were not possible for the beetle product. Gas chromatographic and peak enhancement studies were performed using the log of the retention time against the chain length of standard n-alkanes and n-alkenes for comparison with the beetle secretion. These studies indi cated that both normal and branched, alkanes and alkenes were not present. The standards ranged from C5-C30 in length. These retention value comparisons to the internal standards showed that the carbon number of the male pro thoracic femora secretion exceeded 30 carbons and a molecular weight above 400. Discussion Evidence for the existence of a male-produced aggre gation pheromone attractive to both sexes is reported. This was confirmed by the responses exhibited within a multiple-choice olfactometer. In this system, attraction to male odors over a distance was demonstrated. Both sexes responded significantly to male globules and 150- day-old males with large prothoracic femoral globules as compared to whcle-wheat flour controls. Furthermore, as presented in Section V, the ultrastructure of cells in the setiferous puncture exocrine gland of Tribolium eastaneum is consistent with the hypothesis that they are indeed involved in pheromone production. It appears that this lipid contains the pheromone based upon the female and male age and relative attractiveness response. The lipid is transported to the setiferous puncture by cuti- cular secretory ducts, where the pheromone presumably evaporates, during calling, resulting in the passage of more pheromone from the storage reservoirs. Ryan and 0'Ceallachain (1976, 1977) first reported the presence of a male-produced aggregation pheromone attractive to both sexes of T. confusum. Suzuki and Sugawara (1979) reported the male beetles of T. eastaneum produced an aggregation pheromone. The site of pheromone production was not previously determined by these authors. This is the first report of female and male behavioral responses in T. eastaneum to male-produced pheromonal odors. The responses include leg extension, antennal protraction, and zig-zag locomotion, behavioral responses comparable to those of other Coleoptera (Burkholder and Dicke, 1966; Kuwanara et at., 1975; Barak and Burkholder, 1977; Abdel- Kader and Barak, 1979). Males and females perceive the male-produced pheromone on the day they emerge. 'The sexes differ In that male perception reaches a maximum between 1 and 30 days after eclosion, while female response continues to increase until 30 days post-eclosion. The male pheromone secretion is apparently a continuous process within the gland, but the actual perception of the odors differs between sexes. Stuart and Satir (1968) found trail pheromone production in the termine, Zootermopsis spp.3 to be a continuous process throughout adult life. Observations on sexually active beetles indicate that there is a correlation between the amount of pheromone released from the exocrine gland and the onset of sexual maturity. Each species produces and perceives pheromones at characteristic ages that are related to sexual maturity (Shorey, 1974). Barratt (197*0 found pheromone production in Stegobium panioeum (L.) at 3 to 4 days to be related to female egg production. 0 ’Ceallachain and Ryan (1977) recorded female pheromone production and male responsiveness to be correlated with mating behavior in T. eonfusum. The ability of T. eastaneum males to produce pheromone and to stimulate response to that of 0 to 12 hr females, is con sistent with their ability to mate 0 to 3 hrs after eclosion (Dawson, 1964). Good (1936) recorded T. eastaneum males to be fertile for over 3 years. This is harmonious with 84 the present study where females demonstrated a strong attractiveness to odors from 150-day-old males at "0-days post-eclosion. In hydrocarbon bioassay studies in T. confusion, Keville and Kannowski (1975) reported that 1-pentadecene, n-hexade- cane and 1-heptadecene act as sex stimulants when exposed to 2 to 2.5 mg of each hydrocarbon. Suzuki et al., (1975) obtained strong repellency using the same compounds in lower amounts (1 to 10 yg) in T. eonfusum. These levels of biological activity appear to be high based upon the amount of compound used in the assay. Suzuki and Sugawara (1979) found attractancy to the male-produced aggregation pheromone in T. eastaneum using 3*2-32 ng of isolated pure pheromone material. In the present study, both male and female attractancy was obtained using ea. 60 ng of crude male aggregation pheromone. Since most pheromones are produced by the insect in nanogram and pieogram amounts (Tumlinson and Heath, 1976), a more sound biologically active model for pheromone