Subflexa

Members of the Heliothis genus of noctuid moths have been the subjects of considerable study in our laboratory over the past 10 years not only because of their economic importance, but also because of the ability to hybridize two of the species, H. subflexa and H. virescens. Since members of this genus use variations of the same types of compounds for pheromone communication a commonality in the biosynthetic capability of these insects seems indicated (Table I). 

Figure 1. Percentage of (Z)-11-tetradecen-l-ol acetate (500 ng) converted to alcohol by the action of acetate esterase (solid bars) and subsequently to aldehyde (checked bars) via the oxidase in the intact pheromone glands of H. subflexa. H. virescens and H. zea (n - 10, each species).

However, the sixteen carbon compounds are present in a ratio that closely approximates that of H. virescens. Thus, it appears that females of H. zea do not have the enzyme system required for partial B-oxidation of the sixteen carbon precursors. Females of both H. virescens and H. subflexa are capable of performing this chain shortening step. However, substantially less of the 16-carbon compounds are chain shortened to 14-carbon molecules in the glands of H. subflexa than H. virescens (see 5, 35). Pheromone blend... 

Figure 4. Effect on oxidase activity of soaking cell-free cuticle of H. subflexa. H. virescens. and H. zea in organic solvents for 10 min prior to incubation in (Z)-11-tetradecen-l-ol (500 ng/2/zl hexane for 30 min). All values are relative to that found when using hexane (100%) (n - 8 replications of 5 female equivalents for each species).

Recent research has shown that the pheromone mediated behavior of lepidopterous insects is very complex. The chemical components of the pheromones are usually simple molecules, but complex mixtures involving permutations of geometry, functionality, and chain length are often required to elicit the complicated behavioral repertoire that eventually culminates in mating. To elucidate the chemical and behavioral aspects of this communications system, we have used a combination of methods including collection of the volatiles emitted by the female, analysis by high resolution capillary gas chromatography (GC), and the sequential and temporal analysis of the male s behavioral response to the pheromone blend and components thereof. New liquid phases and state of the art techniques have been developed for capillary GC to separate all the components of a pheromone blend. With these methods the chemical communication systems of Heliothis virescens (F.) and H. subflexa (Gn.) have been analyzed and certain aspects have been elucidated. 

Thus far our investigations have been focused on the chromatographic analysis of pheromones produced and emitted by females and analysis of male behavior evoked by these pheromones. The methods developed to conduct these investigations, using H. virescens and H. subflexa as models, are presented here. These methods are directly applicable to similar investigations of other species. 

Having determined that the high polarity cyano phase and the liquid crystal phase provide the best separation of the compounds likely to be found as components of the pheromone blend of H. virescens and H. subflexa, we analyzed a complex mixture of positional and geometrical isomers of 16 carbon aldehydes, alcohols, and acetates on these two columns (Figure 7). As noted earlier, the elution order of Z- and JE-isomers is opposite on the two phases. Aldehydes elute first on both phases. The alcohols are retained more than the acetates on the high polarity cyano phase, but the elution order of alcohols and acetates is reversed on the liquid crystal phase. While neither phase separated all of the synthetic mixture, the combination of separations obtained on both columns enabled us to pursue the... 

The analyses of the H. subflexa ovipositor extracts (171 revealed eight major components coinciding in retention times on SP-2340 and p-chloro cholesteryl cinnamate capillary columns (Figure 9) with 16 A1, Z9-16 A1, Z11-16 A1, (Z -7-hexadecen-l-ol acetate, (Z7-16 Ac), (Z)-9-hexadecen-l-ol acetate (Z9-16 Ac), (Z)-ll-hexadecen-l-ol acetate, (Zll-16 Ac), Z9-16 OH, and Z11-16 0H. Several other peaks were also variably present but, when present, each composed less than 1% of the total mixture. Mass spectral data confirmed the identity of the major components. 

The differences in the glandular pheromone constituents between H. virescens and H. subflexa are distinct. H. subflexa contains acetates of 16-carbon alcohols not found in H. virescens and does not contain the 14-carbon aldehydes which appear to be unique to H. virescens among Heliothis species studied thus far. It will be interesting to see what blends the hybrids of these species produce and relate this to data from investigations of the genetics of this hybridization. 

