Aquilaria sinensis (Loureiro) Sprenger is an endangered tree species that produces agarwood, which is a highly prized resinous wood that is used in spices, perfumes, and traditional Chinese medicines (Liu et al. 2013). In recent years, A. sinensis has been cultivated in southern China to meet the increasing demands for agarwood (Liu et al. 2013). However, insects have emerged as threats to the large-scale planting of A. sinensis. Among these pests is Heortia vitessoides Moore (Lepidoptera: Crambidae), whose first through fourth larval instars are gregarious feeders with the fifth and final instar being solitary (Chen et al. 2011, Qiao et al. 2013). Heortia vitessoides larvae reportedly can form large feeding groups of hundreds of larvae in a single A. sinensis tree, consuming all leaves within a few days (Qiao et al. 2013).

Several lepidopterans demonstrate strong larval dispersal abilities as they explore new foraging sites (Ramalho et al. 2014, Wangila et al. 2013). In gregarious species, this process can be well organized as larvae produce silk trails, release trail and aggregation pheromones, or follow tactile stimuli to initiate and maintain foraging processions (Pescador-Rubio et al. 2011, Steinbauer 2009). Heortia vitessoides larvae commonly form unidirectional processions on tree branches, which may extend long distances within or between A. sinensis trees (Qiao et al. 2013, C.W. and X.W. unpubl. data). Meanwhile, we observed that some A. sinensis trees growing near these infested trees showed distinctly different levels of damage, even with overlapping canopies and branches. However, the basis of these apparent preferences exhibited by H. vitessoides larvae on A. sinensis is largely unknown and led to this investigation.

In the present study, double-choice tests were conducted to compare the foraging behaviors (aggregation and feeding) of H. vitessoides larvae responding to leaves collected from different A. sinensis plants. Our study aimed to answer two questions: (a) Do H. vitessoides larvae make collective decisions for foraging? (b) Do H. vitessoides larvae have feeding preferences among different geographic sources of A. sinensis?

Materials and Methods

Host plants

In this study, a total of 24 A. sinensis plants were selected in two locations of the Tianlu Lake State Park, Guangzhou, China. These plants were previously introduced from different geographically based populations across southern China and showed quantitative variation in morphology (Table 1).

Table 1 Leaf morphological traits (mean ± SE) of each A. sinensis tree used in the double-choice assays.*

* Ten mature leaves of each tree were randomly selected for measurement.

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Three A. sinensis plants, designated as L1-1, L1-2, and L1-3, were selected from the L1 location where the H. vitessoides larvae were collected for the assays. The L1-1 plants were undamaged with no H. vitessoides larvae on leaves or branches when collected for bioassays conducted on 17 June 2015. However, on 3 July 2015, the L1-1 trees at that same location had >90% leaves and branches damaged by H. vitessoides larvae (Fig. 1A, B). On 1 July 2015, L1-2 trees growing next to the L1-1 plants (canopies overlapping) (Fig. 1A) exhibited much less damage (<10% damaged) (Fig. 1B). The L1-3 trees remained undamaged throughout the study, while trees growing around the L1-3 trees showed different levels of damage (Fig. 1C). Leaves of L1-3 were collected on 25 June 2015 and 1 July 2015. Leaves from these trees (L1-1, L1-2, L1-3) were placed in double-choice assays with leaves from 21 additional A. sinensis trees growing in a different location in Tianlu Lake State Park. The second location (N 23°15′15″, S 113°24′51″) was designated as L2. All 21 trees at the L2 location were undamaged and not infested by H. vitessoides larvae as leaves were collected between 17 June and 1 July 2015.

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All leaves were used in the double-choice assays within 8 h after collection. Water on the leaves was wiped with a paper towel just prior to the bioassay, and 10-cm segments of fully expanded mature leaves were placed in the bioassay arenas.

Results

Larval survivorship did not differ significantly (F = 0.78; df = 20, 105; P = 0.7348) among the 21 double-choice assays (Table 2). Mean (± SEM) percentage survivorship ranged from 76.7 ± 8.9 to 96.7 ± 2.5. Survivorship exceeded 85% in all but two of the double-choice assays.

Table 2 Mean (± SEM) survivorship of H. vitessoides larvae in double-choice assays.*

* Statistical comparisons were made among all assays. Means followed by the same lowercase letter are not significantly different at α = 0.05.

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After release in the arenas, larvae massed together to form feeding groups and tended to aggregate and feed on one leaf in the double-choice test. Calculated larval concentration rates from the assays yielded a value >80% in most double-choice tests, with half of the tests showing a completely biased distribution (larval concentration rate value = 100%).

