MORPHOLOGY,HISTOLOGY, AND FINE STRUCTURE Morphology of the Alimentary Canal of rufilabris (: ) Adults in Relation to Microbial Symbionts

1 2, 3 1, 4 S. W. WOOLFOLK, A. C. COHEN, AND G. D. INGLIS

Ann. Entomol. Soc. Am. 97(4):796Ð808 (2004) ABSTRACT A study of the internal morphology of the alimentary canal of Chrysoperla rufilabris (Brumeister) adults in relation to yeast symbionts was conducted using light, scanning, and trans- mission electron and epißuorescence microscopy. The alimentary canal of Þeld-collected adults possessed a single large (Ϸ300Ð400 ␮m in length) diverticulum at the posterior end of the foregut. Although yeast cells (4.0Ð6.5 ␮m) were distributed throughout the alimentary canal, large numbers of blastically dividing cells (i.e., yeast) were observed within the diverticulum. The diverticulum interior was highly convoluted and folded transversely and longitudinally, and yeast cells were observed to accumulate within the folds. Large tracheal trunks were attached to the lateral side of the diverticulum, suggesting a high demand for gas exchange within this organ. The diverticulum was lined with cuticle, and the underlying tissues did not contain large amounts of endoplasmic reticulum, mitochondria, or Golgi complex, indicating that minimal absorption occurred within this gut region. This suggested that the high potential for gas exchange in the diverticulum by the tracheal trunks was primarily to support yeast metabolic activity. All size classes (i.e., 0.1, 4.0, and 10.0 ␮m) of ßuorescence particles ingested by newly eclosed adults eventually ended up in the midgut and hindgut regions, indicating that the foregut and/or diverticulum do not possess an absolute mechanism for retaining particles based on size. However, all size classes of the ßuorescent particles typically persisted within the diverticulum. The evident conßuence between the diverticulum lumen and the gut lumen suggested a free exchange or ßow of ßuids between these regions. The proventriculus (proximal to the diverticulum) was pronounced and consisted of a series of long “hairs,” short “hairs,” and small spine-like structures projecting into the midgut. Large numbers of yeast cells were observed in association with the proventricular hairs, and these hairs may play a role in the retention of yeast cells. The midgut possessed typical absorptive structures (i.e., microvilli), and large numbers of mitochon- dria, rough endoplasmic reticulum, and Golgi complex were observed in midgut epithelial cells. Because evidence indicated no or minimal absorption of nutrients within the diverticulum, it was concluded that nutritional factors provided by the yeast must be transferred to the midgut where absorption occurs. Large numbers of yeast cells enclosed within a well-developed peritrophic matrix were observed in the midgut, suggesting that the yeast themselves may serve as a source of nutrients. Whereas the exact mechanism by which yeast contribute to the nutrition of C. rufilabris adults was not determined, morphological evidence obtained in this study supported the hypothesis that chry- sopids form a mutualistic symbiosis with yeast and that the esophageal diverticulum was a specialized structure for housing them.

