Introduction

Although most adult Lepidoptera of the suborder Glossata visit flowers to feed on nectar, specialized feeding habits have evolved independently in various groups (see Krenn 2010 for a review). Most non-flower-visiting moths and butterflies feed on exposed surfaces that may include the nitrogen-rich juice of decaying fruit, extra-floral nectar, sap, or animal fluids (Büttiker 1967; Büttiker et al. 1996; Bänziger 1970; Krenn et al. 2001; Molleman et al. 2005). Some species, however, are capable of piercing the substrate to gain access to the internal flesh of either plant or animal origin (e.g., Büttiker et al. 1996; Zaspel et al. 2007). The parallel evolution of fruit-feeding led to morphological modifications of the proboscis that are convergent between distantly related families of Lepidoptera (Krenn 2010).

The first broad-scale study that investigated the correlation between proboscis morphology and feeding habits was Krenn et al. (2001). By comparing flower-visiting and non-flower-visiting nymphalid butterflies (64 species), the authors identified proboscis modifications that evolved independently in various butterfly subfamilies and reflected adaptations to novel food resources (decaying fruit or sap). Interestingly, that study showed that proboscis attributes in non-flower-visiting butterflies were similar to those described for non-flower-visiting noctuid moths which feed from the surface and are not capable of piercing (e.g., Bänziger 1970; Büttiker et al. 1996).

Species of the family Noctuidae vary in feeding habits and proboscis morphology (Büttiker 1967; Bänziger 1970; Hendrix et al. 1987; Lingren et al. 1993). The proboscis of nectivorous species is characterized by a relatively simple tip region devoid of ‘spines’ and with few sensilla (Bänziger 1970). In contrast, fruit-piercing moths in the Calpinae and some of the Catocalinae can lacerate the pulp of damaged fruits or even pierce the intact rind (secondary and primary fruitpiercing sensu Bänziger 1982). Several Calyptra (Calpinae) feed on blood and are, therefore, capable of puncturing mammal skin (Bänziger 1989; Zaspel et al. 2007). The probosces of non-flower-visiting noctuids are typically armed with various long ‘spines’ and bear numerous sensilla at the tip region (e.g., Bänziger 1970). The diversity of feeding habits within Noctuidae makes this family an interesting focal group for studies of proboscis morphology.

Fruit-piercing noctuid moths are known to cause considerable damage to cultivated citrus crops (Fay and Halfpapp 2006), and some species feed on grapes in northeastern Brazil (Haji et al. 2001). Given the potential impact of fruit-piercing moths on fruits crops, baseline studies of their feeding habits, morphology, and natural history are important. Therefore the aims of this study are to characterize Neotropical fruit-feeding noctuid moths and compare their proboscis morphology to nectivorous species occurring in the same area. To do so we examined the proboscis of five species in four subfamilies with optical and scanning microscopy to determine if fruit-feeding species possessed correlated morphological characteristics. Our results indicate that two of our focal species possess attributes previously described for fruit-piercing moths, therefore contributing to the understanding of a neotropical moth fauna within agricultural ecosystems.

Materials and Methods

Species sampled

The following species of Noctuidae were examined (classification follows Fibiger & Lafontaine 2005): Alabama argillacea (Hübner, 1823) and Gonodonta bidens Geyer (both Calpinae); Rachiplusia nu (Guenée) (Plusiinae); Mods latipes Hübner (Catocalinae); and Chabuata major (Guenée) (Hadeninae). These species were selected because fruit-feeding habits were known for A. argillacea (Costa Lima 1950) and the genus Gonodonta (Todd 1959), yet their proboscis morphology had not been examined in detail. Bänziger (1982) noted that two species of Mocis in Thailand are capable of piercing soft-skinned fruit, although European Catocalinae feed on tree sap (H. Krenn pers. comm.). Except for R. nu, all species had similar forewing length, used here as a proxy for body size.

Study specimens were obtained from light and pheromone traps, and museum collections (Museu de Ciência e Tecnologia, Pontifícia Universidade Católica do Rio Grande do Sul, and Laboratório de Biologia, Universidade de Caxias do Sul, Brazil). Although an equal number of males and females were examined per species, sample size varied among species (Tables 1 and 2).

Table 1.

Mean height (mm) of ten proboscis sites for five Noctuidae species; number of male and female examined, and number of galeae measured are given for each species.

Table 2.

Measurements of forewing length (FW), total proboscis length and range, length of the distal region that bears sensilla and range.

Scanning electron microscopy

For each species an individual galea of each sex was examined with a Philips XL 30 Scanning Electron Microscope (SEM) to analyze the surface microstructure of the distal region of the proboscis and prepare images (Center of Microscopy and Microanalysis, Pontifícia Universidade Católica-RS). The base of the proboscis and the food canal were not examined with SEM, but are known to contain sensilla trichodea and basiconica (Krenn 2010). The material was dehydrated in alcohol 100% for 15 min, followed by 15 min in acetone, then a critical point dryer Bal-Tec CPD 030 was used to remove all moisture from the samples (Castro 2002). Each galea was mounted on an aluminum stub with a graphite adhesive double-face tape, and sputter-coated with gold and carbon using a Bal-Tec SCD 005 unit.

