Anatomy, ultrastructure, and functional morphology of the metathoracic tracheal defensive glands of the guttata

DOUGLASW. WHITMAN Department of Biological Sciences, Illinois State University, Normal, IL 61 761, U. S.A. JOHANP. J. BILLEN Zoological Institute, University of Leuven, B-3000 Leuven, Belgium DAVIDALSOP Department of Biology, Queens College, Flushing, NY 11367, U.S.A.

AND MURRAYS. BLUM Department of Entomology, University of Georgia, Athens, GA 30602, U. S. A. Received January 4, 1991

WHITMAN,D. W., BILLEN,J. P. J., ALSOP,D., and BLUM,M. S. 1991. Anatomy, ultrastructure, and functional morphology of the metathoracic tracheal defensive glands of the grasshopper Romalea guttata. Can. J. Zool. 69: 2100-2108. In the lubber grasshopper Romalea guttata, the respiratory system produces, stores, and delivers a phenolic defensive secretion. The exudate is secreted by a glandular epithelium surrounding the metathoracic spiracular tracheal trunks. Embedded in the glandular tissue are multiple secretory units, each comprised of a basal secretory cell and an apical duct cell. Secretory cells have numerous mitochondria, a tubular, smooth endoplasmic reticulum, well-developed Golgi bodies, and a microvilli- lined vesicle thought to transfer secretion to the intracellular cuticular duct of a duct cell. Ducts empty into the metathoracic tracheal lumina where the exudate is stored behind the closed metathoracic spiracle. Tactile stimulation elicits secretion discharge, which,begins when all spiracles except the metathoracic pair are closed and the abdomen is compressed. Increased hemostatic and pneumatic pressures drive air and secretion out of the spiracle with an audible hiss. Both metathoracic spiracles discharge simultaneously. The secretion erupts first as a dispersant spray, then as an adherent froth, and finally assumes the form of a slowly evaporating repellent droplet. Discharge force and number vary with eliciting stimuli, volume of stored secretion, and age, disturbance state, and temperature of the . Molting are unable to discharge because the stored exudate is lost with the shed cuticle. The advantages and limitations of a tracheal defensive system are discussed.

WHITMAN,D. W., BILLEN,J. P. J., ALSOP,D., et BLUM,M. S. 1991. Anatomy, ultrastructure, and functional morphology of the metathoracic tracheal defensive glands of the grasshopper Romalea guttata. Can. J. Zool. 69 : 2100-2108. Chez le criquet Romalea guttata, le systkme respiratoire produit, emmagasine et tmet une stcrttion phtnolique de dtfense. Cette exsudation est stcrttte par l'tpithtlium glandulaire qui entoure les trachtes des stigmates mttathoraciques. De nombreuses unitts stcrttrices sont enfouies dans le tissu glandulaire et chacune est constitute d'une cellule basale stcrttrice et d'une cellule apicale canaliculaire. Les cellules stcrttrices comportent de nombreuses mitochondries, un rtticulum endoplasmique agranulaire tubulaire, des appareils de Golgi kndtveloppts et une vtsicule tapisste de microvillositts que l'on croit servir au transfert des stcrttions au canal cuticulaire intracellulaire d'une cellule canaliculaire. Les canaux se vident dans la lumikre de la trachte mttathoracique et la stcrttion s'accumule denikre le stigmate mttathoracique fermt. La stimulation tactile dtclenche le rejet de la stcrttion; au moment du rejet, tous les stigmates sont fermts, l'exception de la paire mttathoracique, et l'abdomen est comprimk. L'augmentation des pressions htmostatique et pneumatique poussent l'air et la stcrttion au-dehors par le stigmate avec un sifflement perceptible. Les deux stigmates mttathoraciques se vident simultantment. La stcrttion sort d'abord cornrne un jet de vapeur, puis sous forme de mousse collante, et finalement sous forme d'une gouttelette rtpulsive qui s'tvapore lentement. La force des Cmissions et leur nombre varient en fonction des stimulus dtclencheurs, du volume de stcrttion emmagasinte, de l'ige, du degrt d'agitation et de la temptrature de l'insecte. Les criquets en ptriode de mue sont incapables d'tmettre des stcrttions parce que les stcrttions accumultes sont rejettes avec la cuticule. Les avantages et les dtsavantages d'un systkme de dtfense trachten font l'objet d'une discussion. [Traduit par la rtdaction]