KEY WORDS Chrysoperla rufilabris, gut, alimentary canal, yeast, symbiosis

LACEWINGS HAVE LONG BEEN recognized as biological the Noctuidae, Pieridae, and Pyralidae (Principi and control agents of pests. They are particu- Canard 1984, Ridgway and Murphy 1984). Several larly effective as predators of aphids, but they also characteristics make larval lacewings effective biolog- attack many other pests such as leafhoppers, ical control agents. They are voracious, excellent thrips, psyllids, whiteßies, mites, and eggs and small searchers and exhibit high dispersal ability (Bond larvae of several lepidopterans, including members of 1980, New 1984, Senft 1997). The Chrysopidae, espe- cially Chrysoperla, are the most commonly deployed 1 Department of Entomology and Plant Pathology, Mississippi State, in augmentation strategies. The Chrysoperla carnea MS 39762. species complex is most commonly used in biological 2 Biological Control and Mass Rearing Research Unit, USDAÐARS, control programs in Europe, and Chrysoperla rufilabris Mississippi State, MS 39762. (Burmeister) is one of the most common taxa used in 3 Current address:Insect Rearing and Diet Institute, P.O. Box 65708, Tucson, AZ 85728Ð5728. North America (Tauber et al. 2000, Daane and Hagen 4 Current address:Agriculture & Agri-Food Canada, 5403Ð1st Ave. 2001, Henry et al. 2001). Improvements in the mass- S, Lethbridge, AB T1J 4B1 Canada (e-mail:[email protected]). rearing methodology (i.e., development of efÞcacious July 2004 WOOLFOLK ET AL.:G UT MORPHOLOGY OF LACEWING ADULTS 797 artiÞcial diets) in the past few years (Cohen 1998, tives were preserved for comprehensive identiÞcation 1999; Cohen and Smith 1998) has coincided with an by using keys provided by Brooks and Barnard (1990), increase in the numbers of Þeld experiments con- Brooks (1994), and Penny et al. (2000). Remaining ducted in a diverse array of agroecosystems (McEwen were processed for light and electron micros- et al. 2001) and facilitated commercial production of copy. lacewings (Van Lenteren et al. 1997) for pest control External Morphology of Alimentary Canal. To ex- and/or research. amine the external morphology of an intact alimentary Recent studies have indicated that the yeast canal, a laboratory-reared C. rufilabris adult was dis- Metschnikowia pulcherrima Pitt & Miller resides in the sected using an Edmund ScientiÞc stereomicroscope alimentary canal of Þeld-collected C. rufilabris adults, (Edmund Industrial Optics, Barrington, NJ) and ob- and yeast cells of M. pulcherrima accumulate within served with a PhotoMicroscope II (Carl Zeiss, Thorn- the diverticulum and foregut of the gut (Woolfolk and wood, NY), and the image was digitized with Auto- Inglis 2003). Hagen and Tassan (1972) postulated that Montage software (Syncroscopy, Frederick, MD). yeast provide essential amino acids, which were either Light Microscopy. Four Þeld-collected C. rufilabris missing or not readily available in the pollen, nectar, adults, collected on separate occasions, were pro- and/or honeydew in the lacewing adult diet. cessed according to standard protocols. Brießy, intact Ickert (1968), Hagen et al. (1970), Bitsch (1984), individuals were Þxed in 10% formaldehyde and de- and Principi and Canard (1984) reported extensive hydrated in a graded ethanol series (50, 75, 95, and tracheation associated with the lateral side of a con- 100%). Dehydrated specimens were placed in xylene spicuously enlarged diverticulum in chrysopid adults. and embedded in Paraplast (A. Brunswick, St. Louis, These studies were restricted to descriptions of the MO). Serial sections of Ϸ5 ␮m were obtained using a external gut structures. Limited information is avail- rotary microtome. Sections were mounted on glass able on the internal morphology and ultrastructure of slides, stained with hematoxylin and eosin, and exam- the digestive tract and associated tissues within lace- ined with a BH light microscope (Olympus America, wing adults. Furthermore, the mechanism by which Melville, NY) at 100 and 400ϫ. yeast may contribute to the nutrition of lacewings is Scanning Electron Microscopy (SEM). Four Þeld- unknown, and morphological information on the ali- collected adults, collected on separate occasions, were mentary canal of chrysopid adults may reveal impor- processed according to standard protocols (Bozzola tant information on the signiÞcance and mecha- and Russell 1992, Nation 1983, Rumph and Turner nism(s) by which yeast symbionts impact chrysopid 1998). Alimentary canals were cryofractured (n ϭ 2) adults. For example, does the absorption of nutrients or excised longitudinally (n ϭ 2). For cryofracturing, produced by yeast occur within the diverticulum? Is the alimentary canal was Þrst separated into four re- the function of the large tracheal trunks to supply gions (i.e., diverticulum, foregut, midgut, and hind- oxygen to and to release carbon dioxide from this gut). Tissues were Þxed in 2.5% glutaraldehyde in 0.1 organ to support both the yeast cells as well as host M potassium buffer (pH 7.2) (hereafter referred to as metabolic processes? buffer), for a minimum of 2 h, rinsed in buffer, post- The hypothesis here was that lacewing adults form Þxed in 2% osmium tetroxide in buffer for a minimum a mutualistic symbiosis with yeast and morphological of 2 h, rinsed in distilled water, dehydrated in a graded characteristics and ultrastructural characteristics of ethanol series (30, 50, 70, 95, and 100%), and fractured. the alimentary canal will reßect the importance of this Each gut region was fractured using a razor blade relationship. SpeciÞc objectives were to 1) observe while immersing the gut region in liquid nitrogen. the prevalence and spatial distribution of yeast cells in During cryofracturing, each region was cross-sec- various regions of the alimentary canal (i.e., foregut, tioned, and where necessary, longitudinal- or oblique- diverticulum, midgut, and hindgut); 2) ascertain sectioned. The cryofractured gut regions were rinsed whether a Þltering and/or retention mechanism exists with 100% ethanol. Gut regions that were to be sec- for yeast cells; and 3) determine whether the internal tioned longitudinally (i.e., noncryofractured samples) gross morphology and ultrastructure of the alimentary were Þxed overnight in 2.5% glutaraldehyde, rinsed in canal and associated tissues will reveal the process by buffer, postÞxed in 2% osmium tetroxide for a mini- which the yeast microßora contributes to the nutrition mum of 2 h, rinsed in distilled water, pinned to a petri of C. rufilabris adults. dish containing wax, and the gut was longitudinally sectioned using a razor blade. Sectioned gut regions were dehydrated in a graded ethanol series (30, 50, 70, Materials and Methods 95, and 100%). Cryofractured samples were critical Insect Collection and Sample Preparation. Chry- point dried, and noncryofractured samples were soperla adults were arbitrarily collected from two sites, chemically dried using hexamethyldisilazane. Samples one in Monroe County (longitude 88.411Њ W; latitude were coated with gold palladium, and examined with 33.740Њ N) and the other in Oktibbeha County (lon- LEO S360 SEM (LEO, Inc., Thornwood, NY) at 15 kV. gitude 88.782Њ W; latitude 33.416Њ N), MS. Sweep nets Transmission Electron Microscopy (TEM). Four and blacklight traps were used. Immediately after col- Þeld-collected adults, collected on separate occasions, lection, adults were placed on ice, transported to the were processed according to standard protocols. laboratory, and stored for 24 h at 5ЊC. Insects were Adults were primary-Þxed in half-strength Kar- examined for taxonomic characters and representa- novskyÕs Þxative in buffer, rinsed in buffer, postÞxed 798 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 4

