Foraging efficiency with an elongated proboscis
Long-proboscid nemestrinids co-occur with several, mostly nectar rewarding flowers that have elongated, cylindrical flower tubes (Goldblatt and Manning 2000). Proboscis and floral tube length vary between different populations within the same species and have been regarded as a reciprocal adaptation between the flower and the fly (Anderson and Johnson 2008; Pauw et al. 2009). Although allometric relationships between body size and proboscis length have already been shown in a previous study (Anderson and Johnson 2008), corolla tube lengths have been regarded as the significant predictor variable for the proboscis length. Nevertheless, given the positive scaling relationship between body and proboscis length in Prosoeca sp., allometry could be regarded as an important factor in generating the proboscis length variations in long-proboscid flies.
Given the high variation in proboscis lengths in Prosoeca sp., a difference in flower handling time was expected between individuals with different proboscis lengths as shown in Fig. 1. However, in detail, this relationship was verified only for the time spent drinking from a flower. Kunte (2007) explained the strong positive relationship between proboscis length and increased handling time as a function of a disproportionate relationship between the cibarial muscles and an elongated proboscis. With a given body size and cibarial muscle mass, longer proboscides should produce more friction to nectar travelling up the proboscis due to their greater food canal surface area. However, in Prosoeca sp., cibarial and pharyngeal muscle masses showed a positive allometric relationship with body size and proboscis length; thus, an efficient nectar uptake rate would be expected due to the increased muscle mass compensating for an increased proboscis length. Nonetheless, drinking time of flies was seen to increase with an elongated proboscis, supporting the contention that longer proboscides do offer some limitations, as suggested by the functional constraint hypothesis. Additionally, longer drinking times could also be influenced by long-proboscid individuals being able to take up the full complement of nectar out of long, narrow flowers, e.g. up to 7 μl per flower (Goldblatt et al. 1995; Manning and Goldblatt 1996).
Surprisingly, the time required to insert a long proboscis into a flower of L. oreogena appeared to be independent of proboscis length. Previous studies of the flower-visiting behaviour of Prosoeca sp. on L. oreogena revealed the importance of the white arrow markings as functional nectar guides to minimise flower handling times. Flies were no longer able to insert their proboscis into flowers with artificially manipulated nectar markings (Hansen et al. 2011). These nectar guides may help to reduce the negative effects of flower handling with an elongated proboscis. Inserting a proboscis of this length into a long-spurred flower could, however, be negatively influenced by wind and vegetation growing between and over host flowers, as was seen for some foraging flies during field experiments. This might explain the large variation observed between minimum (0.16 s) and maximum inserting times (6.6 s). Removing time was consistently rapid across all individuals observed. Flies leave flowers with a fast, forceful movement, possibly to overcome the friction between proboscis and flower, and therefore, no overall differences between individuals could be recorded.
Similar to apid bees, nemestrinid flies can be regarded as high-speed, continuous foragers (Pivnick and McNeil 1985), which should prefer nectar with sugar concentrations between 30 and 50 %. Concentrations in long-spurred flowers have been recorded to be lower than in flowers with short perianth tubes (Plowright 1987). Similar results have been shown for Lapeirousia species in Namaqualand (Goldblatt et al. 1995), and typical flowers visited by Prosoeca sp. only produce nectar with sugar concentrations of 20–30 % (Goldblatt et al. 1995). Both flowers and flies occur in a semi-arid habitat (Manning and Goldblatt 1997), where preferred nectar concentrations are expected to shift to more dilute nectar to counter water loss as predicted by mathematical models (Pivnick and McNeil 1985). Kim et al. (2011b) discovered that optimal nectar concentration is strongly related to the drinking style of foraging insects, being higher for viscous dippers, e.g. bees, than suction feeders, such as long-proboscid Nemestrinids, thus possibly offering an explanation for the relatively dilute concentrations of nectar in Prosoeca sp.’s host plants.
