The Argentine cactus moth, Cactoblastis cactorum Berg (Lepidoptera: Pyralidae), an invasive insect that feeds on species of Opuntia (Cactaceae), was first found in the USA in 1989 in the Florida Keys (Habeck & Bennett 1990; Dickle 1991). Since then, the pest has extended its range northward along the coast of North Carolina (J. Driscoll, North Carolina Department of Agriculture and Consumer Services), and westward to coastal Louisiana (Hight & Carpenter 2009). While C. cactorum currently poses a threat to native species of cactus in the American south and Mexico, the cactus also is widely cultivated as an agricultural crop in Mexico, and there is concern that if the insect is successful in entering Mexico, the caterpillar will have a significant economic impact on the industry. To contain the insect in ecologically sensitive and agricultural areas, research has been directed at developing bio-rational techniques that have proven successful in the mitigation of other pest species. These techniques include the use of sterile insect releases (Hight & Carpenter 2016), trapping with a synthetic sex pheromone to monitor populations in the field (Bloem et al. 2005; Heath et al. 2006; Cibrián-Tovar et al. 2017), and the identification and study of an Argentine larval parasitoid (Apanteles opuntiarum Martínez & Berta; Hymenoptera: Braconidae), a potential biological control agent (Varone et al. 2015). Here we report the results of a pilot study undertaken to determine the feasibility of managing C. cactorum using a relatively novel, eco-sensitive approach that employs the use of a larval pheromone.

Cactoblastis cactorum eclose from an eggstick and the gregarious larvae contain their activity to a circular, silken arena that they construct around the base of their eggstick (Fitzgerald et al. 2016). The cohort of caterpillars sharing a common eggstick, a colony, does not eclose in synchrony but over a period of hours, and the arena serves to keep the neonates from wandering away (Fitzgerald et al. 2016). This allows for the gradual buildup of numbers to a critical mass that facilitates the penetration of the host plant. Fitzgerald et al. (2016) also showed that, although aggregates of as few as 5 caterpillars can successfully penetrate a cladode, laboratory colonies did not begin to excavate the host plant until, on average, 25 caterpillars had aggregated at the base of the eggstick some 15 h after the first of the cohort had eclosed.

Larvae of C. cactorum mark and follow trails that serve to keep the cohort together as they move over the surface of the host plant (Fitzgerald et al. 2015). Systematic dissection and chemical extraction of body components revealed that 1 or more compounds found in the caterpillar's mandibular glands, hereafter referred to as the mandibular gland pheromone, are responsible for eliciting trail-following behavior (Fitzgerald et al. 2014, 2015, 2016). The glands are remarkably large, extending the full length of the caterpillar's body, and have a wet mass equal to approximately 1% of the total body mass in the fifth instar (Fitzgerald et al. 2014). The ducts of the glands are inserted into the apodemes of the adductor muscles of the mandibles, and their exudate is secreted as the caterpillars move about. Of particular relevancy to the study presented here is that the circular arena surrounding the base of the eggstick, to which the neonates confine their pre-excavation activity as described above, is marked with the mandibular pheromone that the neonates deposit along with their silk as they tentatively venture short distances from the eggstick. Furthermore, it is the pheromone rather than the silk that serves to contain the cohort to the base of the eggstick. This was demonstrated by spraying cladodes prior to the eclosion of the caterpillars with the mandibular gland pheromone (Fitzgerald et al. 2016). The result was that caterpillars failed to aggregate at the bases of their eggsticks, instead scattering independently over the surfaces of the cladodes upon eclosion, resulting in a significant loss of individuals, many of which fell from the plant. This suggested the possibility that a control strategy based upon this approach might be applied under field conditions. Thus, the pilot studies reported here were undertaken to determine the extent to which the elicitation of dispersal behavior of neonate caterpillars with the mandibular gland extract effects colony survivorship, and to assess the potential of the technique to serve as an alternative, eco-rational means of managing pest populations of the caterpillar.

Materials and Methods

Results

GREENHOUSE STUDY

Caterpillars eclosed from eggsticks between 26 to 29 Sep. The number of larvae that eclosed from eggsticks attached to cladodes sprayed with mandibular gland pheromone was 44.2 ± 1.5, those sprayed with hexane alone, 43.7 ± 1.3, and unsprayed controls, 49.9 ± 1.4. Three of the 10 cohorts that eclosed on plants treated with the mandibular gland pheromone, all of the cohorts on the cladodes sprayed with hexane-only, and all unsprayed controls penetrated the cladode near the base of the eggstick, and caterpillars entered plants. Caterpillars in the 7 cohorts that did not penetrate the cladode at the base of the eggstick dispersed widely over the surface of the cladode. Survivorship of caterpillars in cladodes spayed with mandibular gland pheromone was 23.7 ± 10.0%, for those sprayed with hexane alone, 85.3 ± 3.6%, and for unsprayed controls, 85.6 ± 3.7% (Fig. 1). A Kruskal-Wallis 1-way ANOVA on ranks followed by Tukey's multiple comparison test showed that there was no significant difference between survivorship in hexane only and control treatments (q = 0.14), but that the pheromone treatment differed significantly from the hexane only treatment (q = 5.1; P < 0.05), and non-sprayed control (q = 4.98; P < 0.05).

