Wettability and morphology of proboscises interweave with hawkmoth evolutionary history.

Hovering hawkmoths expend significant energy while feeding, which should select for greater feeding efficiency. Although increased feeding efficiency has been implicitly assumed, it has never been assessed. We hypothesized that hawkmoths have proboscises specialized for gathering nectar passively. Using contact angle and capillary pressure to evaluate capillary action of the proboscis, we conducted a comparative analysis of wetting and absorption properties for 13 species of hawkmoths. We showed that all 13 species have a hydrophilic proboscis. In contradistinction, the proboscises of all other tested lepidopteran species have a wetting dichotomy with only the distal ∼10% hydrophilic. Longer proboscises are more wettable, suggesting that species of hawkmoths with long proboscises are more efficient at acquiring nectar by the proboscis surface than are species with shorter proboscises. All hawkmoth species also show strong capillary pressure, which, together with the feeding behaviors we observed, ensures that nectar will be delivered to the food canal efficiently. The patterns we found suggest that different subfamilies of hawkmoths use different feeding strategies. Our comparative approach reveals that hawkmoths are unique among Lepidoptera and highlights the importance of considering the physical characteristics of the proboscis to understand the evolution and diversification of hawkmoths.

Haemolymph viscosity in hawkmoths and its implications for hovering flight.

Viscosity determines the resistance of haemolymph flow through the insect body. For flying insects, viscosity is a major physiological parameter limiting flight performance by controlling the flow rate of fuel to the flight muscles, circulating nutrients and rapidly removing metabolic waste products. The more viscous the haemolymph, the greater the metabolic energy needed to pump it through confined spaces. By employing magnetic rotational spectroscopy with nickel nanorods, we showed that viscosity of haemolymph in resting hawkmoths (Sphingidae) depends on wing size non-monotonically. Viscosity increases for small hawkmoths with high wingbeat frequencies, reaches a maximum for middle-sized hawkmoths with moderate wingbeat frequencies, and decreases in large hawkmoths with slower wingbeat frequencies but greater lift. Accordingly, hawkmoths with small and large wings have viscosities approaching that of water, whereas hawkmoths with mid-sized wings have more than twofold greater viscosity. The metabolic demands of flight correlate with significant changes in circulatory strategies via modulation of haemolymph viscosity. Thus, the evolution of hovering flight would require fine-tuned viscosity adjustments to balance the need for the haemolymph to carry more fuel to the flight muscles while decreasing the viscous dissipation associated with its circulation.

Insect antennae: Coupling blood pressure with cuticle deformation to control movement.

Insect antennae are hollow, blood-filled fibers with complex shape. Muscles in the two basal segments control antennal movement, but the rest (flagellum) is muscle-free. The insect can controllably flex, twist, and maneuver its antennae laterally. To explain this behavior, we performed a comparative study of structural and tensile properties of the antennae of Periplaneta americana (American cockroach), Manduca sexta (Carolina hawkmoth), and Vanessa cardui (painted lady butterfly). These antennae demonstrate a range of distinguishable tensile properties, responding either as brittle or strain-adaptive fibers that stiffen when stretched. Scanning electron microscopy and high-speed imaging of antennal breakup during stretching revealed complex coupling of blood pressure and cuticle deformation in antennae. A generalized Lamé theory of solid mechanics was developed to include the force-driven deformation of blood-filled antennal tubes. We validated the theory against experiments with artificial antennae with no adjustable parameters. Blood pressure increased when the insect inflated its antennae or decreased below ambient pressure when an external tensile load was applied to the antenna. The pressure-cuticle coupling can be controlled through changes of the blood volume in the antennal lumen. In insects that do not fill the antennal lumen with blood, this blood pressure control is lacking, and the antennae react only by muscular activation. We suggest that the principles we have discovered for insect antennae apply to other appendages that share a leg-derived ancestry. Our work offers promising new applications for multifunctional fiber-based microfluidics that could transport fluids and be manipulated by the same fluid on demand. STATEMENT OF SIGNIFICANCE: Insect antennae are blood-filled, segmented fibers with muscles in the two basal segments. The long terminal segment is muscle-free but can be flexed. To explain this behavior, we examined structure-function relationships of antennae of cockroaches, hawkmoths, and butterflies. Hawkmoth antennae behaved as brittle fibers, but butterfly and cockroach antennae showed strain-adaptive behavior like fibers that stiffen when stretched. Videomicroscopy of antennal breakup during stretching revealed complex coupling of blood pressure and cuticle deformation. Our solid mechanics model explains this behavior. Because antennae are leg-derived appendages, we suggest that the principles we found apply to other appendages of leg-derived ancestry. Our work offers new applications for multifunctional fiber-based microfluidics that could transport fluids and be manipulated by the fluid on demand.