Suction pump design
Like in Lepidoptera (Monaenkova et al. 2012), the proboscis of Prosoeca represents a two-level fluidic system, combining a nanosponge with a straw. The mouthpart morphology suggests that Nemestrinidae are suction feeders that rely on pressure gradients within the proboscis (Karolyi et al. 2012). While feeding, on the first microlevel, paired labella at the tip of the proboscis function as a nanosponge. Rutted with a pseudothracheal system, they take in liquid via capillary action, filling the distal part of the food canal (Kingsolver and Daniel 1979). On the second microlevel, the food canal works as a pump-operated drinking straw, transporting fluids to the mouth. However, sucking up nectar from long, narrow floral tubes through a long, straw-like proboscis requires an enhanced pumping organ. Nearly all insects feature a pharyngeal pump, while a cibarial pump is developed only in insects with true sucking mouthparts (Peters 2003).
The suction pump of Prosoeca sp. is here hypothesised to combine the systaltic motion of two pumping organs regulated by two valves, each controlled by dilator muscles, dividing the feeding process into three functional phases as shown in Fig. 5. During the first phase, the functional mouth valve opens, and the cibarial pump is extended by the cibarial dilator. Due to the emerging partial vacuum in the cibarial chamber, nectar is sucked from the food canal into the cibarium (Fig. 5a). In the second phase, relaxation of the cibarial pump and combined contraction of the pharyngeal dilators draw nectar through the now opened cibarial–pharyngeal valve into the pharyngeal chamber (Fig. 5b). During the last phase, the pharyngeal dilator muscles relieve tension, and the compressor muscles push the nectar into the oesopharynx. Finally, dorsal and lateral dilators open the distal oesophagus valve to the midgut (Fig. 5c). According to this model, Prosoeca is able to efficiently suck up viscous nectar using two well-coordinated suction pumps.
Additionally, two paired lateral muscles in the head (Fig. 3e, f) most likely provide a supporting function. Contraction of the cibarial and pharyngeal dilators exerts a force on the suction pump. Simultaneous contraction of the cibarial retractor and protractor holds the pump stationary by working antagonistically against the massive dilator muscles.
Although this feeding model has been hypothesised based on 3D reconstructions acquired from microCT scans, similar systems have been described in detail for short-proboscid Tabanidae (Bonhag 1951), Bombyliidae (Szucsich and Krenn 2000) and female mosquitoes (Kim et al. 2011a; Kim et al. 2012; Snodgrass 1959). Compared to Tabanidae, a massive posterior compressor and an additional pharyngeal dilator exist in Prosoeca sp., enhancing the pharyngeal pump.
The morphological adaptation of a two-pump system has been explained by Kim et al. (2011a). Although a rectangular path is inevitable to connect the food canal within the orthognathe proboscis with the midgut, it also leads to energy loss along the curved path. Therefore, an additional pump is necessary to regulate the flow effectively. Similarly to mosquitoes (Snodgrass 1959) and based on the morphological studies presented here, nemestrinid flies appear to be able to suck liquid efficiently using the phase-shifted motion of cibarial and pharyngeal pump in conjunction with two valves.
Compared to nemestrinid flies, the suction pump of Lepidoptera consists of only a main buccal lumen, formed by the epi- and hypopharynx, operated by several dilator and compressor muscles (Davis and Hildebrand 2006; Eberhard and Krenn 2005). In addition, a cibarial upstream valve controls the entrance to the actual pump, and a posterior valve regulates the influx to the oesophagus. In Nymphalidae, the cibarial oral valve also regulates the outflow of saliva into the proboscis (Eberhard and Krenn 2005). In contrast, the hypopharynx of Prosoeca sp. is part of the proximal proboscis, housing the salivary duct, while the epipharynx provides the lining for the food canal (Karolyi et al. 2012).
Relationship between muscle volume and proboscis length
Mechanical properties of the feeding apparatus, like the musculature needed to maintain a constant pressure gradient, are potentially limiting factors in nectar-sucking insects. Detailed considerations about biophysical and biomechanical properties of the feeding mechanism are given by Kingsolver and Daniel (1979). However, May (1985) indicated that nectar flow is not steady in butterflies and that the pressure drop (i.e. the pressure difference between the proximal and distal end of the feeding channel) is variable during nectar feeding. He further noted that butterflies with a longer proboscis have a higher energy intake rate due to greater pressure drops and greater uptake rates at any given nectar concentration.