perception was demonstrated in the present study. After the crude globule has been further purified, it is likely that perception to the odors will be at pieogram levels. Generally, flour beetles are graminiverous and inhabit farinaceous material that is densely packed. Therefore, a pheromone of low volatity would be expected based upon the active space maintained by the insect. Th i s would indicate a molecule with a very high carbon number (> 20) and molecular weight above 300 (Wilson and Bossert, 196 3)- Gas chromatographic analyses of the crude aggregation pheromone when compared to internal standards revealed such a compound in T. eastaneum. Apparently the pheromone gland produces several volatile fractions together with other components with a greater volatity. The low volatile components probably act as carriers for the more volatile fractions which would provide a greater control over the rate of release. Furthermore, the results presented show that on the basis of well established gas chromatograph techniques, the administration of T. eastaneum male pro thoracic femoral secretion cannot be detected with sufficient sensitivity using packed columns. In the present study, the male-produced prothoracic leg secretion is perceived by olfactory stimuli in both sexes. The synthesis of the male aggregation pheromone in T. eastaneum could lead to a useful tool for monitoring and control of populations in stored grain. In that both sexes are attracted from a distance, this pheromone could prove to be of practical value in a pest management program. 86 ANALOGOUS SEXUALLY DIMORPHIC MALE SETIFEROUS STRUCTURES Introduction Secondary sex characters, traits which are not directly related to reproduction, have proven to be useful tools for taxonomists since they provide an easy sex identification. In insects, particularly the Coleoptera, these unique morphological traits include differences in elytron char acteristics, leg structures, punctures or pits, pronotal ornamentation (i.e., Lamellicorns), body color or size, mandible variations, etc. Generally, secondary sex char acters play a significant function in mating behavior in animals where there is no parental care of offspring (Ward and Humphries, 1977; Otte, 1979). One particular type of trait, a setiferous puncture, is a secondary sex character found in several male insects. Presently, this structure has been observed on a variety of anatomical locations in numerous families of the Coleoptera: Mitidulidae (Murray, 1905), Staphylinidae (Casey, 1905), Tenebrionidae (Hinton, 19^2; Triplehorn, 1952; Hope, 1953; Sokoloff, 1972; Halstead, 1963), Leiodidae (Sheeler, 1979), Cleridae (G. Ekis, personal communication), and Anthribidae (B. Valentine, personal communication) 86 87 although little emphasis has been placed upon the function of this hairy structure. The sub-basal setiferous pur:ire on the male prothoracic femora in Tribolium castaneum (Hinton, 19^2) was found to play an exciting role in the regulation of population density and mating behavior through a sophisticated aggregation pheromone chemical communication system (Faustini, 1979). Considering the few known details of this structure, a comparative study of sexual dimorphic male setiferous punctures found in other Coleoptera was undertaken. I report here through scanning electron microscopy, the ultrastructure of these analogous morphological characters from eight families of the Coleoptera and suggest a possible functional role. Materials and Methods Scanning Electron Microscopy Prior to SEM preparation, some of the specimens were cleaned in a hexane bath for about 35 min to dissolve the setiferous secretion. These specimens will be noted in the text. Male beetles were mounted on Cambridge stubs using a fast drying silver colloidal paint. Preparations were coated with 200 I of a 60:40 gold:palladium mixture applied by sublimation under vacuum, using a Hummer I 83 sputter coater. Specimens were viewed on a Hitachi S-500 scanning electron microscope with an accelerating volt..... ;• of 20 kV. Insects Many of the insects for this study were obtained from private or museum collections. These included the following families: (1) Staphylinidae, obtained from Mr. Larry Watrous; (2) Leiodidae, Mr. Quentin Wheeler; (3) Cleridae, Dr. Ginter Ekis; (4) Anthribidae, Dr. Barry Valentine; and (5) Tenebrionidae, Dr. Charles A. Triplehorn. Live specimens were obtained from the OSU Extension Service. These included: (1) Dermestidae, Ptimidae, Tenebrionidae, Lucanidae and Scarabaeidae, Dr. James Sargent. Scanning Electron Microscopy Results Super family Tenebrionoidea The homologous setiferous structure