Fig. 1. Alimentary canal of a C. rufilabris adult. Cp, crop; Dv, diverticulum; Fb, food bolus; Hd, head; Im, ileum; Mg, midgut; Mp, Malpighian tubules; Pv, proventriculus; Rp, rectal pads; Rpo, rectal pouch. Bar, 170 ␮m. in 2% osmium tetroxide for2honice, rinsed in distilled change in the diverticulum of chrysopid adults. To water, dehydrated in a graded ethanol series, and calculate the C-value, four laboratory-reared C. rufi- embedded in low-viscosity epoxy resin (Spurr 1969, labris adult females and four males were dissected, and Bozzola and Russell 1992, Rumph and Turner 1998). the diverticular tracheal trunk diameter and forewing To locate the areas of interest, thick sections (1.0 ␮m) length were measured using a BH light microscope were cut with a glass knife, placed on a microscope (Olympus America). C-values were calculated by di- slide, and stained with toluidine blue. Pale gold to viding the diameter of the tracheal trunk (microme- silver sections were cut with a glass knife, mounted on ters) by the forewing length (millimeters). To com- copper grids, stained with uranyl acetate for 20 min pare tracheal trunk diameters, forewing lengths and followed by lead citrate for 10 min, and examined with C-values between females and males, the t-test pro- JEOL JEM-100CX2 TEM (JOEL USA, Peabody, MA) cedure of Statistical Analyses System software (SAS at 60 kV. Institute 1999) was used. Epifluorescence Microscopy. Ready-to-emerge pu- pae of C. rufilabris were obtained from larvae reared Results on an artiÞcial diet (Cohen and Smith 1998). Pupae were placed in a large, cylindrical cardboard cage (17 Insects and External Morphology. All Þeld-col- cm in diameter) topped with organdy cloth. Within 2 h lected lacewings adults were identiÞed as C. rufilabris. of eclosion, 10 adults were transferred to a similarly Except for the diverticulum, the alimentary canal was shaped but smaller cages (9 cm in diameter) and were a nearly straight tube, which included anteriorly a allowed to ingest sugar (i.e., sucrose) water paste (1:1) buccal cavity, a short pharynx, and esophagus, fol- containing either 0.1-, 4.0-, or 10.0-␮m-diameter Fluo- lowed by an elongated crop that extended laterally as Spheres microsphere (Molecular Probes, Eugene, a conspicuously large diverticulum (Fig. 1). An en- OR), respectively. After Ϸ20 h, adults were sacriÞced; larged proventriculus was observed anterior to the the intact alimentary canals were dissected, placed on midgut. The midgut typically contained a delineated the microscope slides, and examined with an Axioskop food bolus within a peritrophic matrix (PM). The II epißuorescence microscope (Carl Zeiss), and im- ileum was relatively short followed by the rectum in ages were digitized with Auto-Montage software using which rectal pads were typically observed. a Syncroscopy digital microscope (Syncroscopy USA, Internal Morphology. Large populations of yeast Frederick, MD). The location and relative density of were observed in the foregut particularly in the prox- the microspheres of different diameters in the various imity of the opening to the diverticulum. The foregut gut regions were recorded. was folded internally, and the yeast cells typically Measurement of C-Value. The C-value is an index were observed between these folds (Fig. 2A and B). used by Canard et al. (1990) to estimate the relative Food particles and bacteria also were typically de- degree to which the tracheal trunk supports gas ex- tected. In the crop region of the foregut, highly scle- July 2004 WOOLFOLK ET AL.:G UT MORPHOLOGY OF LACEWING ADULTS 799