The behavioral responses of males of H. virescens and H. subflexa to semiochemicals have been of considerable interest to us for reasons already mentioned. Two assay systems were employed to analyze both precourtship and close-range courtship behaviors (18). The first, used in the analysis of male activation, orientation, and initial analysis of courtship interactions, consisted of a 1.5 x 0.5 x 0.5-m plexiglass wind tunnel through which air was pulled at a constant rate. In our initial... 

Figure 9. Analysis of H. subflexa ovipositor extract (1.5 female equivalent) on 66 m SP-2340 (A) and 30 m cholesteryl cinnamate capillary columns (B). S indicates the saturated compound.

As indicated earlier H. subflexa has recently become the subject of numerous pest control studies due to the potential for population control of H. virescens by a sterile hybrid release program. However, these two species maintain reproductive isolation despite broadly overlapping ranges and intersecting reproductive periods. Hence, barriers to interspecific gene flow are most likely due to differences in their respective sex pheromone communication systems and, as indicated earlier, there are considerable differences in the pheromone gland contents of each. 

The synthetic blend identical in composition to the H. subflexa gland extract (.see earlier) formulated in either polyethylene vials (30 mg of mixture) or on filter paper (75 ng) was as effective in capturing males as either females or gland extracts in cone traps (19). Additionally, only H. subflexa males were captured, while traps in the same fields baited with H. virescens or H. zea females or synthetic blends (10) also captured only conspecific males. 

Although many aspects of both the male and female reproductive behaviors of H. subflexa are similar to those of H. virescens, small differences do exist (11,18). Flight tunnel studies of the semiochemically induced behavioral interactions between H. subflexa and H. virescens indicated that males of the two species respond quite differently to the naturally released sex pheromone of the other species. 

Comparison of sticky trap captures of H. subflexa males using different blends.a b c e... 

The blend of pheromone components identified from H. subflexa female gland extracts has two components in common with H. virescens, Z9-16 A1 and Z11-16 A1 and three not found in H. virescens, (Z)-7-hexadecen-l-ol acetate (Z7-16 Ac), ( >-9-hexa-decen-l-ol acetate (Z9-16 Ac) and (Z)-ll-hexadecen-l-ol acetate (Zll-16 Ac). Field studies (Table III) indicate that at the very least, the two aldehydes plus Zll-16 Ac are of importance to the capture of male H. subflexa. Hence, the absence of these three acetate components from the H. virescens pheromone ( 10), and different ratios of Z9-16 A1 to Z11-16 A1 probably account for the semiochemical isolation between H. subflexa males and H. virescens females. However, the release of pheromone components by female H. virescens such as 14 A1 and Z9-14 A1 may be disorienting to male H. subflexa and cannot be discarded as a possible mechanism for reproductive isolation. 

VICKERS, N.J., Defining a synthetic pheromone blend attractive to male Heliothis subflexa under wind tunnel conditions. J. Chem. EcoL, 2002, 28, 1255-1267. 

Teal, RE.A., Heath, R.R., Tumlinson, J.H. McLaughlin, J.R. 1981. Identification of a sex pheromone of Heliothis subflexa (GN.)(Lepidoptera Noctuidae) and field trapping studies using different blends of components. J. Chem. Ecol 7 1011—1022. 

LaChance LE (1984) Hybrid sterility eupyrene sperm production and abnormalities in the backcross generations of interspecific hybrids between Heliothis subflexa and H, virescens (Lepidoptera Noctuidae). Ann Entomol Soc Am 77 93-101... 

Our studies on H. zea. H. virescens and H. subflexa have shown that the amount of pheromone present in the gland, as indicated by the titer of the major component, (Z)-11-hexadecenal, increases in a linear fashion for at least the first 90 min after injection of PBAN (Fig. 2). Furthermore, as indicated in Figure 3, the ratio of components present in gland extracts obtained at 15 min. intervals after injection of PBAN remains constant and is not different from the naturally produced ratio. Other studies... 

Figure 2. Amounts of Z11-16 AL, the pheromone component present in greatest amount, present in extracts of pheromone glands of virgin females of Heliothis zea. H. virescens and H. subflexa obtained at different times after injection of 0.5 pmole of PBAN in 10 of water during the photophase (n-10 each data point), Extracts were analyzed by capillary GC using Supelcowax 10 and SPBl columns (30m X 0.25mm id).

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