in the confused flour beetle, Tribolium confusum DuVal (Plate VIII) differs from T. castaneum (Herbst) (Plate III) in the diameter of the femoral puncture, sensilla morphology, sensilla number/ puncture, and amount of secretion formed. The helical sensillae (Callahan, 1975) in T. confusum are relatively long and rigid in appearance (Plate VIII-D) . Although the setiferous puncture is found on the pro-, meso- and metathoracic femora, there is a gradual reduction in PLATE VIII Explanation of Figures A Prothoracic femoral (PF) puncture (SP) in Tvibolium aonfusum (2000x) B Mesothoracic femoral puncture (MSF) in T. aonfusum (2000x) C Metathoracic femoral puncture (MTF) in T. aonfusum (2000x). Note the gradual reduction in puncture size and sensilla number when compared to the pro- and mesothoracic setiferous punctures. D Prothoracic setiferous puncture (PF), helical sensillae (HS) and setiferous secretion (S) in T. aonfusum. Secretion appears absent in the meso- and metathoracic punctures. PLATE VIII 91 puncture size and sensilla number (Plate VIII-ASB,C). The mean number of sensilla in the setiferous puncture decreases from 12.72 ± 2.2642 sensillae/puncture (prothoracic femor)j to 8.96 ± 1.0935 (mesothorax), and 4.28 ± 1.1 metathoracic femora. In older males of T . confusum^ accumulation of secretion is not observed (Plate III-B, V-D) and the secretion appears absent in the meso- and metathor acic punctures. In males of Alobates movio (P.) a setiferous structure is present on the mentum (Plate IX). The seta in this structure appear long and elastic. A surface tension which binds these structures together is created by a secretion which coats their length (Plate IX-B). An analogous structure to that observed in the above insects occurs on the first abdominal sternite in Triorophus sp. males (Plate IX-C,D). A dense secretion coats the sensilla within this central region of this structure. The cuticle also appears to be covered with a secretion. A sparse number of sensilla are observed along the margin of the setiferous structure. Close examination reveals that the cuticle in this region is not covered by a secretory product as observed in Plate IX-D. In the long-headed flour beetle, Latheticus oryzae Waterhouse a setiferous puncture appears on the labium of males fPlate X-A.BK Numerous sensillar structures arise *•; 'I t ^ v' r ’:v‘ r‘ “ ‘' c* ■v-u-r. ,'11 •? v ° t ^ ;1 3,bov° h0 r' 1 3.r> 0 PLATE IX Explanation of Figures A Setiferous puncture (SP) on the mentum of Alobates mor-to male. B Enlargement of mentum structure showing secretion (S) covering sensillae. C Setiferous structure on the first abdominal sternite in Triorophus sp. male. D Enlargement of the Triorophus structure shows 'accumulation of a secretion along the length of the sensillae. 92 PLATE IX 93 PLATE X Explanation of Figures A Setiferous puncture on the labium of Lathetious ovyzae male. B Enlargement of L. ovyzae male structure which exemplifies a secretion (S) coating the sensillae. C Setiferous puncture on the metathoracic tibia (MT) of Sootobaenus pavallelus male. D Setiferous puncture on the metathoracic femora (MF) of S. pavallelus male. 94 PLATE x of the cuticle. A smaller number of sensillae are observed around the margin of this structure. A secretion (Plaw X-B) can be readily observed coating the sensillae within the setiferous puncture. This structure, under high magnifica tion, appears to be analogous to the setiferous structure observed on the prothoracic femora of T. castaneum. Setiferous structures are present on the metathoracic tibia and femur of Scotobaenus parallelus LeConte males (Plate X-C,D). The tibial structure is reduced .in size when compared to the femoral patch of setae. Prior to examination, this specimen was cleaned in a hexane solvent bath in an attempt to observe surface pores with either structure. However, due to the high density of sensillae cuticular pores could not be observed. In the forked fungus beetle, Bolitothsvus cornutus (Panzer), a patch of setae occur on the pro-, meso- and metathoracic femora of the male (Plate XI-A). A dense patch of setae also are found on the mesosternite, second and third abdominal sternites (Plate XI-B,D). Upon close examination a secretion which coats these corregated sensillae (Callahan, 1975) is associated with each of these patches (Plate XI-C). All of these structures are absent in the female (Plate XII). PLATE XI Explanation of Figures A Setiferous puncture (SP) meso- and metathoracic femurs in Bolitotherus aornutus male. B Setiferour puncture on the metasternite of B. aornutus male (circle). C Enlargement of metasternite setiferous puncture showing secretion (S) coating sensillae. D Setiferous punctures (SP) on the second, third and fourth abdominal sternites in B. aornutus male. 97 PLATE XI 98 PLATE XII Explanation of Figures A Absence of meso- and metathoracic setiferous punctures in the female B. aornutus. B Absence of second, third and fourth abdominal sternite setiferous punctures in the female B. aornutus. 