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Anatomy and histology of the male reproductive system of the fire ant, Solenopsis invicta Buren (Hymenoptera : Formicidae)
Int. J. Insect MorphoL & Embryol., Vo|. 13, No. 4, pp. 283 to 294, 1984.
Printed in Great Britain.
0020- 7322/84$3.00+ .00 © 1984PergamonPressLtd.
ANATOMY A N D HISTOLOGY OF THE MALE REPRODUCTIVE SYSTEM OF THE FIRE ANT, SOLENOPSIS INVICTA BUREN (HYMENOPTERA • FORMICIDAE)* DAVID E. BALL']" and S. B. V1NSON Department of Entomology, Texas A&M University, College Station, Texas 77843, U.S.A. ( A c c e p t e d 13 M a r c h 1984)
A b s t r a c t - - T h e male reproductive system of the fire ant, Solenopsis invicta Buren (Hymenoptera : Formicidae), consists of the testes, vasa efferentia, vasa deferentia, seminal vesicles, accessory glands, ejaculatory duct, wedge, aedeagal bladder, and external genitalia. The testes in newly eclosed males appear as 4 large white lobes filled with packets of sperm. Each lobe of the testes contains only one follicle. As the testes degenerate, the maturing sperm migrate through the vasa efferentia and vasa deferentia into the seminal vesicles. The seminal vesicles attach to the accessory glands, which are lined with secretory columnar epithelium. The posterior ends of the accessory glands taper and unite to form the ejaculatory duct. A sclerotized wedge is found at the junction of the accessory glands and the ejaculatory duct. An aedeagal bladder, joining the ejaculatory duct posterior to the wedge, is lined with s q u a m o u s epithelium enveloped by heavy musculature. The ejaculatory duct continues posteriorly to form a distal aedeagus surrounded by 3 pairs of valves, comprising the external genitalia.
Index descriptors (in addition to those in title): Epithelium; scanning electron microscopy; maturation; seminal vesicle; accessory glands; aedeagal bladder. INTRODUCTION
THE CONTROL of the fire ant, Solenopsis invicta Buren, has been the subject of considerable research over the last 2 decades. However, little emphasis has been placed on non-chemical forms of control, such as disrupting the reproductive processes, genetic manipulation, or a sterile male release program. Successful use of these techniques would require an extensive knowledge of the anatomy and histology of the male reproductive system. Studies of the male reproductive system have been conducted on at least one species in nearly all the subfamilies of ants. However, to review all the available literature would require a separate monograph, therefore, we will primarily confine our review to the Myrmicinae and refer to the other groups where applicable. Forbes (1954) reviewed the early studies done on the male reproductive system in ants and conducted one of the first extensive anatomical and histological examinations of the male reproductive system of Camponotus pennsylvanicus DeGeer. Janet (1902) described the male reproductive system of Myrmica rubra L. which was reinvestigated by Trakimas
*This manuscript is approved as TA 18703 by the Director of the Texas Agricultural Experiment Station in cooperation with A . R . S . / U . S . D . A . This research was supported by the Texas Department of Agriculture lnteragency agreement IAC (82-83) 0982. t T h i s research was conducted by D.E.B. as part of the requirement for the Ph.D. degree. 283
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(1967). Tice (1967) examined the histology of the male reproductive system of the black imported fire ant, Solenopsis saevissima richteri Forel. According to Janet (1902), the male reproductive system consists of the testes, vasa deferentia, seminal vesicles, ejaculatory duct, and external genitalia. However, additional reproductive structures have been discovered since then. A posteriorly located aedeagal bladder has been found in S. s. richteri (Tice, 1967), M. rubra (Trakimas, 1967) and in other subfamily genera. The aedeagal bladder and a chitinous wedge in the ejaculatory duct were first discovered by Clausen (1938). An aedeagal gland, was briefly mentioned by Trakimas (1967). Distinct accessory glands were found in Dorylus labiatus Shuck (Mukerjee, 1927), Eciton hamatum (Fabr.) (Forbes, 1958) and Neivamyrmex harrisi (Halderman) (Forbes and Do-Van-Quy, 1965). Recently, the nomenclature of Snodgrass (1935, 1956) was used to identify the accessory glands as the more correct term for the seminal vesicles in Formica polyctena Foerst (Jeantet, 1972) and many other genera of ants including S. invicta (Hung and Vinson, 1975). The purpose of this study was to examine the developmental anatomy and histology of the male reproductive system of the imported fire ant from eclosion to after mating. MATERIALS
AND
METHODS
Colonies with male pupae and alates were collected during the summer m o n t h s from fields at College Station, Texas. The colonies were excavated and transported to the laboratory where the ants and brood were flooded out with water (Jouvenaz et al., 1977). The ants were transferred to plastic shoe boxes (Vinson and Robeau, 1974) and fed a mixture of insects and honey water. The emerged male alates in each colony were removed and destroyed. The newly emerging males were transferred daily to small plastic dishes with ca 50 worker nestmates since previous work showed that males lived longer when maintained with workers (Ball and Vinson, unpublished data). In this way, newly emerged males and males of known age were obtained. Additional males were collected flying from the surface of the m o u n d prior to mating (preflight) and after returning (postflight). Sterile males (Hung et al., 1974) at least 2 weeks old were collected from laboratory colonies. After maintaining the colonies for the required length of time, 5 males were randomly selected from each age group and the entire reproductive system was dissected in distilled water by spreading the terminal gastral sclerites with microforceps and carefully pulling the claspers. After measuring the intact glands with a calibrated ocular micrometer, the entire reproductive system was placed in Zenker's fixative for 12 hr. Following dehydration, clearing and embedding in paraffin, 10 lam sections were cut and stained. H e m a t o x y l i n - e o s i n , Schiff's reagent and Mallory's triple staining procedures as outlined by H u m a s o n (1962) were used to identify mucus and cytoplasmic elements, nuclei, and thymonucleic acids. Cytochemical identification of proteins by mercury - bromophenol blue staining and acid mucopolysaccharides by the alcian blue procedure were also used as described by Pearse (1968). Scanning electron microscopy was used to observe any unusual morphological differences in the reproductive systems of fertile and sterile males. The reproductive systems from 5 preflight and 5 sterile males were compared. The entire reproductive system was immersed in 3% gluteraldehyde in a 0.1M sodium phosphate buffer solution (pH 7.2) at 4°C for 1 hr. The specimens were then rinsed 4 times for 10 min each in the buffer solution at 4°C followed by postfixation in a room temperature solution of 1% osmium tetroxide for 1 hr. After rinsing again 4 times for 10 min each in buffer at 4°C, the specimens were placed in a series of ethanol baths (50%, 70%, 90%, 95%, and 3 times in 100% ethanol) for 10 rain each at room temperature. The specimens were then transferred to Reikert capsules and placed in a critical point dryer where ethanol was replaced by CO2. All specimens were attached to aluminum stubs with glue formulated by mixing 16 gm of Foromvar (Electron Microscopy Sciences, Forth Washington, Pa.) with 60 ml of ethylene dichloride. A 200 A thickness of g o l d - palladium was evaporated onto each sample and the specimens were viewed at 12.5 kV, using a JEOL JSM-25S scanning electron microscope. Transmission electron microscopy was used to examine the histology of the aedeagal bladder and the ultrastructure of the accessory glands. Entire gasters and dissected accessory glands were fixed in 3% gluteraldehyde/l.5% paraformaldehyde/3% Acrolein in a 0. I M sodium cacodylate buffer solution (pH 7.2) for 2 hr at 4°C. The specimens were then rinsed 4 times for 15 min each in buffer followed by postfixation in 2% osmium tetroxide for 1 hr. After rinsing as before, the samples were dehydrated in an ethanol series, rinsed in 100% propylene oxide 5 times i n l hr and infiltrated with Epon araldite resin. The specimens were then transferred to fresh resin, vacuum aspirated for 15 min and heated for 3 days in a 70°C oven. The resulting blocks were sectioned (500 A), stained with toluidine blue and viewed on a Zeiss 10C/CR electron microscope at 80 kV.
