Volatile Chemicals of Adults and Nymphs of the Eucalyptus Pest, Thaumastocoris peregrinus (Heteroptera: Thaumastocoridae)

1 Laboratório de Semioquı́micos, Departamento de Quı́mica, Universidade Federal do Paraná (UFPR), Centro Politécnico, 81531-990, Curitiba, PR, Brazil 2 Laboratório de Entomologia Florestal, Embrapa Florestas, Estrada da Ribeira, Guaraituba, 83411 000 Colombo, PR, Brazil 3 Invasive Insect and Behavior Laboratory, ARS Biocontrol, USDA, Agricultural Research Center-West, B-007, Room 313, Beltsville, MD 20705, USA


Introduction
Thaumastocoris peregrinus Carpintero and Dellapé (Heteroptera: Thaumastocoridae) is an introduced pest of nonnative Eucalyptus plantations in various countries in Southern Hemisphere (e.g., South Africa, Argentina, Uruguay, and Brazil) [1][2][3]. In 2005, it was first found in Buenos Aires, Argentina, on Eucalyptus viminalis, E. tereticornis, and E. camaldulensis [1]. In Brazil, T. peregrinus was first found in 2008, on a hybrid clone of E. grandis x E. urophylla in São Francisco de Assis, Rio Grande do Sul, and on E. camaldulensis trees in Jaguariaúna, São Paulo [4]. Initial studies on life history of T. peregrinus were done in Australia [5]; however, no investigation was performed on semiochemicals from these insects.
Heteropteran nymphs and adults characteristically produce allomones for defense; typically, the defensive secretions of nymphs are produced in dorsal abdominal glands (DAGs) [6]. The contents of DAGs are shed along with the exuviae each time the nymph molts, and extraction of exuviae is a convenient method to obtain the DAG secretion [7]. Adult heteropterans characteristically possess metathoracic scent glands from which they release irritating secretions [6]. However, examination of T. peregrinus adults by one of us (JRA) revealed that the metathoracic glands are vestigial (unpublished data). On the other hand, adults and nymphs of these unusual bugs possess a rectal organ, similar to that described for plant bugs (Miridae) [8] that is everted when the insects are disturbed. The Thaumastocoris rectal organ has a glandular appearance and instantly sticks the insects to the substrate when the insects are disturbed and can as quickly be released (JRA, personal observation) ( Figure 1). Pheromones are known for members of several heteropteran families [9], but the semiochemicals of T. peregrinus and other thaumastocorids are completely unknown. Therefore, the volatile chemical compounds present in the exuviae for the five nymphal instars, and both adult sexes were identified and quantified. or females of the same emergence date were grouped in Petri dishes (5 cm of diameter) containing gel and a leaf disc until the extraction. Fifth instar nymphs were grouped in cages provisioned as above to obtain mated males and females for extraction. Couples were formed within 2 days of emergence, and extractions of adults were performed only after eggs were present, which confirmed the mated status of adults.

Extraction of T. peregrinus Exuviae (1st-5th Instar).
Exuviae were extracted with 180 µL of hexane for 24 hours. Each extraction was made with the exuviae available in that day, with a minimum of 12 and maximum of 24 exuviae. At least three repetitions were made for each instar, consisting of at least 45 exuviae in total. After extraction, tridecane (ca. 10 ppm) was added to each sample as an internal standard (IS); the final concentration of the IS was calculated for each extract. Extracts were concentrated and analyzed using a gas chromatograph (GC-2010-Shimadzu) and a gas chromatograph coupled with a mass spectrometer (GC-MS-QP 2010 Plus-Shimadzu). The detected compounds were quantified based on the area of the IS. The GC was equipped with a RTX-5 column (30 m × 0.25 mm i.d. and 0.25 mm film thickness; Restek, Bellefonte, PA, USA). One µL of extract was injected into the GC using the splitless mode with injector temperature at 250 • C. The column oven temperature was maintained at 50 • C for 1 min, then raised to 250 • C at a rate of 7 • C/min, and maintained in 250 • C for 10 min. Helium was used as carrier gas at a column head pressure of 170 kPa. The same parameters were used for all analyses.