99 PLATE XII 101 Superfamily Staphylinoidea In the staphylinid, Homoeotarsus gastrolobium bias lev Graves (Casey), a setiferous puncture located above the frons, on the dorsal region of the head is found in the males (Plate XIII). Each sensilla arises from a single pit as observed in a clean specimen (Plate XIII-B). However, a secretion can be observed binding these sensillae together in an uncleaned specimen (Plate XIII-C) On the second abdominal sternite of another Staphylinid male, Neobisnius sp.3 a setiferous puncture occurs (Plate XIV). Upon close examination, a secretion can be observed binding the sensillae within this structure (Plate XIV-B). Bifovear setiferous structures are located on the first abdominal sternite in the male leiodid, Anisotoma bifoveata Wheeler (Melsheimer) (Plate XV-C,D). The cuticle around this structure is devoid of setae. High magnification disclosed a secretory product coating the sensilla within this setiferous structure (Plate XV-D). Superfamily Bostriahoidea In Ptinidae, a setiferous puncture is located on the metasternal disc on the shiny spider beetle, Gibbium psylloides (Czenpinski) males (Plate XV-A,B), Due to the density of the plumose seta within this structure, cuticular pores could not be observed. PLATE XIII Explanation of Figures A Setiferous puncture (SP) on the dorsal region of the head on Homoeotarsus gastrolobium bicolor male. B Setiferous puncture after treatment with distilled hexane. C Setiferous puncture untreated with solvent showing sensillae coated with secretion (S). 102 PLATE xiii 103 PLATE XIV Explanation of Figures A Setiferous puncture on the second abdominal sternite on Neobisnius sp. male. B Enlargement of the setiferous structure showing secretion (S) coating sensillae. 104 PLATE XIV 105 PLATE XV Explanation of Figures A Setiferous puncture on the metasternal disc in Gibbium psylloides male. Area marked with circle is shown in fig. B. B Enlargement of G. psylloides male setiferous structure. C Bifovear setiferous structures on the first abdominal sternite in Anisotoma bifoveata male. Area marked with circle is shown in fig. D. D Enlargement of a male setiferous structure in A. bifoveata showing a secretion (S). 106 PLATE XV 107 108 Superfamily Dermestoidea In the male larder beetle, Dermestes lardarius L., setiferous punctures occur on both the third and fourth abdominal sternltes (Plate XVI). Despite the dense setae which cover the body of this insect, an absence of setae occurs on the cuticle outlying these structures (Plate XVI- A). A major pore is located within the center of each puncture (Plate XVI-B,C), whereby a secretion is produced as observed in an uncleaned specimen (Plate XVI-D). A smaller pore is associated with each sensilla arising from the setiferous puncture. Superfamily Cleroidea In five species of Clerids, setiferous punctures occur on the metathoracic tibia in the males (Plates XVII- XIX). Within each species, clusters of secretory ducts can be found interdispersed between the sensillae in the setiferous puncture. A slightly higher degree of secretory duct complexity occurs among each species. Nonetheless, each of these structures appear analogous to that found in T. castaneum. Super family Curculionoidea In the Anthribid Ptychoderes nebulosus (Olivier), a setiferous puncture on the first abdominal sternite occurs in the male (Plate XX-A,B). Enlargement of this structure reveals a dense secretion coating one sensillae and cuticle. PLATE XVI Explanation of Figures A Setiferous punctures on the third and fourth abdominal sternite in Dermestes lardarius male. B Enlargement of third abdominal sternite setiferous puncture with major central pore (MP). Note the small pores associated with each sensillae. C Enlargement of major pore treated with distilled hexane. D Untreated setiferous puncture showing secretion (S) exuding from major pore in male. 109 PLATE XVI 110 ______ a PLATE XVII Explanation of Figures A Setiferous puncture (SP) on the metathoracic tibia (MT) in Polonium literatum male. B Enlargement of secondary ducts (SD) or pores interspersed between sensillae within P. literatum setiferous puncture. C Setiferous puncture (SP) on the metathoracic tibia (MT) in P. lampyroides male. D Enlargement of secondary ducts found within the setiferous structure in P. lampyroides. Ill PLATE XVII 112 PLATE XVIII Explanation of Figures A Setiferous puncture (SP) on the metathroacic tibia (MT) in Iehnea elongata. B Enlargement of secondary ducts (SD) found inter dispersed between sensillae in I. elongata. C Metathoracic tibia (MT) setiferous puncture (SP) in Pytiaera aoronata male. D Enlargement of secondary ducts found within the P. aoronata setiferous structure. 