Anatomy and Histologyof the Male ReproductiveSystem of the Fire Ant RESULTS
AND
285
DISCUSSION
Anatomy
The reproductive system of the fire ant, S. invicta consists of the testes and the testicular capsule, vasa efferentia, vasa deferentia, seminal vesicles, accessory glands, a sclerotized wedge, ejaculatory duct, aedeagal bladder, and external genitalia (Figs. 1, 2). In newly enclosed males the 4 follicles of the immature testes were seen as large white lobes. Four distinct follicles are also reported for M . rubra (Janet, 1902) and S. s. ,ichteri (Tice, 1967). The testes taper at the proximal end and form the vasa effe~entia which join into a short collecting duct, the vas deferens (Fig. 3). The vasa deferentia continue into the anterior tip of the seminal vesicles. The studies by Hung and Vinson (1975) and Tice (1967) do not discuss the morphological separation of the vasa efferentia from the vasa deferentia, as described in this study. The testes, vasa efferentia and vasa deferentia are enveloped in a thin fibrillar capsule, which was only found in fertile males. The 4 testes measure 2.39 _ 0.44 mm (SD) (n = 12) in length and 1.37 _+ 0.49 mm (SD) (n = 12) in width in the late pupal stage and slowly degenerated forming 4 amber-colored lobes (Fig. 4). Glancey et al. (1976) observed that the testes obtained a maximum size of ca 1.2 mm by midway through the pupal stage and gradually atrophied to form 4 ambercolored bodies 11 days after eclosion. We found that the testes were connected by a short duct, the vas deferens, to the sperm-filled seminal vesicles. A small amount of sperm was also seen in the accessory glands of several preflight males. Sterile males lack testes (Hung et al., 1974) (Fig. 5) and therefore produce no sperm. The empty seminal vesicles of immature males were 2.10 _ 0.18 mm (SD) (n = 12) in length and 0.14 _+0.03 mm (SD) (n = 12) in width. As the males matured, the length of the sperm-filled seminal vesicles remained the same, while the width increased to 0.18 _+ 0.07 mm (SD) (n = 12). The seminal vesicles curve ventrally and enter the anteromedial region of the accessory glands, which are large, muscular, peanut-shaped lobes (Figs. 1, 2, 6). The accessory glands in mature males measure 1.12 _ 0.06 mm (SD) (n = 12) in length and 0.53 _+ 0.06 mm (SD) (n = 12) in width. Glancey et al. (1976) found that the accessory glands in S. invicta increase in size as the males mature. However, the sample size was not mentioned in their report. Our study showed that there was no apparent physical change in the size of the accessory glands from the time of eclosion to 12 days old, and the average dimensions of the lobes from preflight, postflight and sterile males were approximately equal (n = 30). The accessory glands taper posteriorly and unite to form the ejaculatory duct in the area of a sclerotized V-shaped wedge. At the posterior tip of the wedge, is the opening to the aedeagal bladder. The bladder, measuring ca 1.5 mm in length and ca 0.5 mm in width, consists of a large ovoid sac covered with heavy musculature positioned in the anterior region of the external genitalia (Fig. 7). The ejaculatory duct continues posteriorly to form the aedeagus, which is surrounded by the external genitalia (Figs. I, 2). The external genitalia consist of 3 pairs of valves; the laminae paramerales (outer valve), volsellae (middle valves), and the laminae aedeagales (inner valve), which are surrounded anteriorly by the lamina annularis (basal ring). The outer valves surround the aedeagal bladder, wedge, ejaculatory duct, aedeagus, and the middle and inner valves. A small distal lobe, the paramere, is located on the lateral surface of the outer valve. Each of the paired middle valves consists of a large basal sclerite, the lamina volsellaris, and two dorsal portions; the lateral cuspis volsellaris and a median digitus volsellaris. The inner valves appear as thin paddle-shaped plates, and are positioned on both sides of the ejaculatory duct and the aedeagus. The basal ring is divided into a dorsal oval sclerite and a ventral
286
DAVIDE. BAILand S. B. VINSON
band. The configuration of the external genitalia described here is in agreement with that by Tice (1967). • '~"
/ IMMATUREM A L E ~ DEGENERATED i TESTIS OF
"~'
/ ~,i/
~b /I I \ i
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Fit;. 1. Dorsal view of male reproductive system. × 43.