Introduction through the metathoracic spiracles (Fig. 1) with an audible hiss Only two groups of possess a respiratory-derived (Morse 1907; Duncan 1924; Roth and Eisner 1962). chemical defensive system. Certain blaberid cockroaches eject The products of these tracheal glands have been studied only a defensive secretion from the second pair of abdominal in R. guttata. In this species, the water-based secretion is spiracles (Roth and Stay 1958), and two closely related spe- exceedingly complex, containing over 50 compounds. Major cies of New World romaleid grasshoppers, Romalea guttata constituents are synthesized de novo and include phenolics, ( = microptera) and eques (Rhen and Grant 1959, quinones, and an allenic sesquiterpenoid, romallenone (Mein- 1961), possess a thoracic defensive mechanism. In both wald et al. 1968; Eisner et al. 1971; Jones et al. 1986). Other grasshopper species, the tracheal trunks leading to the meta- constituents are obtained from the diet (alkyl sulfides from thoracic spiracles are specialized for the storage and discharge wild onion and nepetalactone metabolites from catnip) (Jones of secretion produced in an overlying glandular epithelium. et al. 1989; Blum et al. 1990). Man-made compounds may be During predator encounters, the secretion is forcibly ejected sequestered as well; the herbicide breakdown product, 2,5 -

Printed in Canada 1 ImprimC au Canada WHITMAN ET AL. 2101

postero-directed nerves exiting the metathoracic ganglion were severed. Two days later, six operated and six sham-operated individ- uals were each squeezed at the head and the degree of abdominal contraction and secretory discharge was noted. For anatomical studies grasshoppers were killed or immobilized by refrigeration and dissected under Ringer's solution. For light micros- copy studies, grasshopper tracheae were fixed in alcoholic Bouin's (Brasil's) fluid and stained with haematoxylin and eosin. For trans- mission electron microscopy studies, the tracheal glands of cold- anesthetized grasshoppers were dissected and immediately fixed in cold 2% glutaraldehyde, buffered with 0.05 M sodium cacodylate (pH = 7.3) in 0.15 M sucrose. After postfixation in 2% osmium tetroxide in the same buffer, tissues were dehydrated in a graded acetone series and embedded in AralditeB. Thin sections from both sexes were stained with uranyl acetate followed by lead citrate, then examined with a Philips EM 400 electron microscope. Semithin sections stained with thionin and methylene blue were also examined by light microscopy. To investigate stimuli influencing secretion release, we performed the following routine on 40 adult ground-foraging grasshoppers encountered in the field between 13:00 and 15:OO (T,,, -30°C): the experimenter (i) advanced to within 30 cm of grasshopper; (ii) rapidly moved an open hand toward grasshopper (five times in 10 s); (iii) lightly touched grasshopper (five times in 10 s); (iv) roughly poked insect, knocking it off balance (five times); (v) held insect and squeezed one antenna; (vi) held insect and squeezed one front leg; (vii) squeezed head; and (viii) squeezed insect's anterior abdomen. All grasshoppers received all eight stimuli, and approximately 5 s separated different stimuli. Any secretion discharge was recorded. Molting, mating, and ovipositing adults were also disturbed by squeezing their thoraces or heads. For volumetric studies, discharge was collected into capillary tubes and measured.

Results The respiratory system of Romalea guttata, as in other grasshoppers (Uvarov 1966), consists of 10 pairs of spiracles and various interconnecting tracheae, air sacs, and tracheoles. FIG. 1. Foamy defensive secretion emerging from Romalea guttata Each spiracle possesses a sclerotized or membranous valve metathoracic spiracles. serving to regulate air flow into and out of the body. The spiracles differ in size, gegree of sclerotization, and develop- dichlorophenol was reported to occur in the secretion of ment of a surrounding cuticular plate (peritreme) (Fig. 2). The Romalea (Eisner et al. 1971). anterior mesothoracic spiracles (Ms) are the largest and are The phenolic products of Romalea tracheal glands are well- hidden from view beneath the posteroventral flanges of the known defensive agents in both plants and (Blum prothorax. They are located in a membranous field immediately 198 1; Rodriquez et al. 1984). The secretion from,Romalea anterior to the sclerotized anterior border of the mesopleuron, repels ants (Eisner et al. 1971; Jones et al. 1987), and the have well-sclerotized spiracular valves, and a large, posteriorly secretion of the related T. eques is repellent to grasshopper directed protuberance (P) that presumably prevents occlusion mice (Whitman et al. 1985). The metathoracic secretions of of the spiracular opening by the overlying pronotum (Fig. 3). both Taeniopoda and Romalea females also function as con- The metathoracic spiracles (Mt) are slightly smaller than the tact sexual pheromones, eliciting mating behavior in males mesothoracic spiracles and are located directly above the (Whitman 1982; D. W. Whitman, unpublished data). mesocoxal cavities. They lack a protuberance and are situated Although the composition and function of the defensive in a rigid peritremal ring located in a deep notch in the secretion in Romalea are well studied, almost nothing is known posterior border of the mesothoracic pleurites (Fig. 4). Two about the morphology or operation of the glands. This paper tiny cup-shaped sclerites function as a valve, opening to describes the microstructure and macrostructure of the glands, produce a narrow vertical slit. The valves are held apart (open) examines their functional morphology, discusses stimuli that by a basal sclerotized band and brought together (closed) by a elicit secretory ejection, and speculates on the benefits of a muscle originating on a knob protruding inward from the base respiratory-derived defensive system. of the peritreme and inserting centrally on the band. The Methods remaining eight pairs of abdominal spiracles are much smaller Grasshoppers for this study were collected in Athens, Georgia, and and, except for the first abdominal spiracles (A), which lie just Highland County, Florida. Ventilatory and discharge mechanisms anterior to the tympanum, open near the anterior ventral edge were observed in grasshoppers restrained to wooden blocks by straps. of their respective abdominal tergites. To investigate nervous control of abdominal contraction, transverse All tracheal trunks are covered by a thin nonglandular epi- slits were cut in the venters of cold-narcotized adult females, and all thelium, except for the metathoracic pair, which in both sexes 2102 CAN. J. ZOOL. VOL. 69, 1991

