Alarm Pheromone System of Stink Bug Piezodorus guildinii ( Heteroptera : Pentatomidae )

Os compostos utilizados pelo percevejo da soja Piezodorus guildinii como feromônio de alarme foram caracterizados através da análise da composição química das secreções da glândula metatorácica dos insetos adultos. Além dos hidrocarbonetos característicos, ( E)-2-hexenal e ( E)-4-oxo-2-hexenal foram detectados como constituintes majoritár ios. Tais compostos já foram previamente descritos como feromônio de alarme em outras espécies de pentatomídeos.


Introduction
Piezodorus guildinii is a member of the stinkbug complex in soybeans that is economically important pests in Brazil 1,2 .In our investigation on the chemical ecology related to the Pentatomidae family, we have started to elucidate the chemical communication system of P. guildinii 3 and we have reported previously that methyl 2,6,10trimethyldodecanoate and methyl 2,6,10-trimethyltridecanoate are male-produced sex pheromones of this species 3,4 .Field experiments are now in progress to determine the potential for utilizing these compounds in an integrated pest management program.
An important characteristic of adult heteropterans is the fact that when molested, they usually retaliate by discharging volatiles secretions (defensive chemicals / alarm pheromones) from their ventral methatoracic glands (MTG) 5 .This intriguing mechanism has led to the isolation and identification of several defensive compounds released by a number of heteropteran species [5][6][7][8] .
The P. guildinii metathoracic scent gland complex is similar of that reported by Nezara viridula by Gilby and Waterhouse 9 .The adult scent gland complex of both males and females consists of a median ventral metathoracic scent reservoir, which is orange-yellow in color, and paired colorless lateral glands sometimes called accessory glands.The lateral glands discharge through ducts into the reservoir, which also receives secretions from the gland cells which form its epithelium.The glands open to the exterior on the ventral surface 9 .
The main purpose of the present work was to characterize the chemical composition of the metathoracic glands of P. guildinii and to identify the compounds that elicit dispersive behavior in other members of the species.

Extraction
An adult P. guildinii was pinned in a Petri dish with the dorsal side up.The dissection process consisted of cutting the dorsal abdominal edges of the insect cuticle up to the metathoracic region and under the scutelum.The dorsal abdominal cuticule was pulled back and the viscera were removed.The scent gland complex, located at the ventral abdominal metathoracic region, could be reached and removed with the aid of a small surgical scissors.The gland reservoir was removed and immersed in ~100 µL analytical grade hexane distilled from CaH 2 and stored at -20 o C. One sample of dorsal abdominal gland (DAG) secretion of E. heros was prepared by immersing the second-instar nymphs in hexane (~100 µL) for 10 minutes and then transferring the extract directly to a clean conical bottom vial.

Chemical analyses
Samples were analyzed (~ 2 µL of the extract) by gas chromatography (GC) on a HP-5890 Series II (FID detector; 270 o C) in splitless mode (250 o C; 1.5 min).The HP-1 bonded methyl silicone column (25m x 0.25mm x 0.2µm) was operated at 50 o C for 2 min to 220 o C at 7 o C/min, using helium as carrier gas (1.0 mL/min).Electron impact mass spectra were obtained at 70 eV using a Shimadzu QP-5000 GC-MS equipped with a DB-5 column (30m x 0.25 mm x 0.25 µm J & W Scientific) in splitless mode (250 o C; 1.5 min) and with an interface temperature of 200 o C. The temperature program and linear velocity of the carrier gas (He) was the same as described above.The IR refer to films and were measured on a Bomem M-102 spectrometer.The 1 H-RMN spectra were recorded at 200 MHz on a Bruker AC 200 spectrometer (CDCl 3 ).The 13 C-NMR spectra were recorded at 50 MHz on a Bruker AC 200.Column chromatography was carried out on columns packed with Merck Kieselgel 60, Art.-Nr 7734.(E)-2-hexenal, (E)-2-octenal, n-undecane, n-dodecane, n-tridec-1-ene, n-tridecane standards, and hexane were obtained from Aldrich Chemical Co.