Histology The follicles are enveloped in a thin capsule, which consists of long fibers resembling connective tissue. Fusiform nuclei are visible throughout the capsule layers, although distinct cellular membranes were not revealed with any of the staining procedures (Fig. 3). Forbes (1954) also found a fibrous capsule covering the testes and vas deferens in C. pennsylvanicus, but he stated that the capsule was composed of tracheal branches with fusiform nuclei. Sperm in each follicle of the testes were arranged in oval packets in the black pupal stage and newly eclosed males (Fig. 3). Each packet contains several hundred sperm with the heads oriented toward one axis of the packet. A thin membrane and one or more nuclei are occasionally seen along the periphery of the sperm packets stained with Schiff's reagent. Hematoxylin - eosin showed dense concentrations of DNA, verifying the identity of the peripheral nuclei. The sperm packets were more concentrated toward the center of each follicle with loose, mature sperm visible along the inner periphery of the testicular wall. No meiotic stages were seen in any sections of the testes. Gradually, the sperm packets became less predominant in the follicles until shortly after the 7th day only 1 0 - 15
Anatomy and Histology of the Male Reproductive System of the Fire Ant
FJ~. 2. Ventral view of male reproductive system, x 43.
Fx(~. 3. Diagram of a section through testis and ducts.
287
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DAVID E. BALL and S. B. VINSON
Fl(;. 4. Micrograph of a whole mount of seminal vesicle and degenerated testes of a fertile male Fl(;. 5. Micrograph of a whole mount of seminal vesicle of a sterile male.
Anatomy and Histology of the Male Reproductive System of the Fire Ant
FI(;. 6. Diagram of a section through seminal vesicle and accessory gland.
FI(;. 7. Micrograph of a longitudinal section of posterior end of male gaster. Anterior end at top and ventral side at right. AB - aedeagal bladder; M - muscle; SgG = subgenital plate gland; W wedge.
289
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DAVIDE. BAILand S. B. VJNSON
sperm packets per follicle were visible. At approximately 10 days after eclosion the testes were degenerated and formed 4 amber-colored bodies attached to the tip of the seminal vesicles by the apparent remains of the vas deferens. The histological separation of the vasa efferentia and vas deferens was indiscernible at this development stage, altl~ough the close association of the connecting duct to the seminal vesicles and the degenerated testes suggested that it was the structurally reduced vas deferens, since the 4 vasa efferentia were not visible. The remnants of the capsule were still seen as a thin layer surrounding the vas deferens and degenerated testes. Both the vasa efferentia and vasa deferentia in immature males are lined with short columnar epithelium attached to a distinct basement membrane surrounded by several layers of muscle fibers. Sterile males lacked the vasa efferentia and vasa deferentia. The columnar epithelium of the vas deferens continues into the seminal vesicles. The seminal vesicles serve as the storage area for mature sperm prior to mating. The columnar epithelial cells lining the vesicles are slightly taller than the cells found in the vasa efferentia and vasa deferentia and have basally located nuclei. As the seminal vesicles filled with sperm during maturation of the male, the epithelial cells became more cuboidal, probably owing to the compression of the cells by the stored sperm. At the connection of the seminal vesicles to the accessory glands, the epithelial cells are elongated and appear to form a distinct valve, which may prevent sperm from entering the accessory glands (Fig. 6). However, some sperm were observed in the accessory glands of preflight males, although this may have been the result of dissection trauma. The epithelium of the seminal vesicle is attached to a basement membrane, surrounded by 1 or 2 layers of muscular fibers, which are thicker in the posterior region of the vesicle closest to the accessory gland. The epithelium of the accessory gland is composed of simple columnar cells on a distinct basement membrane, surrounded by 2 or 3 layers of muscle fibers (Fig. 6). The basally located nucleus in each cell is spherical and the cytoplasm and lumen of each lobe in mature males are filled with numerous basophilic granules, surrounded by a slight amount of acidophilic material. Mallory's triple stain showed an increase in the mucosal concentration in the lumen of the gland as the males matured. Deep blue staining with the m e r c u r y - bromophenol blue technique indicated high concentrations of protein in the distal region of the epithelial cells, the lumen of the gland and vesicles in the supranuclear cytoplasm. Transmission electron microscopy showed that the vesicles were polymorphic in shape and bounded by a membrane (Fig. 8). The material in the vesicles appeared as non-membranous coalesced material when viewed at higher magnification (see insert, Fig. 8). The fibrillar particles comprising the coalesced material could also be seen in the cytoplasm of the vesicles. The vesicles appeared to be released into the lumen of the gland by the rupturing of the epithelial cells. The lumen of the gland in preflight males was filled with these vesicles (Fig. 9). A major portion of these vesicles was voided during the mating flight, since the lumen of each lobe in postflight males was nearly emptied (Fig. 10) and the epithelial cells were greatly reduced in size and number. Pupal and newly eclosed males had considerably less proteinaceous activity in the gland than mature males. Long fibrillar processes extending from the epithelial cells into the lumen of the gland were revealed with hematoxylin - eosin in males at least 5 days post-eclosion. The fibrillar extensions were probably a result of the rupturing of the cells during the secretory processes. The alcian blue staining procedure showed no significant concentration of acid mucopolysaccharides in the accessory gland. However, sperm entering the gland in preflight males immediately before the mating flight showed a
Anatomy and Histologyof the Male ReproductiveSystem of the Fire Ant
291
FIG. 8. Micrograph of accessorygland vesicles. Insert--coalesced material and fibrillar partictes. characteristic clear blue-green color indicating a slight a m o u n t o f acid mucopolysaccharide. The accessory glands unite to form a common ejaculatory duct in the region of the sclerotized wedge. The ejaculatory duct is lined with columnar epithelium at the junction of the accessory glands, and there appeared to be a gradu~il transition from the epithelium of the accessory gland to the ejaculatory duct. Midway in the duct, the cells gradually shorten to form cuboidal epithelium, which continues into the aedeagus. The epithelium is attached to a basement membrane, enveloped by an inner layer of longitudinal muscle fibers and an outer layer of circular muscle fibers. The arms of the V-shaped sclerotized wedge arise from the ventrolateral epithelium of the ejaculatory duct (Figs. 1, 2, 7). The posterior tip of the wedge is not attached to the duct wall and appeared to extend freely in the lumen. A wedge has been found in the male reproductive system of representative species of the Ponerinae (Hagopian, 1963), Dorylinae (Forbes, 1958; Forbes and Do-Van-Quy, 1965; Ford and Forbes, 1980), Myrmicinae (Tice, 1967; Trakimas, 1967) and Formicinae (Forbes, 1954). Although a
292
DAVID E. BALL and S. B. VINSON
FIc~. 9. Micrograph of a section of accessory gland of a preflight male, x 100. FI(~. I0. Micrograph of a section of accessory gland of a postflighl male. × 100.