Fig. 1.

Percent survival of caterpillars in cohorts of Cactoblastis cactorum on plants sprayed with caterpillar extract (gray bar), solvent-only (white bar), or unsprayed (black bar) for 4 separate experiments. Experiment 1 = laboratory study; experiment 2 = greenhouse study; experiment 3 = field study 1; experiment 4 = field study 2.

Discussion

Although sex pheromones have been employed widely to survey and manage populations of phytophagous insects, trail pheromones have been studied little in either of these contexts, and the conventional wisdom has been that, in comparison to sex pheromones, they hold little promise as agents for pest management. Targeting the stage with the greatest reproductive value, the pre-ovipositional female, and preventing her insemination offers the most direct and efficient means of curbing the spread of an insect. Furthermore, compared with their use of sex pheromones as adults, the larvae of relatively few major plant pests are known to employ trail pheromones. As a consequence, the study reported here is 1 of only a few to explore the possibility of using trail pheromones as control agents.

The pre-eminent insect trail followers are ants, and considerable research has been conducted to identify and synthesize their trail pheromones (Holldobler & Wilson 1990). Several investigations also have been undertaken to determine the potential of trail pheromones for managing populations of various pest species. Studies of 2 economically important invasive species, the Argentine ant Linepithema humile (Mayr) (Hymenoptera: Formicidae) (Suckling et al. 2008, 2010a, 2011; Tanaka et al. 2009; Nishisue et al. 2010) and the red imported fire ant Solenopsis invicta Buren (Hymenoptera: Formicidae) (Suckling 2010b, 2012), showed that exposure to synthetic trail pheromone interfered with the ability of the ants to follow their authentic trails, and negatively impacted the insects' foraging behavior. However, there was no indication in these studies that such disruption caused direct mortality, or resulted in a reduction of population size even when the ants were exposed to the synthetic pheromone for the duration of 2 consecutive field seasons (Nishisue et al. 2010). In another study, Sunamura et al. (2011) showed that combining insecticide baits with trail pheromone dispensers resulted in better control of ants than did baits alone. Choe et al. (2014) obtained similar results by mixing trail pheromone with an insecticide. Studies by Westermann et al. (2014) suggested that long term disruption of foraging behavior of the Argentine ant with a synthetic trail pheromone might reduce their competitive ability, leading to collapse of populations in favor of less harmful, indigenous species. However, the difficulty of achieving significant control of ants by any of these techniques is attributable to the fact that colonies of ants are not fully dependent on their communal trail system for survival, but also can search independently and orient directly to food odors (Suckling et al. 2010a).

In contrast to studies with ants, the results reported here for C. cactorum, and a previous study of tent caterpillars (Fitzgerald 2008), indicate that trail marking caterpillars are more vulnerable to colony collapse when their trail systems are disrupted. For the 4 experiments reported here, the average survivorship of C. cactorum larvae from colonies on cladodes sprayed with mandibular gland pheromone was approximately 15% compared with 80% on unsprayed control plants. Similarly, losses of colonies of the tent caterpillars, Malacosoma dis-stria Hübner and Malacosoma americanum (F.) (Lepidoptera: Lasiocampidae), on trees sprayed with a 1 ppm formulation of the trail pheromone mimic 5β-cholestan-3-one, resulted in the loss of 83% of the former colonies and 87% of the latter colonies (Fitzgerald 2008). As with C. cactorum, new eclosed caterpillars of both tent caterpillar species dispersed independently instead of aggregating en masse on their egg mass, their normal behavior. Although specific causes of C. cactorum larval loss in our study were not determined, tent caterpillar larvae typically perished by losing purchase on the branches of the host tree and falling to the ground (Fitzgerald 2008). A previously reported laboratory study suggested that falls from the plant also may constitute a major mortality factor for C. cactorum (Fitzgerald et al. 2016). When moving over the surface of the host plant en masse, the collective spinning efforts of a cohort of social caterpillars allows them to lay down a broad mat of silk that adheres tightly to the plant and engages their crochets, affording them secure purchase on the often smooth and slippery surfaces of the plant. In contrast, a single strand of silk produced by an isolated individual affords the caterpillar more tenuous purchase (Fitzgerald 1993, 1995).

