Ring T. Cardé, Agenor Mafra-Neto, Robert T. Staten1 and L.P.S. Kuenen2
Department of Entomology, University of California, Riverside, California 92521, USA
Abstract - Field wind tunnels set out over cotton were used to gauge the efficacy and mechanisms of mating disruption in the pink bollworm, Pectinophora gossypiella. Shin-Etsu rope formulation (PBW-Rope) containing synthetic pheromone was hand applied at the standard, commercial-use density of 1 000 dispensers/ha. Laboratory-reared males were marked and released at dusk at the downwind end of the wind tunnels. Efficacy of disruption was estimated from hourly catches throughout the night in both pheromone-baited traps in treatment and check (disruptant-free) tunnels. Exposure of males to pheromone prior to release allowed the contribution of sensory adaptation/habituation to disruption of attraction to be estimated. Male flight tracks as well as other behaviors near Shin-Etsu ropes and pheromone-baited rubber septa were videotaped. Analyses of data revealed that several mechanisms contributed to disruption of attraction: sensory adaptation/habituation, competition, camouflage, advancement in time of the males' window of pheromone response, and arrestment of upwind anemotaxis.
Key words - pheromone, mating disruption, habituation, sensory adaptation, orientation, pink bollworm, Pectinophora gossypiella
In most cotton-growing regions of the world the pink bollworm (Pectinophora gossypiella) is a key pest. Its larval habitus of internal feeding makes foliar sprays of insecticide generally ineffective and consequently in many management programs numerous aerial sprays of insecticide are directed against the adult. Such applications frequently occur as often as weekly, from the setting of the first cotton bolls until close to harvest. Not unexpectedly, these sprays often trigger outbreaks of secondary pests which otherwise would have been regulated by natural enemies. The availability since 1978 of synthetic pheromone formulated to disrupt mating of P. gossypiella has fostered alternative management strategies (Baker et al. 1990). These include the application at the beginning of flowering (“pin-square stage”) of dispensers releasing pheromone over the remainder of the growing season. The Shin-Etsu plastic “rope” dispenser (PBW-Rope®), for example, is typically deployed at the pin-square stage at the base of the cotton plant at a rate of 1 000/ha with a total application of 75 g of pheromone per hectare. Alternatively, catch of males in sentinel, pheromone-baited traps can be used to decide whether a population is dense enough to warrant an application of pheromone. If so, formulations with a longevity of generally 7 to 10 days are applied aerially. The Scentry hollow fiber (NoMate®), is an example of a low rate, short-life formulation: aerial application of 10 000 fibers/ha deposits a total of 2.8 g of pheromone per hectare, mainly on the upper portion of the cotton canopy. As measured by an electroantennogram-based system (Färbert et al. 1997), these two formulations generate different airborne concentrations of pheromone: the Shin-Etsu ropes emit about 1000-fold more pheromone near and within the canopy than the Scentry fibers.
These disparate deployment strategies entail considerable differences in application costs: season-long dispensers have a high, one-time expenditure. Aerially applied formulations cost less per application than hand-applied, long-life dispensers, but the former approach requires season-long monitoring with traps, and, typically, multiple applications.
A number of mechanisms have been proposed as contributing to mating disruption (Bartell 1982; Cardé 1990; Minks & Cardé 1988; Cardé & Minks 1995). When the disruptant is a copy of the natural blend, three mechanisms are believed to predominate. (1) Either sensory adaptation of the peripheral receptors or habituation of response at the central nervous system level either eliminates pheromone response or impairs mate finding performance. (2) In competition males spend time flying upwind along plumes from point sources of synthetic pheromone. Therefore, males have less time available to locate females. The reduction achieved in the proportion of females mating depends on the ratio of artificial pheromone sources to calling females and the comparative attractiveness of synthetic sources and females. (3) Formulated pheromone can camouflage pheromone plumes from calling females; the greater distance a male is from the female the more likely it is that the female's plume will be rendered imperceptible amongst the background of synthetic pheromone. The omnipresence of pheromone also may blur the plume's fine-scale structure, thereby lowering a male's probability of locating the pheromone's source (Mafra-Neto & Cardé 1994; Vickers & Baker 1994).