Extraction of T. peregrinus Adults.
Extractions were made with mated and virgin males and females of different ages (3-9, 10-21, 22-34 days old), according to availability of insects. Quantified extracts were compared for virgin males and females (3-9 days), virgin and mated males (10-21 and 21-34 days old), and mated males and females (10-20 and 21-34 days old). There were at least two repetitions per treatment, with a minimum of 15 insects extracted in total. In both experiments, insects were separated by sex in glass Erlenmeyer flasks. The flasks with insects were put in a freezer for one hour so that they died with the rectal organ exposed while "glued" to the glass. Thus, the adults were extracted as complete adults with their rectal organ exposed. The extraction was made between 11:00 AM and 16:00 PM using 150 µL of double distilled HPLC-grade hexane for 10 minutes, then 150 µL of a tridecane solution was added as an IS. The samples were concentrated before injection into a GC-2010, a GC-MS-QP 2010 Plus, and a GC-Fourier transform infrared spectroscopy (GC-FTIR) (GC-2010 coupled to a DiscovIR-GC-Shimadzu). In the infrared analysis, the GC was operated in the splitless mode, and equipped with a DB-5 (0.25 µm, 0.25 m × 30 m) (J&W Scientific, Folsom, CA, EUA) capillary column with helium carrier gas. The column oven was maintained at 50 • C for 1 min and then increased to 250 • C at 7 • C/min to 250 • C. A liquid-nitrogen-cooled photoconductive mercury-cadmium-telluride (MCT) detector was used with FT-IR resolution of 8 cm −1 . As for nymphal extracts, the final concentration of the IS was calculated Psyche 3 for each extract, and extracted compounds were quantified based on the area of the IS.