FIG.2. Thoracic region of R. guttata, showing mesothoracic (Ms), metathoracic (Mt), and first abdominal (A) spiracles. The prothoracic flange has been cut away to expose the mesothoracic spiracle. FIG. 3. The mesothoracic spiracle of R. guttata, showing the protuberance (P). FIG. 4. The metathoracic spiracle of R. guttata.

is surrounded by a thick glandular layer. This glandular epithe- are associated with a long sinuous duct (d) that connects the lium is clearly visible with the naked eye as a dull milky or secretory vesicle fV) with the tracheal lumen (Fig. 10). Ducts yellowish covering. Nonglandular tracheae have a translucent- lie 20-50 Fm apart and average 1200/mm2 (Fig. 11). silvery appearance. Electron microscopy shows that the tracheal trunks display Tracheal morphology varies somewhat among individuals; the well-known cuticular pattern of spiral taenidia (t), a thin however, there are generally four tracheal trunks (TI, T,, T,, electron-dense epicuticle (e), and a multilayered procuticle (p) and T,) arising from each metathoracic spiracle, with a fifth (Figs. 12-14). Glandular tracheae are surrounded by a thick (Ti) joining T2 a short distance below the spiracular opening epithelium containing apical epidermal cells (EC), basal (Figs. 5 and 6). All metathoracic trunks, except for T,, are long secretory cells (SC), and thin duct cells (DC) (Figs. 12 and - and highly convoluted, with coils that extend into the body 13); nonglandular tracheae possess a thin epithelium of squa- cavity from between the mesothoracic and metathoracic mous epidermal cells (Fig. 14). Epidermal cells in glandular muscles. The nonglandular ends of the trunks insert into tracheae generally contain numerous mitochondria (M) and membranous air sacs, TI with a large ventrolateral metathoracic dark-staining polymorphic nuclei (EN) and show cytoplasmic sac, Ti with a small lateral metathoracic sac, T, with a large extensions that penetrate between the secretory cells. Epidermal mesometathoracic sac lying immediately above the spiracles, cell membrahes bordering overlying cuticle are sometimes and T, to two large dorsolateral sacs, one extending anteriorly microvillate. into the prothorax and the other posteriorly into the second Duct cells are long and thin with a fairly electron-dense abdominal segment. No valves were observed at the tracheal cytoplasm and contain an intracellular epicuticular duct (d) - air sac junctures; however, tracheal diameters are sharply (id. ca. 0.5 ~m)(Figs. 13 and 16). Secretory cells generally reduced prior to insertion into the sacs. Because the five contain large rounded nuclei (SN) (ca. 15 Fm in diameter) and metathoracic trunks are normally partially filled with secretion, show basal infoldings (Fig. 12). Secretory cell cytoplasm is they are bypassed for respiratory purposes and air is supplied characterized by an extensive smooth tubular endoplasmic to the metathoracic sacs and terminal nonglandular tracheae by reticulum, well-developed Golgi apparatus (GA), an abundance tracheal trunks and other interconnections from the meso- of mitochondria (M), and an apical secretory vesicle with a thoracic and abdominal spiracles. microvillar lining (mv) (Figs. 15 and 16). The microvilli may surround an extensive electron-lucent area or reach the center Gland histology and ultrastructure of the vesicle (Fig. 16). Serial sections showed that each Light microscopy revealed that nonglandular tracheal trunks secretory cell is associated with one duct cell, each duct cell are surrounded by a (ca. 5 ~m)single-layered squamous deeply invaginates a secretory cell and ends at a secretory epithelium with scattered nuclei (Fig. 7). In contrast, the vesicle, the lumen of each secretory vesicle surrounds a glandular epithelium covering the metathoracic spiracular cuticular duct of a duct cell, and the sinuous duct opens into tracheal trunks is cuboidal(25-100 ~m)and densely nucleated the tracheal lumen. (Fig. 8). Constrictor muscles are absent. Studies of serial cross sections revealed three cell types. Epidermal cells lay against Functional aspects of metathoracic gland operation the tracheal cuticle and have small, dark-staining polymorphic In Romalea, respiration involves a repetitive series of nuclei (EN) (Fig. 9). Secretory cells (SC) are cuboidal to ventilatory movements and spiracular valve openings. Resting columnar (ca. 35 x 85 ~m)and rest against the basal mem- animals begin air intake with the expansion of the abdomen brane (bm). They possess large, rounded, lightly staining basal timed with the opening of the mesothoracic and first abdominal nuclei and an apical secretory vesicle (Fig. 9). Duct cells are spiracles. As these close and the abdomen contracts, the apically situated, have small, dark-staining nuclei (DN), and posterior five spiracular valves open. Thus, air is drawn into WHITMAN ET AL. 2103