Figure 1.

Schematic representation of the proboscis showing the measured sites I (base) to 10 (tip). Black line represents the total length, and dashed line indicates the region that bears sensillae and tearing hooks/erectible barbs. High quality figures are available online.

Figure 2.

Mean width in ten sites along galea total length showing differential decline in width for five Noctuidae species. High quality figures are available online.

Figure 3.

Distal region of the proboscis of Alabama argillacea. St, erectible sensilla styloconica; lgl, legulae of the galea linkage (rasping spines of Bänziger 1970). High quality figures are available online.

Figure 4.

Distal region of the proboscis of Gonodonta bidens. St, erectible sensilla styloconica; Tr, sensilla trichodea; lgl, legulae of the galea linkage (rasping spines of Bänziger 1970); th, tearing hooks; eb, erectible barbs. High quality figures are available online.

Figure 5.

Distal region of the proboscis of Rachiplusia nu. St, sensilla styloconica; Ba, sensilla basiconica. High quality figures are available online.

Results

As males and females within species did not differ in any parameters measured (results not shown) the sexes were pooled for comparisons between species (Tables 1 and 2, Figure 2). It is verified here for the first time that the galea of fruit-piercing moths has an even height along its length, and that this proboscis attribute differs between fruitpiercing and nectivore species. Mean height was not uniform along the length of the proboscis within species, with the base being taller than the tip (Table 1). All species showed a significant decrease in galea height between sites 1–2 and, except for C. major, between sites 9–10. Nonetheless, the rate of decrease from base to tip differed among species (Table 1, Figure 2). In fruit-piercing A. argillacea and C. major the galea height also decreased significantly between sites 2–3, after which height decreased rather gradually along the proboscis length with a sharp decrease between sites 9–10. Although a steady decrease in height was also observed in nectar feeding species, significant decreases in height were observed between sites 8-9-10 in R. nu and C. major. An unusual enlargement was found between sites 3–4 in C. major, which was maintained in sites 4–5. The galea of M. latipes showed the most even decrease in height among the three studied nectivores. These results showed that the rate in which the galea decreased in height from base to tip varied slightly within Calpinae, and more strongly between fruit-piercing and nectivores.

Figure 6.

Distal region of the proboscis of Mods latipes. St, sensilla styloconica; lgl, legulae of the galea linkage (rasping spines of Bänziger 1970). High quality figures are available online.

Figure 7.

Distal region of the proboscis of Chabuata major. St, pluricarinate sensilla styloconica; lgl, legulae of the galea linkage (rasping spines of Bänziger 1970). High quality figures are available online.

Galea height varied between species (Table 1). Despite the small difference in wing length (Table 2), the galea of the fruit-piercing A. argillacea and G. bidens differed in height at all sites except 1 and 10. The nectivore C. major had a significantly taller galea than all other species at both the base and tip, and along the proboscis length (i.e., a more robust overall aspect). Finally, given the small galea height, the proboscis of R. nu was clearly the most delicate among all five species.

Both total proboscis length, and length of the distal region varied between species (Table 2). The proboscis of nectivores was slightly longer than that of fruit-feeding A. argillacea and G. bidens. On average R. nu had the longest proboscis, and A. argillacea the shortest. The length of the distal region bearing sensilla and/or spines differed significantly among the four species, including the fruit-piercing A. argillacea and G. bidens. However, the length of the distal region was similar in A. argillacea and G. bidens, and much longer than in the nectivores C. major and M. latipes (approximately 2x and 3x longer, respectively).

The general morphology of the distal region varied among the five species, with the two fruit-piercing calpines most similar to each other (Figure 3–7). The dorsal legulae of the galeal linkage (terminology from Büttiker et al. 1996; ‘rasping spines’ in Bänziger 1970) are spine-like in A. argillacea (Figure 3) and G. bidens (Figure 4), being longer in A. argillacea than in G. bidens. In contrast, they are broad in flower-visiting species (Figure 5–7). Alabama argillacea and G. bidens had a larger number of erectible sensilla styloconica on the distal region compared to R. nu (Figure 5) and M. latipes (Figure 6). The distal proboscis of fruit-piercing G. bidens was more complex than A. argillacea. In addition to sensilla styloconica, Figure 4 shows that G. bidens also has sensilla trichodea, ‘tearing hooks’ and ‘erectible barbs’ (terminology from Bänziger 1970). Interestingly, the proboscis of flower-visiting C. major (Figure 7) is heavily textured and has many pluricarinate sensilla styloconica (terminology from Petr & Stewart 2004).

Discussion

The independent evolution of fruit-feeding in Lepidoptera produced interesting modifications of the proboscis (e.g., Noctuidae and Nymphalidae; Büttiker 1967; Bänziger 1970; Krenn et al. 2001; Krenn 2010). Within Noctuidae, members of Calpinae have proboscis and behavioral adaptations that enable them to pierce fruit skin, and fruit-feeding seems to have preceded the remarkable habit of using animal blood and tears as a nutritional resources (Büttiker 1967; Büttiker et al. 1996; Bänziger 1970, 2007; Hilgartner et al. 2007). Such work provided the impetus for our study, particularly in light of the potential negative impact of fruit-piercing moths on agricultural crops (see Haji et al. 2001).