Other mechanisms may contribute to disruption. The presence of formulated pheromone could advance the timing of a male's rhythm of response, so that he initiates upwind search before females call (Cardé et al. 1993). Males may become “arrested” in high concentrations of pheromone (e.g., Baker & Cardé 1979), failing to proceed upwind.
The proposed mechanisms need not be mutually exclusive; rather, their effects may be additive or even synergistic. For example, if the formulation consists of point sources of pheromone attractive to males, competition should be a principal disruptive mechanism. Disruption levels could be enhanced, however, by a precocious response of males to the artificial sources of pheromone before the initiation of female calling. Males that have responded to these artificial plumes may be either less apt or able to navigate a female's plume later in the night when natural calling commences. This example also underscores that the type of formulation (its emission characteristics, distribution, and position in the crop) dictates the mechanisms of disruption, the interactions among mechanisms, and therefore the efficacy of mating disruption. A formulation's efficacy also can be contingent on a pest's population density, which of course varies between sites (Cardé & Minks 1995).
Laboratory bioassays with many moth species (e.g., Traynier 1970; Bartell & Lawrence 1973; Sanders 1996) have provided indirect evidence that each of these mechanisms could contribute to mating disruption in the field, but the extent to which such laboratory observations equate to behavior of free-ranging males in the field remains inferential. To duplicate in bioassays the range of conditions that a native male would encounter in a crop treated with disruptant and to have a record of a male's prior exposure to pheromone is an intractable problem; indeed, we simply do not know enough about the behavior of males in the field to allow us to recreate these parameters in a bioassay. We have attempted to mimic field conditions by employing large, walk-in wind tunnels set out over cotton plants in cotton fields (Cardé et al. 1993). We observed the plume-following behavior of individual moths released either in tunnels set in cotton fields treated with disruptant or in check tunnels placed in fields free of disruptant. A second valuable approach used an electroantennogram-based system to measure the airborne concentrations of pheromone in treated cotton fields over fractions of a second (Färbert et al. 1997). Measurements determined the spatial pattern of airborne pheromone generated by a given formulation as well as a formulation's longevity of emission. A combination of these two approaches has shown promise in establishing how mating disruption works (Cardé et al. 1993).
Our field wind tunnels have a working section that is 6.2 m long, 2.5 m wide and 1.85 m high. Air is pulled through the tunnel at 0.7 to 0.8 m/s by a fan mounted at one end; the air drawn into the tunnel is laminarized by a sheet of Hexel® cells. The tunnel consists of a frame over which a sheet of nearly clear polyethylene is set out at dusk and removed at dawn. The ground serves as the tunnel's floor and the tunnel is positioned lengthwise along two rows of cotton. Tunnels can be moved readily to new sites.
Levels of attraction and its disruption are assessed in several ways. Laboratory- reared males are marked to indicate day of release and treatment with color-coded fluorescent powders. Males are released at ground level at the tunnel's downwind end immediately after sunset. Levels of attraction are gauged by catches in two pheromone-baited sticky traps (Delta), each positioned at the upwind end of one of the two rows of cotton, just below the top of the canopy. Males flying along a plume to one of the two monitoring traps must navigate a 6 m course through and around cotton foliage. Before release males can be pre-exposed to pheromone. Trap catches are tallied hourly from sunset to dawn.
When males are released into a tunnel just after sunset, many are immediately attracted to the traps, well before their normal time of attraction in the field (e.g., Beasley & Adams 1994; Schouest & Miller 1994). Such an alteration in the timing of attraction raises the possibility that the navigation behaviors of released and wild males are not comparable; this is an especial concern if a male's history of exposure prior to release is a dependent variable. Therefore, in one test we used an alternative release strategy designed to mimic a natural pattern of exposure to field conditions. Males are released at dusk into screen cages 2 x 0.75 x 0.5 m high set over two rows of cotton. The screen cage is positioned across the width of the tunnel's downwind end. On the following night at dusk the cage's upwind and downwind sides are removed, and the cage is hoisted on cables to the wind tunnel's ceiling. This procedure allows males during the day before assay to enter fissures in the soil (Flint et al. 1975), where they presumably encounter relatively little pheromone. Males thus are “exposed” for 24 hr in the field to disruptant formulations before bioassay. This method also eliminates handling of males immediately prior to release, a procedure which may alter the timing of response to pheromone.