FIG. 5. Adult female R. guttata, showing metathoracic spiracle and associated glandular trachea. FIG. 6. Romalea guttata, sagittal section of the three thoracic and first two abdominal segments. Glandular tracheae (T,, T,, T,', T,, T,) are shown in white; air sacs are stippled. sp, position of the spiracle.

the anterior body region, moves posteriorly through the insect, viduals were warmed and then induced to discharge. As their and exits at the abdomen. The metathoracic spiracular valves abdomens contracted, blood and tissue emerged from the open serve no role in respiration and in undisturbed remain wounds. Although the grasshoppers ejected some secretion, the shut. They function as a cap, prevepting evaporation or leakage forceful ejection characteristic of unoperated grasshoppers was of the defensive secretion that fills the tracheal lumen. The lacking. When the wounds were sealed with plastic shields and secretion is stored immediately behind the spiracle; by gently hot dental wax, the insects regained their ability to forcefully opening the valve in a narcotized grasshopper and inserting a discharge. Clearly the open wounds prevented the hemostatic sliver of filter paper, the secretion is quickly drawn out by pressurization necessary for maximum secretory ejection. capillary action. To determine if pneumatic pressure was important for In disturbed animals, a process opposite to ventilation secretory discharge, all spiracles of freshly killed grasshoppers occurs. All spiracular valves close except the metathoracic pair, were sealed with hot dental wax, except for the metathoracic and the abdomen contracts longitudinally, laterally, and dorso- pair. When the abdomens of these dead insects were forcefully ventrally. This raises the hemostatic pressure and compresses squeezed, secretion emerged from the metathoracic spiracles in the numerous internal air sacs that communicate with the ends a volume and manner comparable to live, intact individuals. of the secretion-filled tracheae, forcing air and secretion out Conversely, when freshly killed grasshoppers with unsealed through the metathoracic spiracles with a hissing sound audible spiracles were squeezed,-only a little secretion quietly oozed for at least 10 m. Left and right glands always fire simulta- out. neously (Fig. I). The valves flutter during discharge, creating When nerves exiting the metathoracic ganglia were severed, a narrow slit that tends to eject the secretion first as a fine adults became unable to contract their abdomens and forcefully spray and then as an adherent foam. Very little secretion is expel secretion. Sham-operated adults retained the ability to needed to generate an audible hiss; the spiracular valves need contract their abdomens and expel secretion, suggesting that only be moist. Freshly molted individuals lack secretion and neural signals from the ventral nerve cord control abdominal cannot produce the sound, and 6-h-old adults have barely contraction. enough liquid (<0.1 kL) to produce a detectable hiss. Metathoracic secretion Following secretory ejection, the abdomen immediately ex- The metathoracic tracheal defensive secretion is a watery pands, creating a vacuum in the tracheal system. Since the odorous liquid (pH 5.5-6.5), rarely containing suspended oily metathoracic valves often remain partially open at this time, droplets or cellular debris. It possesses surface-active properties some of the secretion is drawn back into the tracheal lumen. that favor the formation of bubbles as it is ejected through the Thus, with repeated abdominal compressions, secretion flows spiracle; when air was gently blown through a fine tube back and forth through the spiracles, allowing supplementary inserted into a droplet of secretion, the liquid was transformed spraying, frothing, and sound production. Grasshoppers void of into a mass of foam. secretion can be made to inhale and discharge water by holding water droplets against the