Anatomy and Histology of the Male Reproductive System of the Fire Ant
293
specific function has yet to be determined, we felt that the wedge may have been used as a structural support for the connection of the accessory glands with the ejaculatory duct and, as Hagopian (1963) suggested, as a directing mechanism for the sperm and accessory gland secretions. The aedeagal bladder enters the ejaculatory duct near the posterior tip of the wedge. The aedeagal bladder, enclosed by the external genitalia, is lined with a single layer of squamous epithelial cells surrounded by heavy muscular fibers (Fig. 7). The lumen of the bladder contains an amorphous mass with numerous acidophilic granules. These findings are in agreement with those of Tice (1967) and Trakimas (1967). The material was found in approximately equal amounts in preflight, postflight and sterile males, but was absent from one-day-old males. Further studies using males of known ages showed that the gland material began appearing at 3 days post-eclosion, indicating that the material was either being deposited or manufactured in the bladder after adult emergence. However, secretory activity by the cells lining the lumen was not evident histologically. A slight amount of protein, indicated by a light-blue staining with m e r c u r y - bromophenol blue, was seen throughout the lumen of the gland. None of the other staining procedures was positive, except hematoxylin-eosin, which revealed the nuclei in the squamous epithelium. A large mass of glandular cells was found on the inner surface of the 9th sternite. H/311dobler and Engel-Siegel (1982) have found similar groupings of cells, which they identify as the "subgenital plate gland" in representative species of every subfamily of ants. Although S. invicta was not examined in their report, we have determined that the group of cells in the fire ant were morphologically similar (Fig. 7). Tice (1967) also described a subgenital gland in the black fire ant, S. saevissima richteri, similar to that in S. invicta. The subgenital plate gland in S. invicta is composed of columnar epithelium with basally located nuclei. The cells have long slender ducts arising from the supranuclear surface of the cell membrane extending into the cuticle of the ant. REFERENCES CI.AUStN, R. 1938. Untersuchungen tiber den m~innlichen Copulationsapparat der Ameisen, speciell der Formicinae. Mitteil. Schweizer. Entomol. Ges. 17: 2 3 3 - 346. FORBES, J. 1954. The anatomy and histology of the male reproductive of Camponotus penns.vlvania DeGeer (Formicidae, Hymenoptera). J. Morphol. 95:523 - 56. FORBFS, J. 1958. The male reproductive system of the army ant, Eciton hamatum Fabricius. Proc. IOth Int. Congr. Entomol. I: 593 96. FORB,~S, J. and D. Do-VAN-QtJv. 1965. The anatomy and histology of the male reproductive system of the legionary ant, Neivamyrmex harrisi (Haldeman) (Hymenoptera, Formicidae). J. N. Y. Entomol. Sot'. 73: 9 5 - 1 1 1 . FORD, F. C. and J. Form:s. 1980. Anatomy of the male reproductive systems of the adults and pupae of two doryline ants, Dor.vlus (Anomma) wilverthi Emery and D. (A.) nigricans llliger. J. N. Y. Entomol. Soc. 88:133 - 42. GI Ant'fir, B. M., M. K. VANDENBUR(iHand M. K. Sl. RostA£n. 1976. Testes degeneration in the red imported fire ant, Solenopsis invicta. J. Ga. Entomol. Sot +. I!: 83 - 8 . HA',.;OPI.XN, M. 1963. An anatomical and histological study of the male ponerine ant, Rhytidoponera metallica F. Smith (Formicidae, Hymenoptera). Dissertation, Fordham Univ., Univ. Microfilms, Ann Arbor, Mich. HOtlDOait:r, B. and H. ENC;H-StI¢;tl. 1982. Tergal and sternal glands in male ants. Psyche 89:113 32. HtJM,XSON, G. L. 1962. Animal Tissue Technology. W. H. Freeman and Co., San Francisco, California. Ht N,.;, A. C. F., S. B. VtNSONand J. W. St MMt rl IN. 1974. Male sterility in the red imported fire ant, Solenopsis invicta. Ann. Entomol. Sot +. Amer. 6"/: 909 12. HUNt.;, A. C. F. and S. B. VtNSON. 1975. Notes on the male reproductive system in ants (Hymenoptera : Formicidae). J. N. Y. Entomol. Sot'. 83: 192- 97.
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JANET, C. 1902. Anatomie du Gaster de la Myrmica rubra. Georges Carr~ et C. Naud, Paris. JEAm'EL A-Y. 1972. Recherches histophysiologiques sur le d~veloppement post-embryonnaire et le cycle annuel de Formica (Hym~nopt~re). II. Donn~es cytophysiologiques sur les glandes annexes males de Formica polyctena Foerst. Ann. Sci. Nat. Zool. 14:285 - 303. JOUVENAZ, D. P., G. E. ALrEN, W. A. BANKS and D. P. WOJClK. 1977. A survey for pathogens of fire ants, Solenopsis spp. in the southeastern United States. Fla. EntOmol. 60:275 - 79. MUKERJEE, D. 1927. Digestive and reproductive systems of the male artt Dorylus labiatus Schuck. J. Proc. Asiatic Soc. Bengal (n.s.) 22:87 - 92. PEARSE, A. G. E. 1968. Histochemistry: Theoretical and Applied. 3rd ed. Little, Brown and Co., Boston. SNODGRASS, R. E. 1935. Principles oflnsect Morphology. McGraw-Hill, N. Y. SNODGRASS, R. E. 1956. Anatomy o f the Honey Bee. Comstock Publ. Co. Inc., Ithaca, N.Y. TICE, M. C. 1967. The a n a t o m y and histology of some of the systems of the male of the imported fire ant, Solenopsis saevissima richteri Forel (Hymenoptera : Formicidae). Dissertation, F o r d h a m Univ., Univ. Microfilms, A n n Arbor, Michigan. TRAKJMAS, W. B. 1967. An anatomical and histological study of the male myrmicine ant, Myrmica rubra L. (Hymenoptera : Formicidae). Dissertation, F o r d h a m Univ., Univ. Microfilms, A n n Arbor, Michigan. VINSON, S. B. and R. ROBEAU. 1974. Insect growth regulator effects on colonies of the imported fire ant. J. Econ. Entomol. 67: 5 8 4 - 87.