Elastocapillary effect in self-repair of proboscises of butterflies and moths.

HYPOTHESIS: Self-repair in living organisms, without tissue regeneration or regrowth, is rare. Recent discovery that butterflies can self-repair the proboscis after the two halves (galeae) have been separated raised a question about the physical mechanism allowing them to reunite the parts. We discovered that butterflies pump saliva during repair of their proboscises. We then hypothesized that saliva spreading along the food canal of the proboscis would create capillary forces capable of bringing the galeae together. EXPERIMENT: To test the hypothesis, we distinguished capillary forces from muscular action of the galeae by sedating butterflies and video tracking retraction of the saliva menisci during galeal separation. To theoretically show capillary adhesion, the elastic moduli of the galeae were measured, and the galeal profiles were extracted from videos as a function of time. The values were then fitted with a mathematical model based on an augmented Euler-Bernoulli beam theory whereby each galea was treated as a beam bent by capillary forces due to saliva. We also evaluated friction forces that prevented disjoining of the galea at the tip of their separation. FINDINGS: The results showed that butterflies use saliva to repair their proboscises via capillary adhesion, and theoretically supported the role of saliva in providing the necessary capillary forces to bring the galeae together. Tangential shear forces acting parallel to the galea at the tip of their separation are caused primarily by friction between the cuticular linking structures.

Nucleation and Formation of a Primary Clot in Insect Blood.

Blood clotting at wound sites is critical for preventing blood loss and invasion by microorganisms in multicellular animals, especially small insects vulnerable to dehydration. The mechanistic reaction of the clot is the first step in providing scaffolding for the formation of new epithelial and cuticular tissue. The clot, therefore, requires special materials properties. We have developed and used nano-rheological magnetic rotational spectroscopy with nanorods to quantitatively study nucleation of cell aggregates that occurs within fractions of a second. Using larvae of Manduca sexta, we discovered that clot nucleation is a two-step process whereby cell aggregation is the time-limiting step followed by rigidification of the aggregate. Clot nucleation and transformation of viscous blood into a visco-elastic aggregate happens in a few minutes, which is hundreds of times faster than wound plugging and scab formation. This discovery sets a time scale for insect clotting phenomena, establishing a materials metric for the kinetics of biochemical reaction cascades. Combined with biochemical and biomolecular studies, these discoveries can help design fast-working thickeners for vertebrate blood, including human blood, based on clotting principles of insect blood.

Self-assembly of the butterfly proboscis: the role of capillary forces.

The proboscis of butterflies and moths consists of two C-shaped fibres, the galeae, which are united after the insect emerges from the pupa. We observed that proboscis self-assembly is facilitated by discharge of saliva. In contrast with vertebrate saliva, butterfly saliva is not slimy and is an almost inviscid, water-like fluid. Butterfly saliva, therefore, cannot offer any viscoelastic adhesiveness. We hypothesized that capillary forces are responsible for helping butterflies and moths pull and hold their galeae together while uniting them mechanically. Theoretical analysis supported by X-ray micro-computed tomography on columnar liquid bridges suggests that both concave and convex liquid bridges are able to pull the galeae together. Theoretical and experimental analyses of capillary forces acting on natural and artificial proboscises show that these forces are sufficiently high to hold the galeae together.