With only one exception, no significant differences in the number of H. vitessoides larvae aggregated on the respective leaves or in the area consumed by the feeding larvae occurred in choices between L1-1 leaves and each of the eight sources of leaves from the L2 site (trees L2-1 to L2-8) (Tables 3, 4). That one exception was observed with the pairing of L1-1 with L2-3, for which the area consumed was significantly lower with the L1-1 leaf (2.4 ± 1.6 cm²) as compared with the L2-3 leaf (20.7 ± 6.8 cm²) (Table 4). The number of larvae aggregating on L1-2 leaves was significantly lower than the numbers observed on leaves from the L2-9 through L2-12 trees (Table 3), and the amount of leaf area consumed was significantly lower on L1-2 than on the L2-9 through L2-12 leaves (Table 4). Larval numbers and/or leaf area consumed on the L1-3 entry also were significantly lower than those observed for six of nine choice comparisons in those assays (Tables 3, 4).

Table 3 Mean (± SEM) number of H. vitessoides larvae on respective A. sinensis leaves in each double-choice bioassay.*

* Means within each row followed by same lowercase letter are not significantly different at α = 0.05 (paired t test).

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Table 4 Mean (± SEM) area (cm²) of A. sinensis foliage consumed by H. vitessoides larvae in each double-choice assay.*

* Means within a row followed by the same lowercase letter are not significantly different at α = 0.05 (paired t test).

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Discussion

Although A. sinensis as a species is phenotypically and genetically diverse (Huang et al. 2014, Zhao 2007), the species has not been characterized into intraspecific taxa (i.e., subspecies, variety, forma, etc.). Based on leaf morphology, Huang et al. (2014) identified A. sinensis as “large-leaf” and “small-leaf” populations, but each of these groupings possess significant genetic and morphological differences. In our study, leaf morphological traits (Table 1) appeared to provide little connection to the relative preference or nonpreference of H. vitessoides larvae on A. sinensis. Perhaps nutrition levels or concentrations of secondary metabolites in the leaves are involved in the observed behaviors and responses, as described in studies by Jeude and Fordyce (2014), Kumar et al. (2014), and Walker et al. (2014).

Selection of the host plant is effected by both the adult female insects in their ovipositional behavior and the offspring in their feeding preferences and survivorship (Badenes-Pérez et al. 2014a, Bernal et al. 2015, Friberg et al. 2015). Adult oviposition substrate choice reportedly is not always conducive to larval feeding preference (Bernal et al. 2015, Friberg et al. 2015) and, on some occasions, is even harmful to offspring when an improper or poor-quality host was chosen as an ovipositional substrate by the adults (Badenes-Pérez et al. 2014a). In our case, we have not yet assessed ovipositional substrate choice by H. vitessoides because adults of this species rarely mate and oviposit under laboratory conditions (Chen et al. 2011, T.M. and X.W. unpubl. data).

The gregarious habit of first through fourth instars of H. vitessoides appears to serve as a basis for collective larval preferences for certain sources of A. sinensis trees. Our results indicate that a collective decision-making mechanism may be involved in choices or preferences of gregarious H. vitessoides larvae for A. sinensis foliage. Survivorship, food consumption, growth rates, and defensive responses of gregarious lepidopteran species are often improved when compared with solitary-living counterparts (Allen 2010, Campbell and Stastny 2015). Allen (2010) reported that noncohort groups of Euselasia chrysippe Bates (Lepidoptera: Riodinidae) larvae assembled into groups and concluded that the benefit of living in such groups outweighs the costs of intraspecific competition of this species. Likewise, in our study, H. vitessoides larvae formed feeding groups even when originating from different cohorts (e.g., larvae of different cohorts were collected and mixed for the choice assays). Furthermore, we have observed that H. vitessoides larvae in larger groups have lower mortality rates and consume more foliage than solitary larvae (C.W. and X.W. unpubl. data).

Little to no chemical pesticide residue is one of the basic requirements for the production of high-quality agarwood, especially when it is used for medicinal and food-additive purposes. Therefore, it is important to reduce the use of chemical pesticides for H. vitessoides control. Naturally occurring levels of resistance among different sources of A. sinensis trees to its pest H. vitessoides that were observed in this study may serve as initial impetus for studies on the responses observed. A “push–pull” strategy might be suggested, using nonpreferred A. sinensis trees to repel (push) H. vitessoides larvae while the preferred plants attract (pull) larvae. The preferred plants can be treated with chemical pesticides (Badenes-Perez et al. 2014b, Shelton and Nault 2004) and, therefore, become “dead-end traps” for H. vitessoides larvae. Larger-scale screenings of A. sinensis trees for preference or nonpreference by H. vitessoides larvae should be explored further.