Printed in Great Britain.
0020- 7322/84$3.00+ .00 © 1984PergamonPressLtd.
ANATOMY A N D HISTOLOGY OF THE MALE REPRODUCTIVE SYSTEM OF THE FIRE ANT, SOLENOPSIS INVICTA BUREN (HYMENOPTERA • FORMICIDAE)* DAVID E. BALL']" and S. B. V1NSON Department of Entomology, Texas A&M University, College Station, Texas 77843, U.S.A. ( A c c e p t e d 13 M a r c h 1984)
A b s t r a c t - - T h e male reproductive system of the fire ant, Solenopsis invicta Buren (Hymenoptera : Formicidae), consists of the testes, vasa efferentia, vasa deferentia, seminal vesicles, accessory glands, ejaculatory duct, wedge, aedeagal bladder, and external genitalia. The testes in newly eclosed males appear as 4 large white lobes filled with packets of sperm. Each lobe of the testes contains only one follicle. As the testes degenerate, the maturing sperm migrate through the vasa efferentia and vasa deferentia into the seminal vesicles. The seminal vesicles attach to the accessory glands, which are lined with secretory columnar epithelium. The posterior ends of the accessory glands taper and unite to form the ejaculatory duct. A sclerotized wedge is found at the junction of the accessory glands and the ejaculatory duct. An aedeagal bladder, joining the ejaculatory duct posterior to the wedge, is lined with s q u a m o u s epithelium enveloped by heavy musculature. The ejaculatory duct continues posteriorly to form a distal aedeagus surrounded by 3 pairs of valves, comprising the external genitalia.
Index descriptors (in addition to those in title): Epithelium; scanning electron microscopy; maturation; seminal vesicle; accessory glands; aedeagal bladder. INTRODUCTION
THE CONTROL of the fire ant, Solenopsis invicta Buren, has been the subject of considerable research over the last 2 decades. However, little emphasis has been placed on non-chemical forms of control, such as disrupting the reproductive processes, genetic manipulation, or a sterile male release program. Successful use of these techniques would require an extensive knowledge of the anatomy and histology of the male reproductive system. Studies of the male reproductive system have been conducted on at least one species in nearly all the subfamilies of ants. However, to review all the available literature would require a separate monograph, therefore, we will primarily confine our review to the Myrmicinae and refer to the other groups where applicable. Forbes (1954) reviewed the early studies done on the male reproductive system in ants and conducted one of the first extensive anatomical and histological examinations of the male reproductive system of Camponotus pennsylvanicus DeGeer. Janet (1902) described the male reproductive system of Myrmica rubra L. which was reinvestigated by Trakimas
*This manuscript is approved as TA 18703 by the Director of the Texas Agricultural Experiment Station in cooperation with A . R . S . / U . S . D . A . This research was supported by the Texas Department of Agriculture lnteragency agreement IAC (82-83) 0982. t T h i s research was conducted by D.E.B. as part of the requirement for the Ph.D. degree. 283
284
DAVID E. BALL and S. B. V|NSON
(1967). Tice (1967) examined the histology of the male reproductive system of the black imported fire ant, Solenopsis saevissima richteri Forel. According to Janet (1902), the male reproductive system consists of the testes, vasa deferentia, seminal vesicles, ejaculatory duct, and external genitalia. However, additional reproductive structures have been discovered since then. A posteriorly located aedeagal bladder has been found in S. s. richteri (Tice, 1967), M. rubra (Trakimas, 1967) and in other subfamily genera. The aedeagal bladder and a chitinous wedge in the ejaculatory duct were first discovered by Clausen (1938). An aedeagal gland, was briefly mentioned by Trakimas (1967). Distinct accessory glands were found in Dorylus labiatus Shuck (Mukerjee, 1927), Eciton hamatum (Fabr.) (Forbes, 1958) and Neivamyrmex harrisi (Halderman) (Forbes and Do-Van-Quy, 1965). Recently, the nomenclature of Snodgrass (1935, 1956) was used to identify the accessory glands as the more correct term for the seminal vesicles in Formica polyctena Foerst (Jeantet, 1972) and many other genera of ants including S. invicta (Hung and Vinson, 1975). The purpose of this study was to examine the developmental anatomy and histology of the male reproductive system of the imported fire ant from eclosion to after mating. MATERIALS
AND
METHODS
Colonies with male pupae and alates were collected during the summer m o n t h s from fields at College Station, Texas. The colonies were excavated and transported to the laboratory where the ants and brood were flooded out with water (Jouvenaz et al., 1977). The ants were transferred to plastic shoe boxes (Vinson and Robeau, 1974) and fed a mixture of insects and honey water. The emerged male alates in each colony were removed and destroyed. The newly emerging males were transferred daily to small plastic dishes with ca 50 worker nestmates since previous work showed that males lived longer when maintained with workers (Ball and Vinson, unpublished data). In this way, newly emerged males and males of known age were obtained. Additional males were collected flying from the surface of the m o u n d prior to mating (preflight) and after returning (postflight). Sterile males (Hung et al., 1974) at least 2 weeks old were collected from laboratory colonies. After maintaining the colonies for the required length of time, 5 males were randomly selected from each age group and the entire reproductive system was dissected in distilled water by spreading the terminal gastral sclerites with microforceps and carefully pulling the claspers. After measuring the intact glands with a calibrated ocular micrometer, the entire reproductive system was placed in Zenker's fixative for 12 hr. Following dehydration, clearing and embedding in paraffin, 10 lam sections were cut and stained. H e m a t o x y l i n - e o s i n , Schiff's reagent and Mallory's triple staining procedures as outlined by H u m a s o n (1962) were used to identify mucus and cytoplasmic elements, nuclei, and thymonucleic acids. Cytochemical identification of proteins by mercury - bromophenol blue staining and acid mucopolysaccharides by the alcian blue procedure were also used as described by Pearse (1968). Scanning electron microscopy was used to observe any unusual morphological differences in the reproductive systems of fertile and sterile males. The reproductive systems from 5 preflight and 5 sterile males were compared. The entire reproductive system was immersed in 3% gluteraldehyde in a 0.1M sodium phosphate buffer solution (pH 7.2) at 4°C for 1 hr. The specimens were then rinsed 4 times for 10 min each in the buffer solution at 4°C followed by postfixation in a room temperature solution of 1% osmium tetroxide for 1 hr. After rinsing again 4 times for 10 min each in buffer at 4°C, the specimens were placed in a series of ethanol baths (50%, 70%, 90%, 95%, and 3 times in 100% ethanol) for 10 rain each at room temperature. The specimens were then transferred to Reikert capsules and placed in a critical point dryer where ethanol was replaced by CO2. All specimens were attached to aluminum stubs with glue formulated by mixing 16 gm of Foromvar (Electron Microscopy Sciences, Forth Washington, Pa.) with 60 ml of ethylene dichloride. A 200 A thickness of g o l d - palladium was evaporated onto each sample and the specimens were viewed at 12.5 kV, using a JEOL JSM-25S scanning electron microscope. Transmission electron microscopy was used to examine the histology of the aedeagal bladder and the ultrastructure of the accessory glands. Entire gasters and dissected accessory glands were fixed in 3% gluteraldehyde/l.5% paraformaldehyde/3% Acrolein in a 0. I M sodium cacodylate buffer solution (pH 7.2) for 2 hr at 4°C. The specimens were then rinsed 4 times for 15 min each in buffer followed by postfixation in 2% osmium tetroxide for 1 hr. After rinsing as before, the samples were dehydrated in an ethanol series, rinsed in 100% propylene oxide 5 times i n l hr and infiltrated with Epon araldite resin. The specimens were then transferred to fresh resin, vacuum aspirated for 15 min and heated for 3 days in a 70°C oven. The resulting blocks were sectioned (500 A), stained with toluidine blue and viewed on a Zeiss 10C/CR electron microscope at 80 kV.