Effect of curvature on wetting and dewetting of proboscises of butterflies and moths.

Proboscises of butterflies are modelled as elliptical hollow fibres that can be bent into coils. The behaviour of coating films on such complex fibres is investigated to explain the remarkable ability of these insects to control liquid collection after dipping the proboscis into a flower or pressing and mopping it over a food source. By using a thin-film approximation with the air-liquid interface positioned almost parallel to the fibre surface, capillary pressure was estimated from the profile of the fibre surfaces supporting the films. The film is always unstable and the proboscis shape and movements have adaptive value in collecting fluid: coiling and bending of proboscises of butterflies and moths facilitate fluid collection. Some practical applications of this effect are discussed with regard to fibre engineering.

Structural and physical determinants of the proboscis-sucking pump complex in the evolution of fluid-feeding insects.

Fluid-feeding insects have evolved a unique strategy to distribute the labor between a liquid-acquisition device (proboscis) and a sucking pump. We theoretically examined physical constraints associated with coupling of the proboscis and sucking pump into a united functional organ. Classification of fluid feeders with respect to the mechanism of energy dissipation is given by using only two dimensionless parameters that depend on the length and diameter of the proboscis food canal, maximum expansion of the sucking pump chamber, and chamber size. Five species of Lepidoptera - White-headed prominent moth (Symmerista albifrons), White-dotted prominent moth (Nadata gibosa), Monarch butterfly (Danaus plexippus), Carolina sphinx moth (Manduca sexta), and Death's head sphinx moth (Acherontia atropos) - were used to illustrate this classification. The results provide a rationale for categorizing fluid-feeding insects into two groups, depending on whether muscular energy is spent on moving fluid through the proboscis or through the pump. These findings are relevant to understanding energetic costs of evolutionary elaboration and reduction of the mouthparts and insect diversification through development of new habits by fluid-feeding insects in general and by Lepidoptera in particular.

Structure of the lepidopteran proboscis in relation to feeding guild.

Most butterflies and moths (Lepidoptera) use modified mouthparts, the proboscis, to acquire fluids. We quantified the proboscis architecture of five butterfly species in three families to test the hypothesis that proboscis structure relates to feeding guild. We used scanning electron microscopy to elucidate the fine structure of the proboscis of both sexes and to quantify dimensions, cuticular patterns, and the shapes and sizes of sensilla and dorsal legulae. Sexual dimorphism was not detected in the proboscis structure of any species. A hierarchical clustering analysis of overall proboscis architecture reflected lepidopteran phylogeny, but did not produce a distinct group of flower visitors or of puddle visitors within the flower visitors. Specific characters of the proboscis, nonetheless, can indicate flower and nonflower visitors, such as the configuration of sensilla styloconica, width of the lower branches of dorsal legulae, presence or absence of dorsal legulae at the extreme apex, and degree of proboscis tapering. We suggest that the overall proboscis architecture of Lepidoptera reflects a universal structural organization that promotes fluid uptake from droplets and films. On top of this fundamental structural organization, we suggest that the diversity of floral structure has selected for structural adaptations that facilitate entry of the proboscis into floral tubes.

Paradox of the drinking-straw model of the butterfly proboscis.

Fluid-feeding Lepidoptera use an elongated proboscis, conventionally modeled as a drinking straw, to feed from pools and films of liquid. Using the monarch butterfly, Danaus plexippus (Linnaeus), we show that the inherent structural features of the lepidopteran proboscis contradict the basic assumptions of the drinking-straw model. By experimentally characterizing permeability and flow in the proboscis, we show that tapering of the food canal in the drinking region increases resistance, significantly hindering the flow of fluid. The calculated pressure differential required for a suction pump to support flow along the entire proboscis is greater than 1 atm (~101 kPa) when the butterfly feeds from a pool of liquid. We suggest that behavioral strategies employed by butterflies and moths can resolve this paradoxical pressure anomaly. Butterflies can alter the taper, the interlegular spacing and the terminal opening of the food canal, thereby controlling fluid entry and flow, by splaying the galeal tips apart, sliding the galeae along one another, pulsing hemolymph into each galeal lumen, and pressing the proboscis against a substrate. Thus, although physical construction of the proboscis limits its mechanical capabilities, its functionality can be modified and enhanced by behavioral strategies.