Results and Discussion
The chromatograms obtained by analyses of the crude hexane extract of the metathoracic glands secretions of P. guildinii showed seven peaks whose structures could be identified, three of those in higher amount than the other ones (Figure 1).
By comparison of these natural products with those on the computerized mass spectra data base, peaks 1, 3-7 were tentatively identified as (E)-2-hexenal, (E)-2-octenal, nundecane, n-dodecane, n-tridec-1-ene and n-tridecane, respectively.The identifications were confirmed by GC coinjection with commercial standards.EI-MS of compound 2 gave a base peak at m/z 83 along with the molecular ion M +. 112 (11%).This structure was tentatively identified by comparison with spectra from natural products previously identified as pheromones and synthetic standards, as described below.
Defensive secretions from first and second-instar nymphs of stink bugs belonging to the same family as P. guildinii were described by Borges and Aldrich 6 .In immature heteropterans (nymphs), defensive secretions are produced in the dorsal abdominal glands (DAGs) instead of the metathoracic glands and the gland contents usually differ within a species 5,6 .DAG extracts of second-instar nymphs of Euschistus heros contained four compounds, and one of these had a MS consistent with the mass spectrum data of component 2 6 .GC-MS co-injection of the MTG extract of P. guildinii with a DAG extract of E. heros showed that compound 2 had the same retention time and mass spectrum of the compound identified as (E)-4-oxo-2-hexenal [MS m/z (%): 112 (11), M + ; 97 (4), CHOCHCHCOCH 2 + ; 83 (100), CHOCHCHCO + ; 55 (78), CHOCHCH + ].
To confirm this identification, we synthesized (E)-4oxo-2-hexenal 2 in five steps, following the synthetic route shown in Scheme 1.The monoprotected alcohol 9 was obtained after protection of the diol 8 with DHP in 60 % yield 10 .Oxidation of 9 with PCC in methylene chloride afforded the aldehyde 10 in 80% yield 11 .Coupling of this compound with ethylmagnesium bromide 12 yielded the alcohol 11 (65 %) which was deprotected to give diol 12 (88%) by treatment with pTSA in methanol 10 .Oxidation of this compound with PCC supported on Al 2 O 3 in CH 2 Cl 2 afforded the desired product in 77% yield, which was found by GC-MS co-injection to be indistinguishable from the natural product.
Pinder and Staddon 13 described the first synthesis of 2 by employing an acid catalyzed hydrolysis of 2-ethyl-2,5-dimethoxy-2,5-dihydrofuran, which gives a mixture  This synthesis offers a useful alternative to those described in the literature.Therefore, the chemical composition of the metathoracic scent gland secretions of P. guildinii was identified as a mixture of (E)-2-hexenal, (E)-4-oxo-2-hexenal, (E)-2-octenal, nundecane, n-dodecane, n-tridec-1-ene and n-tridecane (Figure 1).This composition is similar in both sexes indicating that these compounds do not play any role, as sexual attractant, together with the ones previously reported 3 .Compounds 1, 4, 5, and 7 are known toxins, irritants, or repellents 15,16 , and are released to the stink bugs in response to disturbance.These facts strongly suggest that they really acts as chemical defenses of the species.Further studies in field will clarify their role in the chemical ecology of P. guildinii.
It is not know how the various secretions components interact.However, it was reported for pentatomid that (E)-2-hexenal and n-tridecane were more effective as repellents to insects when combined than when individually tested 17 .Furthermore, other n-alkanes when combined with (E)-2-hexenal were not as effective deterrents towards other insects as n-tridecane.Hence, n-tridecane appears to be the optimal n-alkane to work synergistically with (E)-2-hexenal to repel insects 16 .The other secretions components of the multicomponent blend of P. guildinii may likewise function in an additive way.
We recently reported that the crude aeration extract of P. guildinii has attracted the egg parasitoids Trissolcus teretis, Telenomus podisi, and Trissolcus urichi to the baited traps 4 .The same phenomenon was observed when the synthetic sex attractant pheromone was employed 3,4 .Parasitoids have been reported to respond to the host's body odor; sex, and aggregation pheromones; salivary constituents; excretory products; webbing; honeydew; body scales; and eggs 7,23 .It has been reported that the egg parasitoid Trissolcus basalis utilizes a defensive substance produced by its host bug as a long-range attractant kairomone 24 .
Therefore, these simple structures found on the metathoracic glands of adults P. guildinii could be used as kairomone to synchronize the parasitoid population at the beginning of the host flight season and pave the way for the development of invaluable tools in integrated pest management programs for this important soybean pest.

Figure 1 .
Figure 1.GC profile of a crude hexane extract sample from metathoracic scent gland of adult males P. guildinii.The peak labeled with an arrow is due to solvent impurity.Peak numbers correspond to compounds identified.
of the cis (major) and trans isomers.Later, Ward and Van Dorp 14 synthesized the same compound by reaction of propanal with the Grignard derivative of 1,1-diethoxy-2propyne, followed by the reduction of the acetylenic bond with Na/NH 3 (l), oxidation with active manganese dioxide and acid hydrolysis.We now developed a synthesis of 2 in good overall yield by an operationally simple strategy.