Anatomy and Histologyof the Male ReproductiveSystem of the Fire Ant RESULTS
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DISCUSSION
Anatomy
The reproductive system of the fire ant, S. invicta consists of the testes and the testicular capsule, vasa efferentia, vasa deferentia, seminal vesicles, accessory glands, a sclerotized wedge, ejaculatory duct, aedeagal bladder, and external genitalia (Figs. 1, 2). In newly enclosed males the 4 follicles of the immature testes were seen as large white lobes. Four distinct follicles are also reported for M . rubra (Janet, 1902) and S. s. ,ichteri (Tice, 1967). The testes taper at the proximal end and form the vasa effe~entia which join into a short collecting duct, the vas deferens (Fig. 3). The vasa deferentia continue into the anterior tip of the seminal vesicles. The studies by Hung and Vinson (1975) and Tice (1967) do not discuss the morphological separation of the vasa efferentia from the vasa deferentia, as described in this study. The testes, vasa efferentia and vasa deferentia are enveloped in a thin fibrillar capsule, which was only found in fertile males. The 4 testes measure 2.39 _ 0.44 mm (SD) (n = 12) in length and 1.37 _+ 0.49 mm (SD) (n = 12) in width in the late pupal stage and slowly degenerated forming 4 amber-colored lobes (Fig. 4). Glancey et al. (1976) observed that the testes obtained a maximum size of ca 1.2 mm by midway through the pupal stage and gradually atrophied to form 4 ambercolored bodies 11 days after eclosion. We found that the testes were connected by a short duct, the vas deferens, to the sperm-filled seminal vesicles. A small amount of sperm was also seen in the accessory glands of several preflight males. Sterile males lack testes (Hung et al., 1974) (Fig. 5) and therefore produce no sperm. The empty seminal vesicles of immature males were 2.10 _ 0.18 mm (SD) (n = 12) in length and 0.14 _+0.03 mm (SD) (n = 12) in width. As the males matured, the length of the sperm-filled seminal vesicles remained the same, while the width increased to 0.18 _+ 0.07 mm (SD) (n = 12). The seminal vesicles curve ventrally and enter the anteromedial region of the accessory glands, which are large, muscular, peanut-shaped lobes (Figs. 1, 2, 6). The accessory glands in mature males measure 1.12 _ 0.06 mm (SD) (n = 12) in length and 0.53 _+ 0.06 mm (SD) (n = 12) in width. Glancey et al. (1976) found that the accessory glands in S. invicta increase in size as the males mature. However, the sample size was not mentioned in their report. Our study showed that there was no apparent physical change in the size of the accessory glands from the time of eclosion to 12 days old, and the average dimensions of the lobes from preflight, postflight and sterile males were approximately equal (n = 30). The accessory glands taper posteriorly and unite to form the ejaculatory duct in the area of a sclerotized V-shaped wedge. At the posterior tip of the wedge, is the opening to the aedeagal bladder. The bladder, measuring ca 1.5 mm in length and ca 0.5 mm in width, consists of a large ovoid sac covered with heavy musculature positioned in the anterior region of the external genitalia (Fig. 7). The ejaculatory duct continues posteriorly to form the aedeagus, which is surrounded by the external genitalia (Figs. I, 2). The external genitalia consist of 3 pairs of valves; the laminae paramerales (outer valve), volsellae (middle valves), and the laminae aedeagales (inner valve), which are surrounded anteriorly by the lamina annularis (basal ring). The outer valves surround the aedeagal bladder, wedge, ejaculatory duct, aedeagus, and the middle and inner valves. A small distal lobe, the paramere, is located on the lateral surface of the outer valve. Each of the paired middle valves consists of a large basal sclerite, the lamina volsellaris, and two dorsal portions; the lateral cuspis volsellaris and a median digitus volsellaris. The inner valves appear as thin paddle-shaped plates, and are positioned on both sides of the ejaculatory duct and the aedeagus. The basal ring is divided into a dorsal oval sclerite and a ventral
286
DAVIDE. BAILand S. B. VINSON
band. The configuration of the external genitalia described here is in agreement with that by Tice (1967). • '~"
/ IMMATUREM A L E ~ DEGENERATED i TESTIS OF
"~'
/ ~,i/
~b /I I \ i
rI
/
Fit;. 1. Dorsal view of male reproductive system. × 43.