Fig. 2. Scanning and transmission electron micrographs of C. rufilabris adult foreguts. (A) High populations of yeast (arrows) in between fold structures within cryofractured foregut. Bar, 50 ␮m. (B) Yeast cells together with food particles and bacteria. Bar, 20 ␮m. (C) Tooth-like structures. Bar, 20 ␮m. (D) Higher magniÞcation micrograph of the highly sclerotized tooth-like structures. Bar, 5 ␮m. (E) Cuticle lining the foregut. Bar, 3 ␮m. rotized tooth-like structures were observed (Fig. 2C that the diverticular folds were conspicuously lined and D). Examination with TEM revealed that the with cuticle (Fig. 5D). There were limited numbers of entire foregut region was lined with cuticle (Fig. 2E), organelles indicative of high metabolic activity in the and we observed no or limited numbers of mitochon- underlying cells of the diverticulum. There was no dria, endoplasmic reticulum, Golgi complex, vacuoles, discrimination between the three size classes of par- and microvilli in the underlying cells of the foregut. All ticles (i.e., 0.1, 4.0, and 10.0 ␮m) on entry into, and on size classes (i.e., 0.1, 4.0, and 10.0 ␮m) of ingested the Þnal distribution within the diverticulum; the ßu- ßuorescence particles were observed in the foregut orescent particles were evenly distributed throughout Ϸ20 h after ingestion. Limited numbers of the particles (Fig. 3B). Soon after ingestion (Ϸ4 h), all size classes were present in the anterior foregut (Fig. 3A), and of ßuorescent particles tended to accumulate within they tended to accumulate where the foregut the diverticulum. However, few particles remained in branched into the diverticulum. the diverticulum after Ϸ20 h (Fig. 3B). The diverticulum of C. rufilabris was pronounced The proventriculus was conical with a wide anterior (Ϸ300Ð400 ␮m in length by 30Ð75 ␮m in diameter) end connected to the foregut, and a narrow posterior containing extensive musculature and was associated end connected to the midgut (Fig. 6A). The proven- with conspicuously large tracheal trunks (Figs. 4A and triculus had eight triangular lips, and each lip had a 5A). The tracheal trunks branched into many trache- group of long hair-like structures that attached to the oles, which attached to the outer wall of the divertic- anterior end of the proventriculus (Fig. 6B, C, and D). ulum (Fig. 4B). Closer observation within the trunk The “hairs” projected into the midgut lumen (Fig. 6C revealed tight spirals of the cuticular intima (i.e., and D). The lumen of the proventriculus consistently taenidia) (Fig. 4C and D). Cryofractured cross sec- contained large numbers of yeast cells (Fig. 6B, C, and tions of the diverticulum revealed conspicuous folding D); also, pollen grains, moth scales, and food particles of the inner surface of this structure (Fig. 5A). Within were commonly observed. Longitudinal muscles lay- the diverticulum, numerous yeast cells were observed ered the inner wall of the proventriculus next to each to aggregate on the surface of, and within, the folds lip (Fig. 7A) with circular muscles around it. Each lip (Fig. 5A and B). In several specimens, pollen grains seemed to have a tooth-like boundary (Fig. 7B). Nu- and insect scales also were observed within the diver- merous yeast cells were observed in between the hair- ticulum (Fig. 5C). Observations with TEM revealed like structures (Fig. 7C and D). The latter were long 800 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 4

Fig. 3. Epißuorescence micrographs showing the distribution of ßuorescence particles (4 ␮m in diameter) within the various gut regions of C. rufilabris adults Ϸ20 h after their ingestion. Bars, 20 ␮m. (A) Foregut. (B) Diverticulum. (C) Anterior midgut. (D) Posterior midgut. (E) Ileum. (F) Rectal pouch. thick hairs or short thin hairs (Fig. 7D). Each long hair epithelial cells of the posterior midgut (Fig. 8C). Nu- branched at its tip (Fig. 7D). Also, longitudinal sec- merous mitochondria, Golgi complex, and rough en- tions revealed spine-like structures in between the doplasmic reticulum, indicative of high metabolic ac- two types of hairs (Fig. 7D), which were located tivity, were observed in the midgut cells (Fig. 8D). In within the inner wall of proventriculus basally to the addition to spherocrystals, dense vesicles and storage long and short hairs. All size classes of ßuorescent particles were observed within the proventriculus vesicles were commonly observed. Cryofractured (Fig. 3C) Ϸ20 h after their ingestion. midguts contained well-developed PM (Fig. 8E), Food materials together with moth scales, yeast which was typically net-like (Fig. 8F). The PM always cells, and pollen grains were present within the midgut enveloped the food bolus within the midgut and rec- (Fig. 8A). Conspicuous microvilli were observed at tum. Large numbers of all three size classes of ßuo- the apical surface of the midgut epithelium (Fig. 8B). rescent particles were observed within the food bolus Large numbers of spherocrystals, which looked like in the midgut (Fig. 3C and D) Ϸ20 h after ingestion concentric rings, were consistently observed in the of diet particles. July 2004 WOOLFOLK ET AL.:G UT MORPHOLOGY OF LACEWING ADULTS 801

Fig. 4. Scanning and transmission electron micrographs of the diverticulum and tracheoles of C. rufilabris adults. (A) Diverticulum showing large tracheal trunks (arrow) attached to the lateral surface. Bar, 200 ␮m. (B) Tracheal trunk (arrow) and tracheoles in between folds, which disappear beneath the surface of the diverticulum. Bar, 50 ␮m. (C) Interior of a large tracheal trunk showing taenidia. Bar, 20 ␮m. (D) Lateral view of taenidia within a tracheal trunk. Bar, 2 ␮m.