In several tests we recorded on video the reactions of males to formulations placed in the cotton canopy. We obtained tracks of males flying upwind and records of male reactions near point sources of formulations.
The formulation used in the following tests was the PBW-Rope containing ca. 75 mg of P. gossypiella pheromone. Ropes were tied around the base of cotton plants, approximately 3 m apart in alternate rows of cotton; this pattern is the standard commercial application rate of 1 000 dispensers/ha.
Measurement of disruption using field wind tunnels
Camouflage of a plume
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Our first use of field wind tunnels (Cardé et al. 1993) showed that rope and fiber formulations severely disrupted male P. gossypiella orientation to sticky traps baited with pheromone, compared to attraction to traps in a check tunnel. A rope formulation in a wind tunnel placed in an untreated cotton field only disrupted trap catch along one side of the tunnel, the row of cotton in which the three dispensers were placed. The other row was free of disruptant dispensers, and many males were caught in its monitoring trap. A wind tunnel with the same 3-rope configuration placed in a field treated with rope, however, had low trap catches that were disrupted equally on both sides of the tunnel.
Measurements of airborne concentrations of pheromone by the electroantennogram system (Färbert et al. 1997) showed that in the 3-rope tunnel in an untreated cotton field, pheromone was present mainly on the treated side of the tunnel (some pheromone, of course, is emitted from the monitoring trap on both sides). Equal concentrations of pheromone, however, were measured along both rows of cotton in the tunnel within a rope-treated field (Cardé et al. 1993). The high level of disruption along the untreated cotton row in this tunnel was due, therefore, to pheromone-laden air from the treated field being drawn into the tunnel and along both rows of cotton. We interpret the high level of disruption documented on the untreated side of the tunnel as being due to a camouflage effect, although it is probable that the additional mechanisms described below augment this process.
Attraction to rope formulation
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The PBW-Rope formulation emits pheromone at substantially higher rates than pink bollworm females. Flint et al. (1993) estimated that a rope exposed in the field for 30 to 60 days releases about 370 ng/min, whereas Haynes et al. (1987) measured mean emission from females at 0.16 ng/min. Given this wide disparity and the arrestment of upwind flight noted in other moth species at high pheromone emission rates (e.g., Baker & Cardé 1979), it was not expected that P. gossypiella males would be routinely attracted to ropes. Males flying upwind along pheromone plumes generated by a rope dispenser which had been field aged for 7 days or from a rubber septum baited with 4 mg of pheromone produce generally comparable flight tracks (Figure 1), except that flight along the plumes from 7-day-old ropes is more torturous and slower than flight along plumes from septa. A rope aged in the field for only 24 hr also evokes upwind flight, but males generally do not approach, or land on, fresh rope formulation, and arrestment of upwind anemotaxis often is followed by flight downwind, and either another flight upwind or flight away from the plume.
Analyses of male activity within 0.5 m of a 7-day-old rope shows that a considerable proportion of released males arrive in its vicinity, wing fan for several min, eventually becoming quiescent. We also have found in late season observations in untreated cotton that an individual, week-old rope is quite attractive. Together these observations of flight tracks to ropes and behavior in their vicinity suggest that competition is a mechanism of disruption in rope-treated fields. The quiescence that ensues after attraction also verifies sensory adaptation/habituation as a factor. It will be of interest to learn if the threshold of response of such males has been raised (Cardé 1990; Mafra-Neto & Baker 1996).
