Aggregation Pheromone Glands of Carpophilus Freemani (Coleoptera: Nitidulidae) and Gland Distribution Among Other Sap Beetles
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6 PHYSIOLOGY, BIOCHEMISTRY, AND TOXICOLOGY Aggregation Pheromone Glands of Carpophilus freemani (Coleoptera: Nitidulidae) and Gland Distribution Among Other Sap Beetles PATRICK F. DOWDI AD ROBERT J. BARTELT2 ational Center for Agricultural Utilization Research, USDA-ARS, 1815 N. University Street, Peoria, IL 61604 Ann. EntomoJ. Soc. Am. 86(4): 464-469 (1993) ABSTRACT The apparent site of aggregation pheromone production in the sap beetle Carpophilus freemani Dobson is described. Small (=100 J-Lm diameter) disks associated with the tracheal system in the abdomen contain the pheromone in this insect. These structures appeared to be single cells with large nuclei. Similar structures were found in Carpophilus hemipteros (L.), C. lugubris Murray, C. antiquus Melsheimer, C. (Uropho ros) humeralis (F.), Stelidota geminata Say, Haptoncus luteolus (Erichson), and Glischro chilus quadrisignatus (Say), suggesting that these structures are a common source of pheromones in sap beetles. KEY WORDS sap beetles, pheromones, pheromone glands PHEROMO E PRODUCTIO in insects can occur at Dobson (Bartelt et al. 1990b), C. hemipterus (L.) diverse sites. Within the Coleoptera, pheromone (Bartelt et al. 1990a), and C. lugubris Murray sources include a variety of tissues. Male Pse (Bartelt et al. 1991). Of these species, C. free laphidae may produce sex pheromones in their mani produces, by far, the most pheromone per antennae (DeMarzo & Vit 1983). Epidermal or adult male (=50-100 ng/d) (Bartelt et al. 1990b). subepidermal glands produce sex pheromones Therefore, C. freemani was chosen for our study in female Diabrotica virgifera LeConte (Chry to determine the site of pheromone production. somelidae) (Lew & Ball 1978) and several spe cies of Trogoderma (Dermestidae) (Hammack et Materials and Methods al. 1973) and male aggregation pheromones in Dermestes ater DeGeer (Dermestidae) (Imai et Insects. C. freemani came from colonies al. 1990) and Tribolium castaneum (Herbst) reared on ~into bean-based diet (Dowd 1987, (Tenebrionidae) (Faustini et al. 1982). Female Dowd & Weber 1991). Other species of sap bee cigarette beetles, Lasioderma serricome (F.) tles examined were reared on similar diets (C. (Anobiidae), contain large, lobed sex pheromone hemipteros, C. antiquus Melsheimer, C. [Uro glands within the body cavity (Levinson et al. phoros] humeralis [F.J, Stelidota geminata Say, 1983). Bark beetles (Scolytidae) appear to pro Haptoncus luteolus [ErichsonJ) or were field col duce aggregation pheromones in the hindgut, to lected (C. lugubris, Glischrochilus quadrisigna which gut microflora may contribute (see review tus [Say]). by Jones 1985). The male boll weevil, Anthono Dissections. Insects were chilled on ice and mus grandis grandis Boheman (Curculionidae), dissected in distilled water held in an ice bath. produces aggregation pheromone in the fat body Because some of the tissues are fragile, we kept (Wiygul et al. 1982). them chilled during dissection. This solidified Sap beetles (Nitidulidae) can be pests of fresh lipid materials and preserved tissue integrity. and stored fruits and vegetables and may vector In initial determinations, the insects were sepa pathogenic microorganisms to these commodi rated into three parts: head and prothorax, meso ties, including those that produce mycotoxins and metathorax, and abdomen. After it was (see review by Dowd [1991]). The male-derived discovered the pheromone was located in the aggregation pheromones of sap beetles have abdomen, it was divided into two parts, the end been recently reported for Carpophilusfreemani of the elytra serving as the line of demarcation. The gut, fat body (found in the anterior portion This article presents the results of research only. Mention of only), reproductive tract including testes, semi a proprietary product does not constitute an endorsement or a recommendation for its use by USDA. nal vesicles, and aedeagus (found in the poste 1 Mycotoxin Research Unit. rior portion only), carcass, and disk-shaped struc 2 Bioactive Constituents Research Unit. tures = 100 /-Lm in diameter that were directly July 1993 DOWD & BARTELT: SAP BEETLE PHEROMONE GLANDS 465 Table 1. Location of pheromone in tissues of male C. freemani adults Tissue Nanograms/insect Body sections Head-prothorax 0.0 ± 0.0 Meso-metathorax 0.0 ± 0.0 Abdomen 4.4 ± 0.8 Anterior abdomen Intact 2.7 ± 0.6 Gut 0.0 ± 0.0 Fat body 0.0 ± 0.0 Carcass 0.2 ± 0.2 "Disks" 2.1 ± 0.9 Posterior abdomen Intact 5.2 ± 1.0 Gut 0.0 ± 0.0 Reproductive 0.0 ± 0.0 Carcass 0.1 ± 0.1 "Disks" 1.3 ± 0.4 Values are for at least four replicates of combined tissues from five individual insects each. Fig. 1. Pheromone-containing disks hom C. free mani. Bar, 100 J..Lm. A 200-p,1 aliquot was reduced in volume to =2 p,l under a stream of nitrogen and