The pyloric region was comprised of large, thick- were 5.8 Ϯ 0.3 and 3.5 Ϯ 0.4, respectively. For all three ened circular muscles (Fig. 9A). Large numbers of measurements, values were larger (t Ն 9.2, df ϭ 6, P Ͻ microorganisms, including bacteria and yeast, were 0.001) for females than males. observed within the lumen of the anterior hindgut (Fig. 9A and B). The hindgut was cuticle-lined (Fig. Discussion 9C), and in several specimens, the extruded PM from the midgut was observed (Fig. 9D). A total of eight We observed yeast cells throughout the alimentary Malpighian tubules arose from the pyloric region (Fig. canal of Þeld-collected C. rufilabris adults. However, 10A), and no evidence for a cryptonephridial arrange- conspicuously large numbers of yeast cells were ment of the Malpighian tubules was observed (Chap- present within the diverticulum, an observation made man 1998). Cryofractured cross sections of the Mal- previously by others (Hagen et al. 1970). Further- pighian tubules showed the luminal surface to be more, we observed that the internal wall of the diver- convoluted (Fig. 10B). The ileum was located imme- ticulum was extensively folded, and yeast cells tended diately posterior to the pyloric region and looked like to aggregate within these folds. The high density of a relatively short narrow tube (Fig. 1). The cuticle yeast that we observed in diverticula is consistent with lining the hindgut produced small spines (Fig. 10C). microbiological observations made in Mississippi by A conspicuous rectal pouch followed the ileum and Woolfolk and Inglis (2003) in which the highest den- contained six rectal pads, arranged radially around the sity of yeast (primarily M. pulcherrima) were isolated pouch (Fig. 10D). After Ϸ20 h, all size classes of from the diverticulum, followed by the foregut, mid- ingested ßuorescent particles were observed in the gut, and hindgut regions of Þeld-collected C. rufilabris pyloric region, in the ileum (Fig. 3E), and rectum (Fig. adults; the density of yeast cells in diverticulum was 2, 3F). However, densities of the particles were greater 18, and 44 times greater than in the foregut, midgut, in the midgut (Fig. 3C and D). and hindgut, respectively. Although we did observe C-Values. The diameter of diverticular tracheal bacterial cells in the foregut and diverticulum in some trunks of female and male adults averaged 70.5 Ϯ 3.2 individuals, microbiological data collected by Wool- mm and 37.8 Ϯ 3.8 ␮m, respectively. The length of folk and Inglis (2003) indicated that bacteria are tran- forewings for females was 12.1 Ϯ 0.25 mm and 10.8 Ϯ sients and not true residents of the alimentary canals 0.3 mm for males. The C-values of females and males of C. rufilabris adults in Þeld environments. 802 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 4

Fig. 5. Scanning and transmission electron micrographs of C. rufilabris adult diverticula. (A) Cryofractured section of a diverticulum showing diverticular folds and the presence of large numbers of yeast cells situated in between the folds. Bar, 100 ␮m. (B) Longitudinal view of the internal surface of the diverticulum containing yeast cells. Bar, 100 ␮m. (C) Pollen grains (arrow) along with yeast cells in a diverticulum. Bar, 50 ␮m. (D) Diverticular folds lined with cuticle, note the presence of a yeast cell (Y) and tracheoles (T) in between folds. Bar, 5 ␮m.

The diverticulum is an out pocketing or pouch typ- our conclusion that minimal absorption occurred ically associated with the foregut. A number of insects within the diverticulum. The above-mentioned evi- form diverticula, including adult tephritid ßies (Chap- dence thus suggests that the high gas exchange pro- man 1998), but the extremely large tracheal trunks vided by the tracheal system associated with the di- articulated with diverticula in chrysopids is unique to verticulum is not to support metabolic activity of cells our knowledge. In C. rufilabris adults, we observed within this region. Coupled with the presence of large that the tracheal trunks were attached laterally to the numbers of yeast cells within the diverticulum would diverticulum, an observation made previously by Ick- strongly suggest that the high requirement for gas ert (1968) and Hagen et al. (1970) for other chry- exchange provided by the tracheal trunks was primar- sopids. The immense size of the tracheal trunks sug- ily to support the activity of the yeast residing there; gested a high rate of gas exchange indicative of all yeasts, including M. pulcherrima, are either obligate elevated metabolic activity. Furthermore, taenidia aerobes or facultative anaerobes (Barnett et al. 1990). were consistently associated with the tracheal trunks Canard et al. (1990) used a C-value to estimate the and tracheoles; the role of taenidia is to strengthen the degree to which oxygen was supplied to the divertic- trachea and to provide elasticity to resist collapse and ulum of European chrysopids. Although they did not compression (Chapman 1998). Despite the presence actually measure oxygen supply in developing the of conspicuously large tracheal trunks, we observed C-value, they showed that the C-value was correlated that the diverticulum was continuously lined with with palynoglycophagy (i.e., species using nectar, cuticle, suggesting that no or minimal absorption of honeydew, and/or pollen as a food substrate). Adults nutrients occurred in this region. Transfer of nutrients possessing a large C-value were typically palynogly- can occur through relatively impermeable linings. For cophagous whereas, adults with a small C-value (Ͻ1) example, the lining of the crop of Periplaneta is per- were generally predaceous; predatory insects do not meable to free fatty acids (Chapman 1998). However, typically form symbioses with microorganisms (Buch- in C. rufilabris adults, we did not observe signiÞcant ner 1965). We observed C-values ranging from 2.9 to numbers of organelles (e.g., mitochondria, rough en- 6.1 for Þeld-collected C. rufilabris adults, consistent doplasmic reticulum, and Golgi complex) in the tis- with the palynoglycophagous nature of this insect. sues underlying the diverticulum or crop reinforcing The correlation between oxygen exchange and pa- July 2004 WOOLFOLK ET AL.:G UT MORPHOLOGY OF LACEWING ADULTS 803