Histology The follicles are enveloped in a thin capsule, which consists of long fibers resembling connective tissue. Fusiform nuclei are visible throughout the capsule layers, although distinct cellular membranes were not revealed with any of the staining procedures (Fig. 3). Forbes (1954) also found a fibrous capsule covering the testes and vas deferens in C. pennsylvanicus, but he stated that the capsule was composed of tracheal branches with fusiform nuclei. Sperm in each follicle of the testes were arranged in oval packets in the black pupal stage and newly eclosed males (Fig. 3). Each packet contains several hundred sperm with the heads oriented toward one axis of the packet. A thin membrane and one or more nuclei are occasionally seen along the periphery of the sperm packets stained with Schiff's reagent. Hematoxylin - eosin showed dense concentrations of DNA, verifying the identity of the peripheral nuclei. The sperm packets were more concentrated toward the center of each follicle with loose, mature sperm visible along the inner periphery of the testicular wall. No meiotic stages were seen in any sections of the testes. Gradually, the sperm packets became less predominant in the follicles until shortly after the 7th day only 1 0 - 15
Anatomy and Histology of the Male Reproductive System of the Fire Ant
FJ~. 2. Ventral view of male reproductive system, x 43.
Fx(~. 3. Diagram of a section through testis and ducts.
287
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DAVID E. BALL and S. B. VINSON
Fl(;. 4. Micrograph of a whole mount of seminal vesicle and degenerated testes of a fertile male Fl(;. 5. Micrograph of a whole mount of seminal vesicle of a sterile male.
Anatomy and Histology of the Male Reproductive System of the Fire Ant
FI(;. 6. Diagram of a section through seminal vesicle and accessory gland.
FI(;. 7. Micrograph of a longitudinal section of posterior end of male gaster. Anterior end at top and ventral side at right. AB - aedeagal bladder; M - muscle; SgG = subgenital plate gland; W wedge.
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DAVIDE. BAILand S. B. VJNSON
sperm packets per follicle were visible. At approximately 10 days after eclosion the testes were degenerated and formed 4 amber-colored bodies attached to the tip of the seminal vesicles by the apparent remains of the vas deferens. The histological separation of the vasa efferentia and vas deferens was indiscernible at this development stage, altl~ough the close association of the connecting duct to the seminal vesicles and the degenerated testes suggested that it was the structurally reduced vas deferens, since the 4 vasa efferentia were not visible. The remnants of the capsule were still seen as a thin layer surrounding the vas deferens and degenerated testes. Both the vasa efferentia and vasa deferentia in immature males are lined with short columnar epithelium attached to a distinct basement membrane surrounded by several layers of muscle fibers. Sterile males lacked the vasa efferentia and vasa deferentia. The columnar epithelium of the vas deferens continues into the seminal vesicles. The seminal vesicles serve as the storage area for mature sperm prior to mating. The columnar epithelial cells lining the vesicles are slightly taller than the cells found in the vasa efferentia and vasa deferentia and have basally located nuclei. As the seminal vesicles filled with sperm during maturation of the male, the epithelial cells became more cuboidal, probably owing to the compression of the cells by the stored sperm. At the connection of the seminal vesicles to the accessory glands, the epithelial cells are elongated and appear to form a distinct valve, which may prevent sperm from entering the accessory glands (Fig. 6). However, some sperm were observed in the accessory glands of preflight males, although this may have been the result of dissection trauma. The epithelium of the seminal vesicle is attached to a basement membrane, surrounded by 1 or 2 layers of muscular fibers, which are thicker in the posterior region of the vesicle closest to the accessory gland. The epithelium of the accessory gland is composed of simple columnar cells on a distinct basement membrane, surrounded by 2 or 3 layers of muscle fibers (Fig. 6). The basally located nucleus in each cell is spherical and the cytoplasm and lumen of each lobe in mature males are filled with numerous basophilic granules, surrounded by a slight amount of acidophilic material. Mallory's triple stain showed an increase in the mucosal concentration in the lumen of the gland as the males matured. Deep blue staining with the m e r c u r y - bromophenol blue technique indicated high concentrations of protein in the distal region of the epithelial cells, the lumen of the gland and vesicles in the supranuclear cytoplasm. Transmission electron microscopy showed that the vesicles were polymorphic in shape and bounded by a membrane (Fig. 8). The material in the vesicles appeared as non-membranous coalesced material when viewed at higher magnification (see insert, Fig. 8). The fibrillar particles comprising the coalesced material could also be seen in the cytoplasm of the vesicles. The vesicles appeared to be released into the lumen of the gland by the rupturing of the epithelial cells. The lumen of the gland in preflight males was filled with these vesicles (Fig. 9). A major portion of these vesicles was voided during the mating flight, since the lumen of each lobe in postflight males was nearly emptied (Fig. 10) and the epithelial cells were greatly reduced in size and number. Pupal and newly eclosed males had considerably less proteinaceous activity in the gland than mature males. Long fibrillar processes extending from the epithelial cells into the lumen of the gland were revealed with hematoxylin - eosin in males at least 5 days post-eclosion. The fibrillar extensions were probably a result of the rupturing of the cells during the secretory processes. The alcian blue staining procedure showed no significant concentration of acid mucopolysaccharides in the accessory gland. However, sperm entering the gland in preflight males immediately before the mating flight showed a
Anatomy and Histologyof the Male ReproductiveSystem of the Fire Ant
291
FIG. 8. Micrograph of accessorygland vesicles. Insert--coalesced material and fibrillar partictes. characteristic clear blue-green color indicating a slight a m o u n t o f acid mucopolysaccharide. The accessory glands unite to form a common ejaculatory duct in the region of the sclerotized wedge. The ejaculatory duct is lined with columnar epithelium at the junction of the accessory glands, and there appeared to be a gradu~il transition from the epithelium of the accessory gland to the ejaculatory duct. Midway in the duct, the cells gradually shorten to form cuboidal epithelium, which continues into the aedeagus. The epithelium is attached to a basement membrane, enveloped by an inner layer of longitudinal muscle fibers and an outer layer of circular muscle fibers. The arms of the V-shaped sclerotized wedge arise from the ventrolateral epithelium of the ejaculatory duct (Figs. 1, 2, 7). The posterior tip of the wedge is not attached to the duct wall and appeared to extend freely in the lumen. A wedge has been found in the male reproductive system of representative species of the Ponerinae (Hagopian, 1963), Dorylinae (Forbes, 1958; Forbes and Do-Van-Quy, 1965; Ford and Forbes, 1980), Myrmicinae (Tice, 1967; Trakimas, 1967) and Formicinae (Forbes, 1954). Although a
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DAVID E. BALL and S. B. VINSON
FIc~. 9. Micrograph of a section of accessory gland of a preflight male, x 100. FI(~. I0. Micrograph of a section of accessory gland of a postflighl male. × 100.