Kunte (2007) hypothesised that insects that depend exclusively on nectar as food source should have a greater cibarial muscle mass to increase the rate of nectar uptake. Our results support this hypothesis, as seen in the positive linear relationship between proboscis length and sucking pump dimensions in Prosoeca sp., indicating that a longer proboscis demands larger pumping organs. These correlations underline the importance of both pumps in nemestrinid flies.
Evolutionary origin of the sucking pump
In various insects with sucking mouthparts, the suction pump has evolved from the cibarium, the pharynx or both (Davis and Hildebrand 2006). Snodgrass (1944, 1935) determined the evolutionary origin of the insect sucking pump as primarily cibarial with a minor pharyngeal component. He further described the frontal ganglion as a stomodaeal–cibarial border, suggesting that all muscles proceeding between clypeus and buccal cavity are cibarial. In contrast, Davis and Hildebrand (2006) postulated a stomodaeal origin of the buccal cavity, since the visceral muscles enveloping the sucking pump are characteristic of the stomodaeum. While the cibarium only forms the pre-oral valve, the main sucking pump is recognized as pharyngeal. In Prosoeca sp., the cibarial and pharyngeal pumps can be clearly assigned by their attached dilator and visceral muscles. Snodgrass (1959) described the two-part suction pump of the female mosquito which included a connectional alimentary canal with two antagonistic muscles, which represented the true mouth, and a pharyngeal pump positioned after the brain. However, in Prosoeca sp., both pumps are located anterior to the brain, and the alimentary canal is modified to the cibarial–pharyngeal valve. The oesophagus traverses the brain, with an additional posterior valve. This is quite similar to the model described for Tabanidae (Bonhag 1951), suggesting that the ancestors of Nemestrinidae might have been blood-sucking insects (Grimaldi and Engel 2005).
Dimensions of the food canal
The feeding mechanism associated with a long proboscis can be regarded as a simple pipe flow system with a laminar nectar flow. The food canal represents a long, thin cone, changing its diameter at a constant rate (Kingsolver and Daniel 1979). Intake of fluids depends on viscosity, and an increasing sugar concentration results in a curvilinear increase in the weight of sucrose per unit time which leads to an increase of viscosity (Heyneman 1983). Therefore, nectar containing 40 % sucrose is six times more viscous than water at the same temperature (Chapman 1998).
The pressure drop created by the suction pump depends also on the morphological characteristics of the proboscis. Furthermore, according to the Hagen–Poiseuille equation, a longer proboscis results in a reduced nectar flow (Kingsolver and Daniel 1979) as long as all other parameters remain constant. However, nectar flow also depends on the fourth power of the radius of the food canal. In order to maintain a constant flow rate within individuals with different proboscis lengths, flies with longer proboscides would require correspondingly increased suction pump dimensions but a relatively small increase of the food tube diameter. Reinforced suction pump musculature has also been recorded for extremely long-proboscid riodinid butterflies (Bauder et al. 2013).
Compared to Lepidoptera with similar proboscis length variations (May 1985; Bauder et al. 2011), the food canal dimensions of Prosoeca sp. are relatively large, indicating that they possibly compensate the excess length of the proboscis with extended food canal dimensions. Flower-visiting insects, like nemestrinid flies, that use energy-expensive feeding techniques benefit from minimised feeding times (Heyneman 1983), which can be achieved with efficient suction pumps and an increased nectar flux through extended food canal dimensions.
The present study has described the flower-visiting behaviour of long-proboscid Prosoeca sp. on flowers of L. oreogena and has verified that individuals with a longer proboscis spent more time drinking from long-spurred flowers. These results suggest that longer proboscid individuals are able to take up more nectar in a single visit and therefore gain a possible advantage over individuals with an average proboscis length. In addition, this study has shown that feeding from elongated floral tubes not only requires an efficient two-part suction pump in the head, but also a positive allometric relationship between proboscis length and suction pump muscle mass. These results indicate that Prosoeca sp. represents a highly adapted and efficient nectar feeder.