Hydrophobic-hydrophilic dichotomy of the butterfly proboscis.

Mouthparts of fluid-feeding insects have unique material properties with no human-engineered analogue: the feeding devices acquire sticky and viscous liquids while remaining clean. We discovered that the external surface of the butterfly proboscis has a sharp boundary separating a hydrophilic drinking region and a hydrophobic non-drinking region. The structural arrangement of the proboscis provides the basis for the wetting dichotomy. Theoretical and experimental analyses show that fluid uptake is associated with enlargement of hydrophilic cuticular structures, the legulae, which link the two halves of the proboscis together. We also show that an elliptical proboscis produces a higher external meniscus than does a cylindrical proboscis of the same circumference. Fluid uptake is additionally facilitated in sap-feeding butterflies that have a proboscis with enlarged chemosensory structures forming a brush near the tip. This structural modification of the proboscis enables sap feeders to exploit films of liquid more efficiently. Structural changes along the proboscis, including increased legular width and presence of a brush-like tip, occur in a wide range of species, suggesting that a wetting dichotomy is widespread in the Lepidoptera.

Butterfly proboscis: combining a drinking straw with a nanosponge facilitated diversification of feeding habits.

The ability of Lepidoptera, or butterflies and moths, to drink liquids from rotting fruit and wet soil, as well as nectar from floral tubes, raises the question of whether the conventional view of the proboscis as a drinking straw can account for the withdrawal of fluids from porous substrates or of films and droplets from floral tubes. We discovered that the proboscis promotes capillary pull of liquids from diverse sources owing to a hierarchical pore structure spanning nano- and microscales. X-ray phase-contrast imaging reveals that Plateau instability causes liquid bridges to form in the food canal, which are transported to the gut by the muscular sucking pump in the head. The dual functionality of the proboscis represents a key innovation for exploiting a vast range of nutritional sources. We suggest that future studies of the adaptive radiation of the Lepidoptera take into account the role played by the structural organization of the proboscis. A transformative two-step model of capillary intake and suctioning can be applied not only to butterflies and moths but also potentially to vast numbers of other insects such as bees and flies.

Predicting occurrence of the fungal symbiote Harpella colonizing black fly larvae in coastal streams of Alabama and Mississippi, USA.

The environmental conditions governing symbioses are poorly known in aquatic systems. Stream conditions associated with the distribution of the black fly (Simuliidae) midgut symbiote Harpella were investigated in southern Alabama and Mississippi streams. Stream conditions that were most useful in predicting the distribution of Harpella spp. in the study area were dissolved oxygen and water temperature. Presence of Harpella species in streams was associated with higher dissolved oxygen and decreased water temperature compared to streams where Harpella spp. was absent. Stream conditions associated with the distribution of Harpella spp. in other regions of the world vary according to conditions other than those elucidated here, indicating that geography, host species, and stream conditions play important roles in the spatial distribution of Harpella species.

Laboratory investigations of trichomycete prevalence, abundance and fecundity in a Smittium-simuliid model.

Smittium, the most speciose genus of the "gut fungi" (Zygomycota: Trichomycetes), is found attached to the hindgut cuticle of larval aquatic Diptera. Smittium spp. colonize several host families (e.g., Smittium culisetae in Chironomidae, Culicidae and Simuliidae), but some species appear to be specific to a single host family (e.g., Smittium morbosum Sweeney in Culicidae). The specificity of Smittium spp. within a host family has been difficult to resolve. This research presents evidence that certain Smittium spp. differentially colonize particular species of black fly (Diptera: Simuliidae) hosts as measured by differences in prevalence, abundance and fecundity. Reasons for this differential occurrence and fecundity in hosts are unclear but might include fungal responses to variations in host morphology, physiology, distribution or behavior. Variable fitness of Smittium spp., within a suite of available hosts, could be a factor in the diversity of this fungal group.