Anatomy and Histology of the Male Reproductive System of the Fire Ant
293
specific function has yet to be determined, we felt that the wedge may have been used as a structural support for the connection of the accessory glands with the ejaculatory duct and, as Hagopian (1963) suggested, as a directing mechanism for the sperm and accessory gland secretions. The aedeagal bladder enters the ejaculatory duct near the posterior tip of the wedge. The aedeagal bladder, enclosed by the external genitalia, is lined with a single layer of squamous epithelial cells surrounded by heavy muscular fibers (Fig. 7). The lumen of the bladder contains an amorphous mass with numerous acidophilic granules. These findings are in agreement with those of Tice (1967) and Trakimas (1967). The material was found in approximately equal amounts in preflight, postflight and sterile males, but was absent from one-day-old males. Further studies using males of known ages showed that the gland material began appearing at 3 days post-eclosion, indicating that the material was either being deposited or manufactured in the bladder after adult emergence. However, secretory activity by the cells lining the lumen was not evident histologically. A slight amount of protein, indicated by a light-blue staining with m e r c u r y - bromophenol blue, was seen throughout the lumen of the gland. None of the other staining procedures was positive, except hematoxylin-eosin, which revealed the nuclei in the squamous epithelium. A large mass of glandular cells was found on the inner surface of the 9th sternite. H/311dobler and Engel-Siegel (1982) have found similar groupings of cells, which they identify as the "subgenital plate gland" in representative species of every subfamily of ants. Although S. invicta was not examined in their report, we have determined that the group of cells in the fire ant were morphologically similar (Fig. 7). Tice (1967) also described a subgenital gland in the black fire ant, S. saevissima richteri, similar to that in S. invicta. The subgenital plate gland in S. invicta is composed of columnar epithelium with basally located nuclei. The cells have long slender ducts arising from the supranuclear surface of the cell membrane extending into the cuticle of the ant. REFERENCES CI.AUStN, R. 1938. Untersuchungen tiber den m~innlichen Copulationsapparat der Ameisen, speciell der Formicinae. Mitteil. Schweizer. Entomol. Ges. 17: 2 3 3 - 346. FORBES, J. 1954. The anatomy and histology of the male reproductive of Camponotus penns.vlvania DeGeer (Formicidae, Hymenoptera). J. Morphol. 95:523 - 56. FORBFS, J. 1958. The male reproductive system of the army ant, Eciton hamatum Fabricius. Proc. IOth Int. Congr. Entomol. I: 593 96. FORB,~S, J. and D. Do-VAN-QtJv. 1965. The anatomy and histology of the male reproductive system of the legionary ant, Neivamyrmex harrisi (Haldeman) (Hymenoptera, Formicidae). J. N. Y. Entomol. Sot'. 73: 9 5 - 1 1 1 . FORD, F. C. and J. Form:s. 1980. Anatomy of the male reproductive systems of the adults and pupae of two doryline ants, Dor.vlus (Anomma) wilverthi Emery and D. (A.) nigricans llliger. J. N. Y. Entomol. Soc. 88:133 - 42. GI Ant'fir, B. M., M. K. VANDENBUR(iHand M. K. Sl. RostA£n. 1976. Testes degeneration in the red imported fire ant, Solenopsis invicta. J. Ga. Entomol. Sot +. I!: 83 - 8 . HA',.;OPI.XN, M. 1963. An anatomical and histological study of the male ponerine ant, Rhytidoponera metallica F. Smith (Formicidae, Hymenoptera). Dissertation, Fordham Univ., Univ. Microfilms, Ann Arbor, Mich. HOtlDOait:r, B. and H. ENC;H-StI¢;tl. 1982. Tergal and sternal glands in male ants. Psyche 89:113 32. HtJM,XSON, G. L. 1962. Animal Tissue Technology. W. H. Freeman and Co., San Francisco, California. Ht N,.;, A. C. F., S. B. VtNSONand J. W. St MMt rl IN. 1974. Male sterility in the red imported fire ant, Solenopsis invicta. Ann. Entomol. Sot +. Amer. 6"/: 909 12. HUNt.;, A. C. F. and S. B. VtNSON. 1975. Notes on the male reproductive system in ants (Hymenoptera : Formicidae). J. N. Y. Entomol. Sot'. 83: 192- 97.
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JANET, C. 1902. Anatomie du Gaster de la Myrmica rubra. Georges Carr~ et C. Naud, Paris. JEAm'EL A-Y. 1972. Recherches histophysiologiques sur le d~veloppement post-embryonnaire et le cycle annuel de Formica (Hym~nopt~re). II. Donn~es cytophysiologiques sur les glandes annexes males de Formica polyctena Foerst. Ann. Sci. Nat. Zool. 14:285 - 303. JOUVENAZ, D. P., G. E. ALrEN, W. A. BANKS and D. P. WOJClK. 1977. A survey for pathogens of fire ants, Solenopsis spp. in the southeastern United States. Fla. EntOmol. 60:275 - 79. MUKERJEE, D. 1927. Digestive and reproductive systems of the male artt Dorylus labiatus Schuck. J. Proc. Asiatic Soc. Bengal (n.s.) 22:87 - 92. PEARSE, A. G. E. 1968. Histochemistry: Theoretical and Applied. 3rd ed. Little, Brown and Co., Boston. SNODGRASS, R. E. 1935. Principles oflnsect Morphology. McGraw-Hill, N. Y. SNODGRASS, R. E. 1956. Anatomy o f the Honey Bee. Comstock Publ. Co. Inc., Ithaca, N.Y. TICE, M. C. 1967. The a n a t o m y and histology of some of the systems of the male of the imported fire ant, Solenopsis saevissima richteri Forel (Hymenoptera : Formicidae). Dissertation, F o r d h a m Univ., Univ. Microfilms, A n n Arbor, Michigan. TRAKJMAS, W. B. 1967. An anatomical and histological study of the male myrmicine ant, Myrmica rubra L. (Hymenoptera : Formicidae). Dissertation, F o r d h a m Univ., Univ. Microfilms, A n n Arbor, Michigan. VINSON, S. B. and R. ROBEAU. 1974. Insect growth regulator effects on colonies of the imported fire ant. J. Econ. Entomol. 67: 5 8 4 - 87.