Figure 1 Representative flight tracks of P. gossypiella males flying to three different pheromone sources in field wind tunnels. Wind flow was 0.7 to 0.8 m/s from the right to the left. Large dots represent the position of the pheromone sources and small dots represent the position of the moth every 0.07 s. Groups of 20 moths were released downwind, and flight within 2 m of the source was recorded from above using a Sony RSC 1050 rotary-shutter video camera. Ambient light was supplemented by a 25 w dark red tungsten lamp directed downward from the tunnel's ceiling. Moths flying upwind usually landed on either the 7-day-old rope or on the rubber septum; however, males exposed to a 1-day-old rope were arrested in flight and terminated the approach. In general, flight tracks were slower and more tortuous as the dose of pheromone in the dispenser was increased
Effects on the rhythm of response
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When males are released from their holding cups at dusk, many of them initiate flight immediately and proceed upwind along plumes generated either from the rope formulation or the two monitoring traps. At the time of year and temperatures of our tests, male attraction would normally commence from about 24:00 to 02:00 (Beasley & Adams 1994; Schouest & Miller 1994). A precocious rhythm raises two questions. First, is the orientation behavior of males released from cups disrupted in the same way as that of males that have spent the previous 24 hr in the treated field where they potentially were exposed to disruptant? Second, can this wind tunnel method be used to evaluate the possibility of formulated pheromone altering the timing of males present in the field?
We compared the timing of attraction of males released at dusk with males of the same age held in large field cages for the previous 24 hr (Figure 2). No disruptant was used in this comparison. A pronounced flush of males in the traps shortly after their release from cups is evident. In contrast, males held in cages for 24 hr exhibit a rhythm of attraction similar to that described for native males (Beasley & Adams 1994; Schouest & Miller 1994), except that a small proportion is attracted between dusk and midnight (an interval when few native males would be attracted) and the peak of male attraction seems advanced to earlier in the night. We suggest that the small advance in time of attraction of some males held for 24 hr in the field is induced by the presence of the two pheromone-baited traps at the tunnel's upwind end. The overall levels of recapture averaged across the three nights of the test are equivalent (37.8% ± 4.1 S.E. for males released at dusk on the night of capture versus 34.5% ± 3.3 S.E. for males caged in the field for 24 hr before release). Such comparability indicates that measuring the disruption of navigation to lures yields an equivalent outcome with either of these release protocols.
Figure 2 Hourly percentages of P. gossypiella males that were either field-exposed for 24 hr or released at dusk. Males were captured in Delta traps baited with 4 mg of pheromone in a rubber septum. Fluorescent-powder-marked P. gossypiella males were held in groups of 50 in closed 1.8-dl containers. Field-exposed males were released at dusk on the night before the test into 1.8 x 0.75 x 0.5 m high screen cages placed across two rows of cotton. The following day the wind tunnel was positioned over the two rows of cotton such that the downwind end of the tunnel was over the screen cage. At dusk the upwind and downwind screen panels of the screen cage holding the 24-h-field-exposed males were removed and the cage was hoisted to the ceiling of the wind tunnel. Simultaneously, the cups with males that had just been brought to the field were introduced into the downwind end of the same wind tunnel and opened, thereby allowing both groups of males to move upwind. The only sources of pheromone in the tunnels were the two Delta traps in the upwind position. The experiment was replicated three times. Bars represent the mean and line bars represent the standard error of the mean (N = 420 for each treatment)
Effects of pre-exposure on subsequent response
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Confinement of males in a holding cup with a 4-mg pheromone rubber septum for 24 hr before their release into tunnels showed that such prior exposure lowered subsequent attraction by about one-half compared to control males (Figure 3). The magnitude of reduction was similar both in the check tunnel with only the two monitoring traps and in the tunnel treated with three ropes. The lower levels of attraction in the three-rope tunnel reflect disruption of attraction on the treated right side, with some spillover of disruptive effect to the tunnel's untreated left half. When the covers of the cups containing the 4-mg lure were removed, many males took flight within 10 to 60 s, and in the check tunnel many of these males proceeded directly upwind to the traps.