Fig. 6. Scanning and transmission electron micrographs of C. rufilabris adult proventriculi. (A) Lateral view showing the conical-shaped (arrow) of the proventriculus. (Note:the structures beneath and to the left of the proventriculus are the diverticulum and midgut, respectively.) Bar, 200 ␮m. (B) Anterior portion of the proventriculus showing the eight groups of long hairs with numerous yeast cells. Bar, 100 ␮m. (C) Anterior portion showing that the proventricular hairs projecting into the lumen of the midgut. Bar, 100 ␮m. (D) Cryofractured section of proventriculus showing the large anterior end and smaller posterior end and the presence of long and short hairs. Bar, 100 ␮m. lynoglycophagy in combination with our Þndings of then transported as a ßux from the diverticulum to large numbers of yeast and minimal host metabolic other regions of the alimentary canal where they are activity in diverticular cells all supported the impor- absorbed; and 2) the yeast themselves could be trans- tance of yeast in the biology of these insects. Canard ferred to another region of the alimentary canal where et al. (1990) reported that the C-values of some pa- they are digested releasing essential nutritional fac- lynoglycophagous chrysopids differed between males tors; yeast ingested by insects can serve as a source of and females, a Þnding that we observed for C. rufila- nutrients (Peng et al., 1984, Byzov et al. 1993). For bris adults; a mean C-value of 3.5 was obtained for both strategies, it would be expected than an absolute males compared with a C-value of 5.8 for females. or partial retention mechanism would exist keeping Reasons for the differences in C-values between sexes yeast in the diverticulum, allowing proliferation. are unknown but may reßect differences in their nu- To test this possibility, we monitored the fate of tritional requirements and digestive ability (Canard three size classes of ßuorescent particles (i.e., 0.1, 4.0, 2001). and 10.0 ␮m) ingested by newly eclosed lacewing Yeast is an excellent source of vitamins and sterols, adults. All size classes ingested by lacewing adults and yeast is are often used as a nutrient supplement in were ephemeral within the foregut and initially ac- insect diets (Cohen 1999). Based on studies in which cumulated within the diverticulum. However after sorbic acid (i.e., a fungistatic agent) was fed to Chry- Ϸ20 h, the majority of the particles of all size class was sopa carnea (Stephens) adults with and without spe- observed in the midgut, suggesting that a retention ciÞc amino acids, Hagen et al. (1970) concluded that mechanism based on size alone does not exist in C. the yeast symbionts in the diverticulum provide valine rufilabris. This was consistent with observations in to adult females, imparting a signiÞcant positive effect which considerable numbers of yeast cells were iso- on fecundity. There are two salient ways in which lated from the midgut region of Þeld-collected C. rufi- yeast proliferating in the diverticulum could impact on labris adults (Woolfolk and Inglis 2003). However, the nutrition of palynoglycophagous chrysopids:1) they noted that yeast populations (i.e., adjusted for the yeast could produce essential nutrients (e.g., differential sizes of the gut regions) were substantially amino acids) that are released from the yeast cells and greater in the diverticulum relative to the midgut, 804 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 4

Fig. 7. Scanning electron and light micrographs of C. rufilabris adult proventriculi. (A) Triangular-shaped lips which serve as the base for hairs, and each lip is attached to the proventricular inner wall, which is strengthened by longitudinal muscles (arrow). Bar, 50 ␮m. (B) Cross section showing the eight triangular lips in which a pointed, tooth-like structure separates each lip (arrow). (Note:yeast cells in between the lips and tooth-like structures.) Bar, 260 ␮m. (C) Numerous yeast cells that have accumulated in between the long hairs. Bar, 20 ␮m. (D) Long hairs (L), short hairs (S), and spine-like (Sp) structures that cover the internal surface of the proventriculus. (Note:yeast in between the hairs and the branched tips of the long hairs ϭarrow.) Bar, 20 ␮m. suggesting that either the viability of these yeast cells unbranched, short, thin hairs. The long hairs may serve was reduced in the midgut and/or a partial Þltering to Þlter large particles such as yeast, and the branched mechanism for yeast exists between the diverticulum tips may facilitate sorting of particles based on size and and midgut. Whether a mechanism exists for differ- consistency. The short hairs may serve to Þlter smaller entially retaining yeast in the diverticulum is uncer- particles. Certainly the conical shape of proventricu- tain, but attachment of yeast cells to the cuticular wall lus in C. rufilabris adult oriented with a large anterior of the diverticulum facilitated by the extensive folding and small posterior end suggested that this structure within this organ may occur. We also observed that the serves to facilitate the transfer of materials to the proventriculus of C. rufilabris adults was well devel- midgut. oped and produced spines and two types of dense Large numbers of yeast cells were observed in the hair-like structures that projected posteriorly into the midgut, supporting the hypothesis that the yeast cells midgut, possibly somehow serving as a yeast-accumu- themselves serve as a nutrient source. In all specimens, lating structure. Furthermore, large numbers of yeast yeast cells were observed within the food bolus sep- cells were typically observed in the proventriculus arated from the midgut epithelial cells by a well-de- embedded within the hairs. In honey bees, a similar veloped PM; a primary function of the PM is to prevent arrangement of hairs and spine-like structures oc- microorganisms from contacting the epithelial surface curred within the proventriculus, and the opening (Chapman 1998). We observed that the PM of C. action of longitudinal muscles of the proventriculus rufilabris adults was net-like, similar to PMs produced allowed yeast to be caught and Þltered (Peng and by other insects, suggesting a Þltration/protection Marston 1986). When the longitudinal muscles relax, function of this structure. For example, the net-like the yeast cells are passed into the midgut by the action PM produced by desert locusts consists of a chitinous of the hairs where they are digested (Peng and Mar- lattice that forms around the microvilli at the anterior ston 1986). We observed that C. rufilabris adults pro- end of the midgut (Chapman 1985). The PM is sufÞ- duced two types of hairs in association with the prov- ciently permeable to allow the passage of enzymes and entriculus:long, thick hairs with branches and catabolic products and serves to regulate the excretion July 2004 WOOLFOLK ET AL.:G UT MORPHOLOGY OF LACEWING ADULTS 805