113 I u , - i - - . i > I______ 111ax a i v i d PLATE XIX Explanation of Figures A Setiferous puncture (SP) of metathoracic tibia (MT) on Tlatynoptera lyaiformis male. B Enlargement of secondary reservoirs (SR) found interdispersed between setiferous sensillae. C Secretory ducts (SD) or tertiary reservoirs found within the secondary reservoirs in P. lyaiformis male. 115 PLATE XIX 116 PLATE XX Explanation of Figures A Setiferous puncture on the first abdominal sternite in Ptychoderes nebulosus male. B Enlargement of P. nebulosus structure showing secretion (S) coating sensillae. C Setiferous puncture on the metathoracic coxa in Phoeotragus heros male. Area marked with circle is shown in fig. D. D Enlargement of P. heros setiferous structure show ing secretion (S) coating sensillae. 117 PLATE XX 118 119 A setiferous puncture located on the metathoracic coxa of Phoetragus heros (F.) males (Plate XX-C,D). Close exa;1 ra tion reveals a secretion coating the sensillae within the puncture. A setiferous puncture occurs on the basal segment of Deuteroorates longieornus (F.) (Plate XXI). Secretory pores, as well as a secretion coating the sensillae, can be observed within the setiferous puncture (Plate XX-B,C). Super family Searabaeoidea Although the setiferous structures observed on the prothoracic femora of the Lamellicorns are not sexually dimorphic a few noteworthy observations can be made. In the Scarab, Onthophagus heaate Panzer, the prothoracic femoral setiferous puncture appears as a dense patch of hairs in a cleaned specimen (Plate XXII-A,B). However, in an uncleaned specimen, scanning electron microscopy revealed this structure to be coated heavily with a globular secretion (Plate XXII-B), In the Lucanid, Pseudolueanus eapreolus (L.), a femoral setiferous puncture appears homologous to that of the Scarabaeidae (Plate XXII-C,D). Also associated with this structure is a secretion which coats the sensillae. Table 19 shows a current list of Coleoptera in which similar sexual dimorphic characters have been found. This list includes seven superfamilies, eight families, twenty- four genera and fifty-four species. PLATE XXI Explanation of Figures A Setiferous puncture (SP) on the basal antennal segment in Deuteroorates longieornus male. B Secretion (S) coating sensillae in D. longieornus. C Secretory pores (SP) found interdispersed within the setiferous puncture in D. longieornus. 120 PLATE XXI 121 _ #a“ *»#», * * ►* *-»«. •; ,- o 3 " ■ * 1 WkwU*&'£ ' V V * ■ i c i y £ * j* * j ^ i **% ^ > v jfm 9 ' PLATE XXII Explanation of Figures A Prothoracic femoral setiferous puncture on Onthophagus heoate male treated with hexane. B Non-solvent treated setiferous structure showing secretion (S) on 0. heoate. C Prothoracic setiferous structure on Pseudoluoanus capreolus male. D Secretion shown at the base of sensillae on P. capreolus. 122 PLATE xxii 123 124 TABLE 19 Superfamily Staphylinoidea Character and Sexual Expression References Staphylinidae: * Homoeotarsus gastrolobium : setiferous puncture above bioolov the frons, on the dorsal region of the head * Neobisnius sp. < ? : setiferous puncture on the 2nd abdominal sternite Leiodidae: * Anisotoma bifoveata : two setiferous punctures on Wheeler, 1979 the 1st abdominal sternite Superfamily Dermestoidea Dermestidae: Demestes frisahii D. peruvianus D. haemorrhoidalis ■ D. lardarius * D. atev D. m a m o v a t u s D. c a m i v o r u s D. mwcinus D. undulatus < Z : setiferous punctures on the Hinton, 1945 D. talpinus 3rd and 4th abdominal ster nite D. laniarius D. mustelinus D. ol-ivieri D. bieolor D. nidrn * Presently have SEM micrographs of these specimens. 