metathoracic spiracles while Factors influencing secretion discharge squeezing and releasing the abdomen. Even when no secretion Individual Romalea varied in their propensity to eject secre- is resorbed, insects can continue to bubble froth because with tion. Insects did not readily discharge during oviposition and every abdominal contraction, additional air is passed through were unable to discharge during molting. Conversely, copulat- the droplet of secretion adhering to the external surface of the ing males and females readily sprayed when molested. When metathoracic spiracle. adults were subjected to increasing levels of threatening stimula- To determine if hemostatic pressure was important for tion (Table l),some individuals discharged when lightly touched, secretion ejection, 25 mm2 of tergal integument was dissected whereas others failed to discharge even when squeezed at the from the abdomens of cold-narcotized Romalea. These indi- head. Females exhibited a significantly lower disturbance 2104 CAN. J. ZOOL. VOL. 69, 1991 threshold than males; when sharply poked, 65% of females discharged versus only 20% of males (p < 0.05; X2). Visual stimulation alone did not elicit secretion discharge. Over 50% of both sexes ejected secretion in response to antenna1 or leg squeezing. Discharge in these cases was sometimes low in force and volume. Conversely, when squeezed at the anterior abdomen, all insects forcefully discharged large amounts of secretion. Temporal and spatial summation of stimuli occurred. The more times an insect was subjected to the same stimulus, the more likely it was to discharge. Lightly squeezing three legs and the antennae elicited more discharges than strongly squeezing just the antennae. A slow hard squeeze was not as effective as a quick grab and a soft squeeze. The force of secretory ejection varied with the eliciting stimuli and the stage, age, disturbance state, and temperature of the grasshopper. Warm (>2g°C) adults sometimes produced a violent and noisy 3-s ejection of secretion when squeezed; up to 50% of the first discharge sprayed into the air, traveling up to 20 cm horizontally. The remainder collected as a 6-mm hemispherical mass of bubbling froth at the spiracular opening and remained intact for up to 10 s (Fig. 1). Normally, however, secretory ejection was not so forceful. In most cases, discharge continued for about 1 s, and approximately 90% of the ex- pelled secretion remained on the grasshopper. Atomized drop- lets traveled only a few centimetres, and the resultant mass of foam reached 3-4'mm in size. After 3-4 s, if there were no additional discharges, the bubbles in the foam dissipated and the secretion assumed the form of a droplet that, depknding on temperature and relative humidity, required up to 10 min to evaporate. The mildest discharge was seen when wild grass- hoppers were -disturbed on a cold night (< 19°C); only a small amount of liquid appeared at the spiracles, with little sound. The number of discharges was inversely related to the force of ejection. With mild stimulation, adult grasshoppers dis- charged up to 30 times, each accompanied by a hissing noise. Conversely, only four or five discharges, occurred when Romalea were strongly squeezed at the thorax, although these discharges were massive. The number of ejections was also related to the volume of secretion stored in the tracheae. In- sects with little secretion were unable to discharge repeatedly. Secretion volume The volume of stored secretion varied with insect stage and age. First- and second-instar Romalea lacked the exudate and third and fourth instars and individuals that had either recently molted or discharged possessed 0.1-0.2 pL/insect; wild fifth instars averaged 0.61 t 0.39 pL (n = 32), adult males 2.56 t 1.54 pL (n = 71), and adult females 4.20 + 1.68 pL (n = 74). Because molting individuals shed the cuticular lining of the tracheal system, the stored secretion is lost at the molt.