Fig. 8. Scanning and transmission electron micrographs of Chrysoperla rufilabris adult midguts. (A) Longitudinal section of the midgut. (Note:in addition to the food materials, large numbers of yeast cells and to a lesser extent, moth scales and pollen grains were observed.) Bar, 200 ␮m. (B) Microvilli (arrow). Bar, 3 ␮m. (C) Spherocrystals. Bar, 3 ␮m. (D) Midgut cells. ER, endoplasmic reticulum; G, Golgi complex, M, mitochondria; S, spherocrystal; SV, storage vesicles; V, dense vesicles. Bar, 1 ␮m. (E) Peritrophic matrix (arrow) enclosing the food bolus. Bar, 100 ␮m. (F) Net-like appearance of the peritrophic matrix. Bar, 20 ␮m. of digestive enzymes (Chapman 1998). Therefore, the midgut and contribute to the nutrition of chrysopid PM would not be expected to preclude the digestion adults warrants study. of yeast in the midgut of chrysopids or other insects; The hindgut of C. rufilabris consisted of a well- digestion of yeast readily occurred in the midgut of developed pylorus, a relatively short and narrow il- honey bees despite the presence of well-developed eum, and a somewhat enlarged rectum that contained PM (Peng et al. 1984). In honey bees, digestion of six rectal pads. We observed that the PM enclosing the yeast commenced with depolymerization of the yeast food bolus was eventually passed to the ileum via the cell wall followed by hydrolysis of yeast proteins pyloric valve where it lost integrity; the spines present (Peng et al. 1984). Whether chrysopid adults possess on the hindgut wall may have functioned to break the enzyme systems necessary to digest yeast, and the down the PM before it is released from the rectum mechanisms by which yeast cells are digested in the (Becker 1978). Fluorescent particles of all size classes 806 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 4

Fig. 9. Scanning and transmission electron micrographs of C. rufilabris adult hindguts. (A) Cross section of the pyloric region. (Note:strong circular muscles surrounding this region and the food bolus.) Bar, 100 ␮m. (B) Yeast and bacterial cells in the lumen of the ileum. Bar, 20 ␮m. (C) Cuticle (arrow) lining the ileum. Bar, 1 ␮m. (D) Extruded peritrophic matrix (arrow) within the ileum. Bar, 1 ␮m. reached the ileum relatively rapidly and accumulated this organ. Therefore, putative nutritional factors pro- in the rectum. Furthermore, we observed what vided by the yeast must be transferred to other regions seemed to be intact yeast cells in the hindgut of of the alimentary canal where digestion and/or ab- Chyrsoperla adults, an observation made previously by sorption occurred. The most likely scenario was that Bozsik (2000). Even though yeast cells were digested yeast cells proliferate in the diverticulum, and nutri- in the midgut of bees, Peng et al. (1984) observed that ents produced by the yeast and/or the yeast them- the feces of honey bees still contained large numbers selves (i.e., as a catabolic source) are transferred to the of apparently undigested yeast cells. Whether the midgut; this is consistent with previous Þndings based yeast was viable was uncertain, but Woolfolk and on microbiological analyses (Woolfolk and Inglis Inglis (2003) recovered few viable yeast cells from the 2003). The proventriculus was found to be a complex hindgut of Þeld-collected C. rufilabris adults, suggest- organ consisting of spines and two types of hair-like ing that most yeast cells did not survive passage structures, whose likely function was to control the through the alimentary canal of lacewings. Among transfer of yeast cells to the midgut. We also fre- insects that possess symbionts in their hindgut, the quently observed pollen grains in the alimentary ca- ileum is usually enlarged (Chapman 1998). That the nals of C. rufilabris adults but their impact on nutrition ileum of C. rufilabris adults was very restricted, cou- of chrysopids was uncertain. The impact of yeast and pled with previous Þndings of small numbers of viable the mechanisms by which symbionts contributed to yeast cells, Þlamentous fungi, and bacteria in this re- the nutrition of lacewing adults warrants study, and gion (Woolfolk and Inglis 2003), would suggest that the Þndings reported here will facilitate such studies. the hindgut of lacewing adults did not play a signiÞ- cant role in the housing of yeast symbionts. In conclusion, the microscopic evidence obtained Acknowledgments in this study indicated that the diverticulum attended We are grateful to the following people:Amanda Law- by exceptionally large tracheal trunks, is a specialized rence (Department of Entomology and Plant Pathology, Mis- structure for yeast proliferation. However, the diver- sissippi State University) for assistance with light micros- ticulum was cuticle-lined, and underlying cells did not copy, and thick- and thin-sectioned preparation for contain sufÞcient numbers of organelles to indicate transmission electron microscopy; David Cross (Department that substantive assimilation of nutrients occurred in of Entomology and Plant Pathology, Mississippi State Uni- July 2004 WOOLFOLK ET AL.:G UT MORPHOLOGY OF LACEWING ADULTS 807