125 TABLE 19 (CONTINUED) Superfamily Bostrichoidea Character and Sexual Expression References Ptinidae: * G-ibb-Lwn psylloides G. boieldieui puncture with erect hairs Hinton, 1941 on the metasternal disc Trigonogenius globulus Pseudeurostus hilleri shallow, oval setiferous Hinton, 1941 puncture on the 5th abdominal sternite Superfamily Cleroidea Cleridae: Pelonium lampyvoides P. lituratum Pytiaera aoronata : setiferous puncture on the metathoracic tibia Platynoptera lyciformis Iahnea elongata Superfamily Cucu.joidea Nitidulidae: Brachypeplus rubidus : setiferous puncture on the Murray, 1864 prosternum Ithypheneus bakeri : two setiferous punctures on the labrum Paromia sp. (?) c f : two setiferous punctures on the submentum and labrum Superfamily Tenebrionoidea Tenebrionidae: * Bolitotherus aomutus cf : setiferous punctures on the Triplehorn, 1952 pro-, meso- and metathoracic femurs; and metasternite, 3rd and 4th abdominal ster- nites TABLE 19 (CONTINUED) Superfamily Tenebrionoidea Character and Sexual Expression References (continued.) Tenebrionidae: * Triorophus sp. : setiferous structure on the 1st abdominal sternite Sootohaenus parallelus setiferous punctures on the metathoracic femur and tibia * Alobates morio J : setiferous puncture on the — mentum * Lathetious ovyzae setiferous puncture on the - labium * Triboliwn oastaneum T. madens T. audax T. freemcmi T. aylindricum setiferous puncture on the Hinton, prothoracic femur T. politum T. waterhousei T. parki T. apioulum T. aloine : setiferous punctures on the Hinton, pro- and mesothoracic femur T. gigcmteum T. anaphe < f : setiferous puncture on the Hinton, prothoracic femur * T. eonfusum setiferous punctures on the Hinton, pro-, meso- and metathoracic femur Superfamily Curculionoidea Anthribidae: Euparius sutesselatus : two setiferous punctures along the medial longitudi nal metasternal groove TABLE 19 (CONTINUED) Superfamily Curculionoidea Character and Sexual Expression References (continued) Anthribidae: Deuterocrates longieornus & : setiferous puncture on the basal antennae segment Phoeotragus heros d : setiferous puncture on the metathoracic coxa Ptychoderes nebulosus ( f : setiferous puncture on the 1st abdominal sternite Superfamily Scarabaeoidea Lucanidae: * Pseudolucanus capreolus Passalidae: Popilius disjunctus ^ : setiferous puncture on the prothoracic femur Scarabaeidae: * Onthophagus hecate Canthon laevis Aphodius prodramus Geotrupes splendidus 128 Discussion For the first time, the ultrastructure of sexually dimorphic setiferous punctures in Coleopterous species have been described in detail. These structures appear to be analogous to that which is found in Tvibolium aastaneum. In T. aastaneum, the secretion exuded from the obscure femoral structure is obviously an aggregation pheromone which attracts both sexes. Of the Tribolium species investigated to date, all have similar femoral setiferous punctures; details, however, are slightly different. Nonetheless, the apparent homology of these structures in each species may reveal insights to the evolutionary relationships among Tribolium species-groups, The setiferous punctures observed in other Coleoptera are of a very peculiar type. It has been reported that these organs are present in a variety of anatomical regions; metathoracic tibia, abdominal sternites, antennal segments, coxae, femora, mouthparts (i.e., mentum, labrum, labium) and head (Murray, 1905j Casey, 1905', Triplehorn, 1952; Halstead, 1963; Wheeler, 1979). Therefore these structures cannot be regarded as homologous. Nevertheless, there are some interesting common features in their morphology, which may assist in the understanding of the evolution of these highly developed and unique structures in the Coleop tera. All of these sexually dimorphic structures are 129 restricted to males only. Many have cuticular pores associated with the puncture, which appear to act as a reservoir for a common secretory duct. In the Cleridae and Anthribidae, small cuticular ductiles enter into a secondary reservoir similar to that observed in T. eastaneum. Various forms of sensillae occur in each structure, all of which extend above the plane of the cuticle. Of primary significance is a secretion associated with many of the setiferous structures. Since most of the specimens were preserved prior to examination, laboratory bioassays could not be conducted to evaluate the role of this secretory material. However, based upon comparisons among other structures, the secretion appears to be a common morphologi cal feature. The primary function of the sub-basal setiferous puncture on the prothoracic femora of T. eastaneum males has been confirmed by detailed observations to be a site of aggre gation pheromone production. This system has probably been selected for as a means of increasing intraspecific competition for a mate in natural habitats, resulting in optimum propagation of the species. Although analogous setiferous structures in other Coleoptera remain to be studied, it seems likely that many will be found to function' as an acute sensory apparatus for the recognition of species individuals. Sexually dimorphic setiferous structures have been found in seven superfamilies, eight families, twenty-? v- genera, and fifty-four