FIG.7. Nonglandular tracheal epithelium, showing the low density of nuclei. FIG. 8. Glandular tracheal epithelium, showing dense nucleation. Arrows point to secretory cell nuclei, which are larger than the nuclei of nonsecretory cells. FIG. 9. Sernithin histological section through glandular tracheal epithelium, showing large basal secretory cells (SC) with conspicuous nuclei and secretory vesicles (arrows). Apical epidermal cells contain smaller, dark-staining nuclei (EN). bm, basal membrane; L, tracheal lumen. FIG. 10. Romalea guttata secretory cell, showing secretory cell nucleus (SN), vesicle (V), draining tubule (d), and duct cell nucleus (DN). FIG. 11. Romalea guttata draining tubules (d), showing spacing of the gland cells. WHITMAN ET AL.

TABLE1. Percentage of adult Romalea guttata discharging defensive secretion in response to various increasingly threatening stimuli (n = 20 for each sex)

Body Hand Antennae Front leg Head Abdomen approach approach Touch Poke squeeze squeeze squeeze squeeze

Male 0 0 0 20 55 60 70 100 Female 0 0 15 65 90 95 100 100

However, secretion replenishment begins immediately; new startling, threatening, and memorable to potential predators, secretion first appears adjacent to the spiracle. In 25-day-old especially when it occurs in conjunction with the visual startle adults, the full length of T, (55 mm in females and 42 mm in display of bright red hind wings, elevated hind legs, and curled males) was filled. abdomen characteristic of these aposematic grasshoppers Discussion (Whitman et al. 1985). Functional morphology of the metathoracic tracheal glands Comparison with other species Our results suggest that the metathoracic tracheal gland Although the MTTG in Romalea is unique, its epidermal (MTTG) functions as follows. The infoldings of the secretory origin and cellular ultrastructure are similar to those of other cell basal plasma membranes imply that materials from the exocrine glands, which are generally epidermally hemolymph are absorbed across the membrane. The presence derived (Blum 1985; Whitman et al. 1990). In Romalea, the of Golgi bodies and smooth endoplasmic reticulum in the glandular tissue is formed by numerous individual units, each secretory cells suggests that synthesis of nonproteinaceous composed of one secretory cell and one duct cell. This secretion products occurs in the secretory cells (Noirot and arrangement, known as a class 3 gland, is common in insects Quennedy 1974). Secretory cell products presumably pass (Noirot and Quennedy 1974): across the microvilli of the secretory vesicle, enter and flow The only other insects known to possess tracheal defensive through the narrow canal of the Quct cell, and are deposited glands are certain blaberid coc'kroaches that produce secretions into the lumina of the metathoracic tracheal trunks. In adults and (or) noise from the second pair of abdominal spiracles this process is apparently continuous because older individuals (Roth and Eisner 1962). The phenomenon is best studied in possess more secretion than younger ones. Thus, over time, a Diploptera punctata, where glandular tissue, surrounding substantial portion of the metathoracic tracheal system becomes tracheae leading to the second abdominal spiracles, produces utilized for secretion storage. a benzoquinone secretion (Eisner 1958; Roth and Stay 1958; Various factors influence secretory discharge. Threatening Roth and Alsop 1978; Baldwin et al. 1990). As in Romalea, tactile (and-possibly visual) stimuli are perceived and centrally the secretion is stored in the tracheal lumen and is ejected with integrated; temporal and heterogeneous summation occurs. air through the spiracles when the roach is disturbed, the Secretory ejection eventuates either as an active stimulus- spiracles remain closed during normal respiration, no valve response (neuromuscular abdominal contraction) or passively isolates the exudate from the rest of the tracheal system, the (from external pressure to the abdomen or thorax). The MTTG tracheae along with the stored secretion are shed at each molt, is devoid of muscles and cannot forcefully discharge secretion and the newly molted individuals lack secretion and require by itself; ejection occurs via pneumatic and hemostatic time to recharge. In contrast to Romalea, either gland can fire pressure. The narrow spiracular orifice initially discharges the independently, and the secretion is ejected as a broadly secretion as a spray. This serves to eject the liquid away from dispersed spray. Despite the similarity of cockroach and lubber the insect, where it might contact the sensitive eyes or noses grasshopper tracheal glands, the narrow taxonomic occurrence of predators, and also disperses and increases the surface area of the respective glands and their disparate anatomical loca- of the secretion, allowing rapid volatilization. Thus, the insect tions clearly imply independent evolution. quickly generates a startling and repellent odorous signal. The Glands associated with the tracheal system are also found in repellent nature of the secretion from T. eques was clearly certain Lepidoptera, Hymenoptera, Trichoptera, and Diptera demonstrated during tests using predatory Onychomys mice (Whitten 1972). They are usually small (c0.1 mm) and are (Whitman et al. 1985). Mice that were sprayed in the face generally located at anastomosis junctures of adjacent abdomi- dropped T. eques and rubbed their faces in the soil. nal tracheal rudiments. Their biological functions are unknown As the force of ejection decreases, tracheal air emerges with (Hinks and Byers 1975; Byers and Hinks 1976). Other grass- the secretion, causing it to froth out of the spiracle with a hoppers produce defensive secretions, but from epidermal hissing noise. The resultant spheres of foam cover both sides glands clearly not homologous to those of Romalea. Some of the grasshopper, assuring contamination of predator mouth- oedipodin grasshoppers evert a middorsal gland through a parts. Because external pressure supplements secretion ejection, transverse slit opening on the promesonotal intersegmental biting predators experience immediate negative feedback. After membrane (Whitman 1990). Certain (e.g., secretory discharge ends, odorous exudate remains on the Phymteus, Poekilocerus, Zonocerus) eject defensive sub- grasshopper for up to 10 min. Olfactorily oriented predators stances from a bilobed gland opening between the first and such as nocturnal mice use this smell to identify and reject T. second abdominal tergites (Whitman 1990). In larvae the eques (Whitman et al. 1985). secretion is normally ejected as a spray, but in adults the The secretion assumes a succession of forms (spray, adher- exudate flows under the wings to the second abdominal ent froth, persistent odorous droplet), providing an olfactory, spiracles, where exhaled air causes it to froth (Fishelson 1960; gustatory, and acoustic multisensory event that is undoubtedly Qureshi and Ahmad 1970). Other Pyrgomorphidae, such as 2106 CAN. J. ZOOL. VOL. 69, 1991 WHITMAN ET AL. 2107