Statistical
Analysis. Statistical analyses were performed using R version 2.13 [10]. To analyze the six main compounds found in the exuviae, the Kruskal-Wallis rank sum test was used followed by a nonparametric multiple comparisons test using the package "pgirmess" in case of significance. Data for the comparison of extracts of virgin and mated adults were tested for normality by the Liliefors and Shapiro-Wilk test. After the normality of the data was confirmed (P > 0.05), we performed a GLM (generalized linear model) procedure following Gaussian distribution, considering that mating status was an independent variable. For all analyses, P values >0.05 were considered not significant.  Table 1). Some of the compounds present in the exuviae of T. peregrinus have been found in other heteropteran species, either as repellents or attractants. For example, benzaldehyde from copulating pairs of Triatoma infestans (Klug, 1834) (Reduviidae) was highly attractive to conspecific females at low doses (0.05-0.1 µg) [11]. In the bed bug, Cimex lectularius (Linnaeus, 1758) (Cimicidae), decanal, (E)-2-octenal, and benzaldehyde are reportedly essential components of the airborne aggregation pheromone [12]. The hexanoic acid is produced in metathoracic scent gland secretions of many bugs (e.g., Scutelleridae: Eurygaster maura (Linnaeus, 1758)), along with (E)-2-hexanal, (E)-2-hexenyl acetate, n-tridecane, octadecanoic acid, and n-dodecane [13]. The alarm pheromone of Leptoglossus zonatus (Dallas, 1852) (Coreidae) adults includes hexyl acetate, hexanol, hexanal, and hexanoic acid [14]. Also, in Japan, a mixture of (E)-2-octenyl acetate and 1-octanol attracted the rice bug, Leptocorisa chinensis Dallas, 1852 (Alydidae) [15]. While the compounds identified here for T. peregrinus nymphs are commonly known exocrine compounds of Heteroptera, the combination of these compounds in these thaumastocorid nymphs is unique compared to the secretions of other heteropteran nymphs [6]. Other heteropterans produce some of these compounds (e.g., Cimex lectularius) but not Psyche all of them combined. The significance of this uniqueness is unknown. In contrast, the molecular ion in MS of compound A was not obvious; however, the base peak at m/z 68 and a fragment at m/z 71 suggested A was an ester similar to B. Although the GC-FTIR spectra of A showed the same characteristic bands for esters that were detected for B, the presence of a band at 3080 cm −1 due to asymmetric stretch of a terminal double bond, demonstrating that A was an unsaturated ester with a terminal double bond. To positively identify the natural products A and B, the twenty-one above-mentioned esters were synthesized. Thus, the major compound B was identified as 3-methylbut-2-en-1-yl butyrate by coinjection of this standard with the natural extract on the three GC columns (RTX-5, RTX-WAX, and HP-1). Identification was based on coelution and MS. Additionally, the minor compound A was identified as 3methylbut-3-en-1-yl butyrate by coinjection of this standard with the natural extract on the different GC columns. Females and males produced the same esters, but their quantities varied by sex and age, particularly for the major compound, 3-methylbut-2-en-1-yl butyrate (Figure 4). Although the concentration of the esters in males increased with age (Table 2), reaching a maximum of approximately 1 µg per insect in 22-day-old mated males, this age difference could not be detected statistically. Only the amount of the major compound (B) of mated males was statistically different from that for mated females (F 1,3 = 10.3, P value =0.048) (GLM). Ester concentrations of virgin males and females were not statistically different (GLM) for either the minor (A) (F 1,4 = 0.6, P value =0.47) or major (B) (F 1,4 = 3.2, P value =0.14) compounds. Likewise, ester concentrations of mated and virgin males (A: F 1,3 = 2.4, P value =0.21; B: F 1,3 = 5.7, P value =0.09), and of mated males and females (minor F 1,3 = 4.5, P value =0.12) were not statistically different (GLM) ( Table 2). The adults of 10-21 days old did not have enough repetitions to be compared. Thus, they were not considered for the concentration analysis.
The biological function(s) of 3-methylbut-2-en-1-yl and 3-methylbut-3-en-1-yl butyrates in T. peregrinus remain to be elucidated. An aggregation function was attributed to the major compound through olfactometer experiments, in which males attracted only males (Gonzalez et al. 2012 this issue); however, we did field tests using delta traps with different concentrations of the major compound, and they all failed to attract insects in the field and in a greenhouse with a T. peregrinus population. Allomones and pheromones known for other heteropterans, such as those described above, undoubtedly originate from the dorsal abdominal glands of nymphs or the metathoracic scent glands that are characteristic of most true bug adults. In T. peregrinus, however, the metathoracic scent glands are vestigial. The butyrates from T. peregrinus appear to be associated with extrusion of the rectal organ ( Figure 1) that has   (3) 0.1 ± 0.1 0 .5 ± 0.3 heretofore only been described within the Heteroptera for plant bugs (Miridae) [8]. Unequivocal verification that the rectal organ tissue is the source of these esters awaits further experimentation. Mated T. peregrinus males produce greater quantities of both esters, especially ester B, compared with virgin males and younger mated males. Moreover, these esters are produced by females, suggesting that these compounds are not involved in aggregating the sexes for mating. Speculating the differences of concentration, these esters could be indicators of sex and age recognition by conspecifics.

Conclusion
Benzaldehyde, octanol, (E)-2-octenol, octanoic acid, decanal, and hexanoic acid were present in the exuviae of T. peregrinus nymphs. Volatiles from adult males and females included 3-methylbut-3-en-1-yl butyrate and 3-methylbut-2-en-1-yl butyrate. Compounds identical or similar to those found in T. peregrinus exuviae and esters identified in the adults were found in other heteropterans with various functions. The possible pheromonal roles of these volatile blends are being studied.