The success of this pilot study of C. cactorum caterpillars can be attributed, most notably, to the stability and persistence of the caterpillars' pheromone, and to the identification and targeting of the most vulnerable stage of the insect's life cycle. Because the mandibular gland pheromone of the cactus caterpillar was applied up to 10 days in advance of caterpillar eclosion, its persistence in active form was essential to its ability to draw caterpillars away from the eggstick and their siblings. Furthermore, studies of cactus caterpillars and tent caterpillars targeted the earliest active stage in the life cycle of the insects, interfering with the ability of neonates to form stable post-eclosion aggregates, essential to the colonies' survival. Cactoblastis cactorum and the tent caterpillar M. americanum present a particularly narrow window of vulnerability. In the case of the tent caterpillar, survivorship rates on treated and control trees did not differ significantly when trees were sprayed with the pheromone mimic after the first instar caterpillars had constructed a tent, even when the pheromone concentration was increased 100-fold (Fitzgerald 2008). While the reason for this is not fully known, the robust and spatially concentrated trail marking of the caterpillars when aggregated appears to allow them to distinguish their authentic trails from surfaces marked with the synthetic pheromone. Similarly, exposure of cactus caterpillar neonates to the mandibular gland pheromone is limited to the brief interval between the time the caterpillars eclose and the time of penetration, because they thereafter confine almost all their activity to the interior of the host. Although the later instars of C. cactorum may mark and follow trails over the surface of cactus when moving between cladodes (Dodd 1940; Pettey 1947), and have been shown to follow pathways marked with mandibular gland extract (Fitzgerald et al. 2015), the extent to which blanket-spraying plants with pheromone might affect the ability of maturing caterpillars to stay together is unknown. Furthermore, even if the pheromone were to interfere with the ability of the cohort to maintain a tight aggregate, such disruption might have little impact on colony survival. Dodd (1940) and Pettey (1947) reported that dispersing colonies of older caterpillars commonly fragment into smaller units that successfully penetrate and tunnel cladodes.

Were this technique to be adapted to manage pest populations of C. cactorum, it would be necessary to formulate the active ingredient with a non-phytotoxic carrier. A largely water-based formulation used to successfully apply the tent caterpillar pheromone in the disruption studies discussed above (Fitzgerald 2008; T. D. F. unpublished), indicate that a similar formulation can be used for the cactus caterpillar. Moreover, while a sprayable formulation might be prepared from whole body larval extract, as in the present study, a preferable approach would require the identification of the bioactive components of the mandibular glands and their synthesis in quantities sufficient for abatement programs. Chemical analysis of the mandibular gland indicates that the fluid consists of a large number of chemically distinct compounds (Fitzgerald et al. 2015). Particularly prominent are a series of 2-acyl-1, 3-cyclohexanediones. Laboratory bioassays show that one of these, 4-hydroxy-2-oleoyl-1, 3-cyclohexanedione, is as effective on a per unit volume basis in eliciting trail following from neonates as whole mandibular gland extract (Fitzgerald et al. 2015). Moreover, the compound is non-volatile and chemically stable, important characteristics of a chemical that needs to be persistent under field conditions. Although a molecule lacking the hydroxyl group in position 4 can be prepared directly from commercially available starting materials, the addition of the hydroxyl group requires 3 additional synthetic steps, significantly increasing the task of its preparation, and potentially making it too costly for use in a control program. While less effective than 4-hydroxy-2-oleoyl-1,3-cyclohexanedione, studies show that sub-components of the compound, as well as other 2-acyl-1, 3-cy-clohexanediones, also elicit trail following responses in neonate C. cactorum, raising the prospect of designing and synthesizing a cost-effective trail following molecule (Fitzgerald et al. 2015). Moreover, bioassays of fractionated mandibular gland extract indicate that other yet unidentified chemical components of the mandibular gland, chemically distinct from the 2-acyl-1, 3-cyclohexanediones, also elicit trail following, though it is not known the extent to which any of these compounds are competitive with whole mandibular gland extract. The capability of these unidentified mandibular gland components to disrupt the collective excavation behavior of neonates has not been investigated.

Particularly problematic for the effective application of this technique for disrupting the establishment of pest populations of caterpillars is the need for effective timing of application. The tent caterpillars represent the ideal situation. These insects have 1 generation per yr, the eggs hatch in close synchrony over large geographic areas, and the date of hatching is highly predictable (Fitzgerald 1995). In contrast, the cactus caterpillar has multiple generations and egg hatching is largely asynchronous. Dodd (1940) and Pettey (1947) recorded winter and summer generations in Australia and South Africa, each with flight periods lasting about 1.5 mo for the winter generation and just over 1 mo for the summer generation. In the southeastern US, there are 3 non-overlapping generations per yr, each flight period lasting approximately 2 mo (Hight & Carpenter 2009). Thus, management of the cactus caterpillar by disrupting the establishment of neonates would require long-term presence of the disruptor pheromone. This would largely limit the applicability of the technique to heavily managed, commercial plantations and, depending on the effective longevity of the pheromone, require repeated application of the chemical. It remains to be determined if the potential ecological benefit of high target specificity and the added market value of an “organically” managed crop would warrant the use of this procedure over conventional approaches involving pesticides.