Dictyophorus and Aularches, apparently eject blood or secre- the secretion is probably prevented by keeping the meta- tion mixed with air, while producing a hissing noise (Grasse thoracic spiracles closed during respiration. 1937; Carpenter 1938). Although the discharge mechanism is The evolutionary origin of the MTTGs is problematic. That unclear, it appears to be different from that found in Romalea. the glands have a narrow taxonomic and geographic distribu- tion, are largely nonfunctional during the vulnerable periods of Evolutionary implications molting and oviposition, are absent or greatly reduced in larvae For Romalea, a defensive gland derived from the respiratory and freshly molted individuals, and can only be discharged system confers certain advantages: (i) the large from both glands simultaneously suggests that the gland or its tracheal trunks function well for secretion storage; (ii) the defense role is a relatively recently evolved phenomenon. A numerous internal air sacs and abdominal contractor muscles more ancestral gland would presumably be distributed across (present in grasshoppers) are suited for pneumatic more taxa and exhibit fewer limitations. An examination of secretory discharge; (iii) coupling secretory ejection with the closely related grasshoppers might clarify the origin of the 'ystem the exudate to be mixed with air' gland. Other (e.g., Chromacris) are aposematic and permitting foaming and rapid volatilization; and (iv) the gregarious (features associated with chemical defense), sug- spiracular valves are well suited to function as spray, foam, gesting that they too might possess tracheal glands. and sound-producing orifices. Thus, with little modification, the basic grasshopper respiratory system may be adapted to Acknowledgements function as an effective secretion storage and ejection device. A defense derived from the respiratory system has also We appreciate the assistance of Larry Orsak, Amy Pasteur, Els Plaum, and Mathew Nadakavukaren. Support was provided imposed certain constraints on Romalea. Because the defensive by National Science Foundation grants DEB 8 1- 1799 and DEB secretion is stored behind closed spiracular valves in the 8 1 17943 to Murray Blum and Clive Jones. This is a contribu- metathoracic tracheae, the insect cannot use these structures for - tion to the program of the Institute of Ecosystem Studies, The respiration; the metathorax must be ventilated by the meso- New ork Botanical Garden. thoracic and abdominal spiracles. The loss of the metathoracic Y trunks for respiration may be compensated for, in part, by the numerous enlarged air sacs in the region. In most insects, the BALDWIN,I. T., DUSENBERY,D..B., and EISNER,T. 1990. Squirting metathoracic tracheae are essential', they supply oxygen to the and refilling: dynamics of p-benzoquinone production in defensive primary flight muscles. But, for Romalea, there may be little glands of Diploptera punctata. J. Chem. Ecol. 16: 2823-2834. BLUM,M. S. 198 1. Chemical defenses of arthropods. Academic Press, cost associated with their loss because Romalea is flightless New York. and relatively sluggish, making high metabolic rates and 1985. Exocrine systems. In Fundamentals of insect physiol- associated metathoracic ventilation less necessary. However, ogy. Edited by M. S. Blum. John Wiley & Sons, New York. pp. this relationship may be viewed in another manner; in terms of 535-579. evolution Romalea may have traded flight for a tracheal BLUM,M. S., SEVERSON,R. F., ARRENDALE,R. F., WHITMAN, defensive capability. D. W., ESCOUBAS,P., ADEYEYE,O., and JONESC. G. 1990. A A defense based on the respiratory system has other generalist herbivore in a specialist mode metabolic, sequestrative, limitations. In Romalea, as in all grasshoppers, the respiratory and defensive consequences. J. Chem. Ecol. 16: 223-244. system is used to increase body volume during molting and BYERS,J. R., and HINKS,C. F. 1976. Fine structure of the midventral oviposition. During these times it becomes unavailable for abdominal tracheal glands in banded woolly bear caterpillars (Arctiidae:Lepidoptera).Can. J. Zool. 54: 1824-1839. secretory ejection. Furthermore, because the tracheal trunks are CARPE~ER,G. D. H. 1938. Audible emission of defensive froth by shed during molting, the defensive secretion stored in the insects. Proc. Zool. Soc. Lond. Ser. A, 108: 243-252. metathoracic trunks is periodically lost. This explains why DUNCAN,C. D. 1924. Spiracles as sound producing organs. Pan.-Pac. Romalea do not readily discharge secretion during oviposition Entomol. 1: 42-43. and are unable to do so during molting or the postmolting EISNER,T. 1958. Spray mechanism of the cockroach Diploptera period when their cuticles are soft. During these very vulnera- punctata. Science (Washington, D.C.), 128: 148-149. ble periods, the secretory defense is virtually nonfunctional. EISNER,T., HENDRY,L. B., PEAKALL,D. B., and MEINWALD,J. Another limitation is that the secretion is always ejected from 197 1. 2,5-Dichlorophenol (from ingested herbicide?) in defensive both metathoracic spiracles simultaneously; some secretion secretion of a grasshopper. Science (Washington, D.C.), 172: may be wasted by being discharged in a direction away from 277-278. a predator. FISHELSON,L. 1960. The biology and behavior of Poekilocerus bufonius Klug, with a special reference to the repellent gland. Eos In theory, the storage of a potentially harmful volatile (Rev. Esp. Entomol.), 36: 41-62. secretion in the respiratory passages could pose a problem for GRASSE,P. P. 1937. L'hCmaphorrhCe, rejet-rCflexe de sang et d'air Romalea. What prevents the secretion from contaminating the par les Acridiens phymatkides. C.R. Hebd. SCanc. Acad. Sci. remainder of the respiratory system is unknown; perhaps the (Paris), 204: 65-67. hydrophobic nature of the internal tracheal walls, coupled with HINKS,C. F., and BYERS,J. R. 1975. A new glandular organ in some a reduction in their diameter, serves as a barrier. Backflow of toxic caterpillars. Experientia, 31: 965-967.