The volume of muscle bundles inside the galeal lumen affects both the width and height of the galea (see Bänziger 1970 and Krenn 2000 for cross-sections). Given that fruitpiercing species forcefully penetrate the substrate upon which they feed, their proboscis is likely to be more robust than that of nectivores. Galea height was more evenly maintained along the proboscis length in fruitpiercing than in nectivore species where the decline in height from base to tip was more pronounced (Table 1). This result complements previous studies showing a small decline in proboscis width in fruitpiercing moths (Bänziger 1970), and suggests larger muscle bundles are needed in fruitpiercing species. We found no significant differences in mean galea height between species in fruit-piercing and nectar-feeding guilds as predicted by Bänziger (1970). For most of the proboscis length (segments 2–8), galea height was significantly different among the five species suggesting that our sample size was too small to separate interspecific differences from those attributed to feeding habits. Despite similarity in proboscis length (Table 2), G. bidens had a significantly more robust proboscis than A. argillacea, thus suggesting G. bidens may be more efficient at piercing fruit than A. argillacea.

Previous studies suggest that the proboscis of fruit-feeding species is generally shorter than nectivores (e.g., Bänziger 1970). Indeed, a long proboscis is not needed for feeding on exposed surfaces, and natural selection likely favored a reduction of proboscis length in fruit-feeding nymphalids (Krenn et al. 2001). Although our sample size was small, we found no significant differences in proboscis length between fruit and nectar-feeding noctuids. Unlike moths that feed on exposed surfaces (sap or damaged fruit), fruit-piercing species must use force to penetrate the fruit rind and also have to reach into the fruit for feeding. Therefore, within Noctuidae we would expect to find species with shorter proboscis that fed on exposed surface (secondary fruit piercing), primary fruitpiercing species with moderately long proboscis, and nectar-feeding species with generally longer proboscis that might be associated with the type flowers they visit (e.g., Whittall and Hodges 2007). We found that neither galea height, nor length alone were good predictors of feeding habits in noctuid moths. A more comprehensive study would be required to compare statistically the proboscis length of primary and secondary fruit-piercing noctuids with that of nectivores.

In butterflies and moths the proboscis tip shows a number of morphological adaptations to fruit-feeding habits, and they differ in surface-feeding species and piercing species (Krenn 2010). In his study of Malaysian calpines (Calyptra and Scoliopteryx), Bänziger (1970) provided descriptions of proboscis morphology and fruit piercing behavior. We identified similar morphological characteristics in South American A. argillacea and G. bidens, such as long and spine-like dorsal legulae of the galeal linkage (Figure 3–4). While the proboscis distal region of both of the fruit-piercing species studied bear a large number of erectible sensilla styloconica, G. bidens also has sensilla trichodea, tearing hooks and erectible barbs. These differences strongly suggest that A. argillacea and G. bidens vary in their fruitpiercing capability, and may target different types of fruit, or fruit condition (intact versus damaged). Although the presence of specialized structures at the proboscis tip is a good predictor of feeding habits, variation in composition of these structures is known among fruit-feeding calpines (e.g., Bänziger 1970; Büttiker et al. 1996). Indeed, based on the proboscis tip morphology Hilgartner et al. (2007) suggested that Malagasy Hemiceratoides hieroglyphica, which feeds at eyes of sleeping birds, seem to be more closely related to fruit-feeding species than tear-drinking moths associated with mammals. The absence or presence of sensilla trichodea, tearing hooks and erectible barbs between A. argillacea and G. bidens may be due to phylogenetic relationships within Noctuidae, but further interpretation of feeding habits and morphological evolution requires more comprehensive sampling and a better resolution of noctuid phylogeny.

We found that C. major (Hadeninae) had characteristics of nectar-feeding species, such as broad galeal legulae, and the sensilla styloconica were noticeably more numerous in this species than in R. nu and M. latipes. Therefore, direct observations on C. major feeding habits would be of interest to determine if it opportunistically exploits resources other than nectar. Given that we found an unusual enlargement of C. major proboscis in sites 4–5, observations of proboscis movement would allow us to pinpoint the location of the usual ‘proboscis bend’ and verify whether that actually corresponds to a constriction in the measured site 3 (see Krenn 2010 for a discussion of the Glossata proboscis groundplan).

All available evidence suggests that the distinctive morphology of the tip region can be used to predict the feeding habits of noctuid species for which behavior and natural history information is unavailable. Given the broad distribution of Calpinae, this becomes particularly useful in areas where fruit-crops are being grown (see Bänziger 1982 for a brief overview). Together with knowledge of noctuid classification, examination of collection specimens should allow us to predict the number of potential fruit-piercing moths that occur in any given area. Such information is useful from the standpoint of agricultural entomology, and it will also provide insight on the ecology of noctuid feeding guilds in Subtropical South America.