species of the Coleoptera. The setiferous puncture in T. aastaneum functions as a site of aggregation pheromone production and is parsimonious in that both sexes are attracted by the same secretory product. Analogous structures present among other Coleop tera possibly play a comparable role based upon common morphological features. SUMMARY Evidence for the existence of a male-produced aggrega tion pheromone attracted to both sexes of Tviboltum eastaneum (Herbst) is reported and the ultrastructure of the secretory site is described. In the present study, using a multiple-choice olfacto meter, both male and female attractancy was obtained using approximately 60 ng of crude male prothoracic setiferous puncture secretion. Both sexes responded significantly to male globules and 150-day-old males with large prothoracic femoral globules. Males and females perceive the pheromone on the day they emerge. Actual perception differs between the sexes: male perception reaches a maximum on day 1 post-eclosion, when tested at < 1, 1 and 30 days; while female response continues to increase until 30 days post- eclosion. Based upon the weight of the secretion, 1.2 ng/ globule, responses by both sexes to the male produced odors are within nanogram levels to elicit biological activity. This was the first report of male and female behavioral responses in T. eastaneum to male-produced phermonal odors. These responses include prothoracic leg extension accompanied by bobbing movements, followed by locomotion, antennal 131 132 protraction and orthokinetic movements to the source of - odor. Using gas liquid chromatographic techniques, the pheromone gland secretion was resolved into two fractions, one highly volatile and the other of low volatility. Glass capillary GLC with high attenuation yielded over 250 com ponents, even though the same product in a packed column could not be resolved. Scanning electron microscopy observations disclosed a globular secretion covering the sub-basal setiferous puncture. Numerous sensillae arise from within the puncture and continue above the plane of the cuticle. Secondary reservoirs are found interdispersed between the sensillae along the cuticular basement of the puncture. Tertiary reservoirs, minute cuticular ducts, are found within the secondary reservoirs. The epidermal cells of the setiferous puncture appear to be active secretory cells. These cells have cuticular secretory ducts, large nuclei, and lipid secretory reser voirs. It appears that the lipid secretion contains the pheromone, based upon the female and male age and relative attractiveness response. The lipid is transported to the puncture by secretory duccs, where the pheromone evaporates, resulting in the passage of more pheromone from the cell storage reservoirs. After release from the exocrine cells, the secretion flows along the surface of the cuticle within the puncture and coats the sensillae. The secretion was shown to accumulate in older beetles. The ultrastructure of the femoral puncture, combined with the presence of a specialized secretory epidermal layer on the inner surface of this structure, implies that the male has a unique exocrine gland that synthesizes and releases a lipid to the surface of the body. A comparative study of sexual dimorphic male setiferous punctures in eight families of the Coleoptera was reported through scanning electron microscopy. These structures appear to be analogous to that which is found in T . eastaneum. Common morphological features include various sensillae forms, cuticular pores associated with the puncture, and a secretory product. Although analogous structures in other Coleoptera remain to be studied, it seems likely that many will be found to function as acute sensory apparatus for the recognition of species individuals. In conclusion, the male-produced prothoracic leg secretion is perceived by olfactory stimuli in both sexes. 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Am., 60: 575-582, APPENDIX A TABLE 20 Light Microscope Solutions Sorensen’s Phosphate buffer 0.1 M, pH 7-2 (SPB) 2.22 g NaH2P04»H20 5.115 g Na2HP0u to 500 ml with distilled H 20 4% Glutaraldehyde 20 ml 8$ Glutaraldehyde 20 ml SPB Osmotically Adjusted SPB 3.34 g Glucose 100 ml SPB 1% Osmic Acid (Osmium tetroxide, OsOn) 0.675 g Glucose 0.25 g OgO4 25 ml SPB Toluidine Blue Stain (2% solution) 10 ml Toluidine blue 10 ml Sodium borate 5 ml Pironine B Spurr Standard Embedding Medium 10 ml Vinylcyclohexane (VCD) 6 ml Diglycidyl ether of polypropyleneglycol (D.E.R. 736) 26 ml Nonenyl succinic anhydride (NSA) 0.4 ml Dimethylaminoethanol (DMAE) 8 Cure schedule (hr) at J0°C 3.4 Pot Life (days) 147