injected into the gas chromatograph as described previously by attached to the trachea (Fig. 1) were separated Bartelt et al. (1990b). The major pheromone com and removed, and the empty carcass was also ponent (2E, 4E, 6E)-5-ethyl-3-methyl-2, 4, 6 saved for analysis. When organs were intermin nonatriene) (Bartelt et al. 1990b) was quantitated gled, care was taken to ensure that "pure" tissue by integrator peak area relative to the internal of each type was isolated, even when not all of a standard. particular organ could be recovered. A 50-pJ Exteriors and frass of C. freemani were also Wiretrol (Drummond Scientific, Broomall, PA) examined for pheromone. Twenty male beetles was particularly useful in transferring dissected were placed in a clean glass vial and allowed to internal organs. Tissues were transferred di defecate for 4 h. The beetles were removed, and rectly to slides for histological examination. For the vial with frass was extracted with 200 p,l of pheromone extraction, tissues were transferred hexane. Beetles were then rinsed as a group for to 5-ml vials prerinsed with hexane. The body 1 min with 200 p,l ofhexane, transferred to 200 p,l sections or tissues from five individuals were of fresh hexane, and extracted for 3 d in the pooled for pheromone analysis. At least four such freezer. Females also were used for comparison groups were analyzed for each step ofdissection. in the 3-d extraction analyses. One p,l of each Each sex was examined separately. Only well rinse was used in initial gas chromatographic fed insects, as indicated by fully expanded abdo analyses (0.5% of total extract). Ifno pheromone mens, were used. was seen at this level, the extracts were further Histology. Unstained mounts were prepared concentrated and analyzed such that =50% ofthe by air-drying the tissues, then fixing for 1 min total extract was evaluated at one time. with methanol. Some tissues were also stained with hematoxylin for 2 min immediately follow Results and Discussion ing fixation with methanol. Slides were then pre served with Permount. The pheromone appeared to be localized pri Extraction and Quantitation of Pheromone. marily in the posterior abdomen (Table 1). Be Hexane (500 p,l) was added to each dissection cause this region is smaller, there is probably sample (which contained up to 100 p,l of water). more pheromone there on a per-weight basis An internal standard ([3E, 5E, 7E]-5-ethyl-7 than in the anterior portion. The pheromone was methyl-3,5,7-undecatriene, 71 ng in 10 p,l hex located mostly in the disk-shaped structures as ane) was added to each sample. Samples were sociated with the trachea (Table 1). Pheromone sonicated for = 15 s, then MgS04 (=100 mg) was detected in the carcass perhaps came from some added to absorb the water. After several hours, of the trachea and attached disks not removed the salt crystals were broken up and the samples during dissection. The relatively low recovery of were resonicated. The clear organic layers (top) pheromone from the disks compared with the were transferred to clean vials, and =50 mg of corresponding whole portions ofthe abdomen is hexane-washed silica gel was added to each sam probably because of loss of disk tissue during ple to remove the nonhydrocarbon components. dissection and tissue separation. 466 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 86, no. 4 Fig. 2. Internal view of posterior, ventral portion of C. freemani abdomen. Digestive and reproduc tive systems have been removed, showing the extensive numbers of disks that were packed around them. Bar, 500 J-Lm. Fig. 3. Fat body (FB) and pheromone containing disks (D) from C. freemani stained with hematoxylin. N, nucleus. All tis sue around the disks is fat body. Bar, 100 J-Lm. July 1993 DOWD & BARTELT: SAP BEETLE PHEROMONE GLANDS 467 Fig. 4. Disk show ing attachment to trachea. The arrow points to the trachea. Bar, 20 f-L. Fig. 5. Clusters of disks attached to tra chea, and their connec tion with the main tra cheal trunk. Bar, 500 f-LID. 468 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 86, no. 4 Fig. 6. Clusters of disks attached to tra chea, and attachment of tracheal trunk to spira cle. The arrow indicates an air bubble issuing from the spiracle. Bar, 500/-Lm. The disk-shaped structures appeared to occur Acknowledgments only in the abdomen of males (the only sex that We thank J. M. Kingsolver and James Pakaluk (Sys produces the pheromone) and appeared to be tematic Entomology Laboratory, USDA-ARS) for spe packed around the rest of the internal organs of cies identifications, and B. Thomas and C. M. Weber the abdominal body cavity (Fig. 2). The other (USDA-ARS, ational Center for Agricultural Utiliza species of sap beetles had similar structures, but tion Research) for technical assistance. only in males. No pheromone was extracted from the feces or from the superficial rinse ofthe males. However, References Cited the extensive extraction of whole insects did Bartelt, R.