Figure 3 Percentages of pre-exposed and naïve P. gossypiella males captured in Delta traps baited with 4 mg of pheromone in a rubber septum. Fluorescent-powder-marked P. gossypiella males were held in groups of 50 for 24 hr prior to test in closed 1.8-dl cups. Cups for naïve males were pheromone-free, whereas the ones for the pre-exposed males contained a rubber septum loaded with 4 mg pheromone, attached with a pin to the internal surface of the container's lid. The two groups of males were held in separate environmental control rooms for 24 hr, and transported to the field in separate, temperature-regulated containers. Males were released in the wind tunnel at ground level at sunset (approx. 20:00). Lids and attached rubber septa were removed and capture of males was tallied hourly until sunrise (approximately 05:00 of the following day).
The check wind tunnel had no source of pheromone other than the two monitoring Delta traps in the upwind end of the tunnel. In the treatment tunnel 3 Shin-Etsu PBW-Ropes were attached 3 m apart to base of cotton plants on the right side of the tunnel. The percentage of naïve males responding in the check wind tunnel establishes the base levels of response against which disruption is measured. The experiment was replicated three times. Bars represent the mean and line bars represent the standard error of the mean (N = 450 for each treatment). For each wind tunnel, bars having no letters in common are significantly different (ANOVA, p = 0.05, LSD comparisons)
This short interval indicates that some males have a very brief refractory period between prior exposure to high concentrations of pheromone and recovery of responsiveness.
Behavioral experiments with the pink bollworm moths in field wind tunnels have verified that when PBW-Ropes are applied to a cotton field, divers mechanisms contribute to mating disruption. Applied at the standard rate, this formulation releases sufficient pheromone to camouflage a pheromone plume within several meters of its point-source origin. This effect is reflected both in reduction in trap catch and in the airborne concentrations of the plume and ambient pheromone as measured by EAG (Cardé et al. 1993).
Direct behavioral observations in a field wind tunnel have established that some males fly upwind to these high-dose formulations, and many of these become arrested in flight. Other males may reach the rope dispenser, walk on it and wing fan nearby; within minutes, many of these males become quiescent within 50 cm of the ropes. Such reactions support both competition and sensory adaptation/habituation as contributing to disruption. Pre-exposure of males to high concentrations of pheromone before release in the tunnel results in a reduction in the proportion of males responding, again supporting a contribution of sensory adaptation/habituation to disruption. This pre-exposure test also documents that some males retain their ability to navigate a pheromone plume to its source. Males that presumably are exposed continually to pheromone in the field appear to advance their rhythm of pheromone response, which contributes to a competition effect and likely increases the probability of those moths subsequently becoming adapted/habituated.
Pink bollworm males in a cotton field treated with rope thus are subject to multiple, interactive mechanisms of disruption. A male's fate may be dependent on factors such as the level of his exposure to pheromone before his rhythm of attraction is expressed and his individual vulnerability to habituation. Our present understanding of the mechanisms of disruption remains fragmentary, in part because many other factors remain to be clarified. The effects of foliage, for example, on the fine-scale distribution of disruptant and on its adsorption and re-release (Färbert et al. 1997) may alter how disruption is achieved. If so, these effects will vary with the growth stage of cotton.
Mating disruption by broadcast application of pheromone clearly regulates pink bollworm populations and thereby the damage that this pest inflicts on cotton. Several commercial formulations achieve this end, but they do so with substantially different application rates and spatial distributions of emitted pheromone. We conclude that an examination of the behavior of males in cotton treated with different types of formulation will show substantial divergences in how disruption of mate finding is achieved. The levels of contribution of the various mechanisms of disruption and their interactions will differ with the dispersion pattern of the formulation, and the spatial distribution of disruptant achieved within and near the canopy. Understanding these behavioral and dispersion processes will allow us to optimize deployment strategies and resultant efficacy.
Acknowledgements
We thank our colleagues at the APHIS Laboratory for excellent technical support. These studies were supported by a grant to R.T.C. from the National Research Initiative of the U.S.D.A.
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