Prevalence of the trichomycete fungus Harpella melusinae (Harpellales: Harpellaceae) in larval black flies (Diptera: Simuliidae) across a heterogeneous environment.

A total of 2063 mid- to late-instar larval black flies were collected from 64 stream sites in South Carolina and screened for the presence of the trichomycete fungus Harpella melusinae. Sixteen of 18 host species were colonized by H. melusinae on at least one occasion. Prevalence of H. melusinae in larvae of Simulium tuberosum cytospecies "A" was highest in acidic streams with low conductivity, whereas H. melusinae colonized larvae of Simulium verecundum most frequently in slower-moving streams. Ecological conditions, therefore, can serve as predictors of the prevalence of H. melusinae. Prevalence in host larvae was significantly lower in the Piedmont ecoregion than in the Mountain ecoregion. Prevalence did not differ in the host species S. verecundum across ecoregions, suggesting that different prevalences among host species might indicate some host preference. The prevalence of H. melusinae differed significantly between two univoltine host species (Simulium venustum and Prosimulium magnum) at the same site but not between two multivoltine host species (S. tuberosum cytospecies "FG" and S. tuberosum cytospecies "CDE"), suggesting that host life history could be important in determining fungal prevalence.

The microdistribution of the trichomycete Smittium culisetae in the hindgut of the black fly host Simulium vittatum.

We examined the distribution of hyphae of the trichomycete fungus Smittium culisetae (Harpellales: Legeriomycetaceae) in the hindgut of a larval black fly (Simulium vittatum, cytospecies IS-7) by analyzing its prevalence and relative abundance. Hyphal prevalence was highest in the posterior colon (93.1%) and rectum (86.3%), with low prevalence (12.0%) in the anterior colon. Relative abundance of hyphae was highest in the posterior colon, followed by the rectum; relative abundance of hyphae in the anterior colon was lower. Hyphae of S. culisetae were not observed in the pylorus. We used a novel method of quantifying the relative abundance of S. culisetae in the host hindgut. The hindgut was observed with an ocular grid, and abundance was expressed as the ratio of grids occupied by hyphae to the number of grids occupied by hindgut.

Zygospores of selected Trichomycetes in larval Diptera of the families Chironomidae and Simuliidae.

The midgut-inhabiting fungi (Harpellaceae) Harpella melusinae and Stachylina pedifer were induced to form zygospores, using an application of a pH 10 potassium hydroxide solution with culture media. The previously unknown zygospores of S. pedifer are borne perpendicular to the zygosporophore, as in Harpella melusinae. The zygospores of the hindgut-inhabiting species Smittium coloradense, borne obliquely to the zygosporophore (in vivo), are described for the first time.

Seasonality of trichomycetes in larval black flies from South Carolina, USA.

Trichomycete fungi are common endobionts of aquatic insect larvae, but little is known of their ecology. In this study, the seasonality of trichomycete colonization of larval black flies (Diptera: Simuliidae) was investigated in three streams in northwestern South Carolina. At least eight species of trichomycetes were found in two species of black flies, and 93.8% of 1819 larval black flies examined contained trichomycetes. Significant differences were found in the seasonal prevalence of Harpella melusinae, Simuliomyces microsporus, and Paramoebidium spp. at one of three sites. At this site, the lowest mean prevalence for H. melusinae occurred in winter (67%) versus the other seasons (96-100%), whereas mean prevalence was lowest in summer for Simuliomyces microsporus (1%) versus the other seasons (2-21%) and lowest in summer for P. spp. (9%) versus the other seasons (45-67%). Significant differences in levels of colonization among seasons were not detected. Conjugations of H. melusinae (representing early stages of sexual reproduction) occurred most frequently in the spring and winter (up to 14% of larvae). Sexual reproduction (represented by zygospores) of Legeriomycetaceae occurred most frequently in the spring and fall (up to 17% of larvae).