Fig. 10. Scanning electron micrographs of C. rufilabris adult hindguts. (A) Malpighian tubules arising from pylorus outer wall. Bar, 100 ␮m. (B) Cryofractured cross section of Malpighian tubules showing the convoluted luminal surface. Bar, 20 ␮m. (C) Spine-like cuticle on the internal surface of the hindgut. Bar, 20 ␮m. (D) Lateral view of the rectal pouch with rectal pads. Bar, 100 ␮m. versity) for assistance with Þeld collection of lacewing adults Bozsik, A. 2000. Nahrungsanalytische untersuchungen an from Monroe County, Mississippi; Terry Schiefer (Depart- einigen mitteleuropa¨ischen chrysopiden-imagines (Neu- ment of Entomology and Plant Pathology, Mississippi State roptera:Chrysopidae). Beitr. Entomol. 50:237Ð246. University) for assistance on the identiÞcation of lacewing Bozzola, J. J., and L. D. Russell. 1992. Electron microscopy: adults; Gerald Baker and Peter Ma (Department of Ento- principles and techniques for biologists. Jones and Bart- mology and Plant Pathology, Mississippi State University) for lett Publishers, Boston, MA. allowing access to the Carl Zeiss PhotoMicroscope II and Brooks, S. J. 1994. A taxonomic review of the common green Carl Zeiss Axioskop II epißuorescence microscope; and Ger- lacewing genus Chrysoperla (Neuroptera:Chrysopidae). ald Baker, Peter Ma, and Dawn Luthe (Department of Bio- Bull. Br. Mus. Nat. Hist. (Entomol.). 63:137Ð210. chemistry and Molecular Biology, Mississippi State Univer- Brooks, S. J., and P. C. Barnard. 1990. The green lacewings sity) for conducting critical reviews of the manuscript. This of the world:a generic review (Neuroptera:Chrysopi- research was funded in part by a Departmental Graduate dae). Bull. Br. Mus. Nat. Hist. (Entomol.). 59:117Ð286. Fellowship to S.W.W. and is Mississippi Agriculture and For- estry Experiment Station contribution J10464. Buchner, P. 1965. Endosymbiosis of with plant mi- croorganisms. Interscience, New York. Byzov, B. A., T. V. Nguyen, and I. P. Babjeva. 1993. Inter- References Cited relationships between yeasts and soil diplopods. Soil Biol. Biochem. 25:1119Ð1126. Barnett, J. A., R. W. Payne, and D. Yarrow. 1990. Yeasts: Canard, M. 2001. Natural food and feeding habits of lace- characteristics and identiÞcation, 2nd ed. Cambridge wings, pp. 116Ð129. In P. K. McEwen, T. R. New, and A. E. University Press, New York Becker, B. 1978. Determination of the rate of peritrophic Whittington [eds.], Lacewings in the crop environment. membranes in some Diptera. J. Insect Physiol. 24:529Ð Cambridge University Press, New York. 533. Canard, M., H. Kokubu, and P. Duelli. 1990. Tracheal Bitsch, J. 1984. Anatomy of adult Chrysopidae, pp. 29Ð36. In trunks supplying air to the foregut and feeding habits in M. Canard, Y. Se´me´ria, and T. R. New [eds.]. Biology of adults of European green lacewing species (Insecta:Neu- Chrysopidae. Dr W. Junk Publishers, The Hague, The roptera:Chrysopidae), pp. 227Ð286. In M. W. Mansell and Netherlands. H. Aspo¨ck [eds.], Advances in neuropterology, Proceed- Bond, A. B. 1980. Optimal foraging in a uniform habitat:the ings of the 3rd International Symposium on Neuropter- search mechanism of the green lacewing. Anim. Behav. ology, South African Department of Agricultural Devel- 28:10Ð19. opment, Pretoria, South Africa. 808 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 4

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