FIG.12. Thick tracheal wall in the glandular region, with apical epidermal and basal secretory cells and taenidial cuticular pattern (Romalea male). Scale bar = 5 Fm. FIG. 13. Detail of apical region, with efferent draining tubules penetrating between epidermal and glandular cells (Romalea female). Scale bar = 1 Fm. FIG. 14. Normal tracheal wall in the nonglandular region, with only a very thin layer of epidermal cells (Romalea female). Scale bar = 5 Fm. FIG.15. Secretory cell cytoplasm containing numerous mitochondria and well-developed Golgi apparatus (Romalea female). Scale bar = 1 Fm. FIG. 16. Detail of secretory vesicle, showing microvillar sheath surrounding cuticular ductule (Romalea male). Scale bar = 1 Fm. d, duct; DC, duct cell; e, epicuticle; EC, epidermal cell; EN, epidermal cell nucleus; GA, Golgi apparatus; M, mitochondria; mv, microvilli; p, procuticle; SC, secretory cell; SN, secretory cell nucleus; t, taenidium. 2108 CAN. J. ZOOL. VOL. 69. 1991

JONES,C. G., HESS,T. A., WHITMAN,D. W., SILK,P. J., and BLUM, ROTH, L. M., and ALSOP, D. W. 1978. Toxins of Blattaria. In M. S. 1986. Idiosyncratic variation in chemical defenses among Arthropod venoms. Handbook of experimental pharmacology individual generalist grasshoppers. J. Chem. Ecol. 12: 749-761. No. 48. Edited by S. Bettini. Springer-Verlag, New York. pp. JONES,C. G., HESS,T. A., WHITMAN,D. W., SILK,P. J., and BLUM, 465487. M. S. 1987. Effects of diet breadth on autogenous chemical defense ROTH,L. M., and EISNER,T. 1962. Chemical defenses of arthropods. of a generalist grasshopper. J. Chem. Ecol. 13: 283-297. Annu. Rev. Entomol. 7: 107-1 36. JONES,C. G., WHITMAN,D. W., COMPTON,S. J., SILK,P. J., and ROTH,L. M., and STAY,B. 1958. The occurrence of para-quinones BLUM,M. S. 1989. Reduction in diet breadth results in sequestra- in some arthropods, with emphasis on the quinone-secreting tion of plant chemicals and increases efficacy of chemical defense tracheal glands of Diploptera punctata (Blattaria). J. Insect Physiol. in a generalist grasshopper. J. Chem. Ecol. 15: 18 1 1-1 822. 1: 305-3 18. MEINWALD,J., ERICKSON,K., HARTSHORN,M., MEINWALD,Y. C., UVAROV,B. 1966. Grasshoppers and locusts. Vol. 1. Cambridge and EISNER,T. 1968. Defensive mechanisms of arthropods. XXIII. University Press, London. An allenic sesquiterpenoid from the grasshopper ~omaleamicro- WHITMAN,D. W. 1982. Grasshopper sexual pheromone: a component ptera. Tetrahedron Lett. 25: 2959-2962. of the defensive secretion in . Physiol. Entomol. MORSE,A. P. 1907. Further researches on North American Acrididae. 7: 111-115. Carnegie Inst. Washington Publ. No. 68. pp. 1-54. 1990. Grasshopper chemical communication. In Biology of NOIROT,C., and QUENNEDEY,A. 1974. Fine structure of insect grasshoppers. Edited by R. F. Chapman and A. Joern. John Wiley epidermal glands. Annu. Rev. Entomol. 19: 61-80. & Sons, New York. pp. 357-391. QLTRESHI,S. A., and AHMAD,I. 1970. Studies on the functional WHITMAN,D. W., BLUM,M. S., and JONES,C. G. 1985. Chemical anatomy and histology of the repellent gland of Poekilocerus pictus defense in Taeniopoda eques (: Acrididae): role of the (F.) (Orthoptera: Pyrgomorphidae). Proc. R. Entomol. Soc. Lond. metathoracic secretion. Ann. Entomol. Soc. Am. 78: 451455. Ser. A, 45: 149-155. WHITMAN,D. W., BLUM,M. S., and ALSOP,D. W. 1990. Allomones: REHN, J. A. G., and GRANT,H. J., JR. 1959. A review of the chemicals for defense. In Arthropod defenses: adaptive mechanisms (Orth. Acrididae) found in America north of Mexico. and strategies of predators and prey. Edited by D. L. Evans and Proc. Acad. Nat. Sci. Phila. 111: 109-271. J. 0. Schmidt. State University of New York Press, Albany, 1961. A monograph of the Orthoptera of North America pp. 289-351. ' (North of Mexico). Vol. 1. Monogr. No. 12, Acad. Nat. Sci. Phila. WHITTEN,J. M, 1972. Comparative anatomy of the tracheal system. pp. 1-257. Annu. Rev. ~ntomol.17: 373402. RODRIQUEZ,E., HEALEY,P. L., and MEHTA, I. (Editors). 1984. Biology and chemistry of plant trichomes. Plenum Publishing Corp., New York.