Article pubs.acs.org/JPCB
The Role of Surface Viscosity in the Escape Mechanism of the Stenus Beetle Alexander A. Dietz, Matthias J. Hofmann, and Hubert Motschmann* Institute of Physical and Theoretical Chemistry, University of Regensburg, D-93053 Regensburg, Germany ABSTRACT: Beetles of the species Stenus comma live and hunt close to ponds and rivers, where they occasionally fall on the water surface. To escape this jeopardized state, the beetle developed a strategy relying on the excretion of a secretion containing the substances stenusine and norstenusine. They reduce surface tension and propel the bug to the saving river bank. These substances were synthesized and analyzed with respect to their equilibrium and dynamic adsorption properties at the air−water interface (pH 7, 23 ± 1 °C). The surface dilatational rheological characteristics in a frequency range from 2 to 500 Hz at molar bulk concentrations of 20.6 mmol L−1 were studied using the oscillating bubble technique. Both alkaloids formed surface viscoelastic adsorption layers. The frequency dependence of the surface dilatational modulus E could successfully be described by the extended Lucassen−van den Tempel model accounting for a nonzero intrinsic surface viscosity κ. The findings confirmed a dual purpose of the spreading alkaloids in the escape mechanism of the Stenus beetle. Next to generating a surface pressure, a transition to surface viscoelastic behavior of the adsorbed layers was observed.
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function in the first place; instead, they evolved for defensive reasons. Especially its uncovered abdomen is vulnerable to microorganisms. Hence, to protect itself from infestation, the beetle distributes the antimicrobial pygidial secrete over its entire body surface.3 Water walking arthropods usually exhibit a higher density than water. Nevertheless they do not sink. Therefore, buoyancy forces must be supplemented by an additional contribution, namely curvature forces, which arise from the displacement of water volume by formation of a meniscus at the contact area between the liquid and the animal’s hydrophobic legs.6 Baudoin developed a parameter to estimate the physical boundaries for surface walking, referred to as Baudoin number Ba = mg/γP, which expresses the ratio between body weight mg and tensile force provided by the surface tension γ along the contact perimeter P.6,9 In order to approach their prey, water walking predators employ their legs to strike the surface. As the meniscus is usually maintained, the latter acts analogous to a paddle. Thereby the beetle experiences a drag proportional to the exposed leg area, enabling the animal’s net propulsion. Accordingly surface locomotion requires deformation of the air−water interface. In the aftermath of secretion liberation a monomolecular surfactant film is spread over the surface, which does not only reduce the equilibrium surface tension γ but may also affect the properties of the air−water interface under dynamic conditions.10 It is conceivable that the deposition of such a monolayer may influence the surface dilatational
INTRODUCTION Some species of the rove beetles utilize an exceptional manner of locomotion on water surfaces, referred to as “skimming”.1 Beetles of the genus Stenus comma inhabit banks of ponds and slow-flowing streams. While hunting for springtails, they occasionally fall onto the water surface.2 As terrestrial bugs they would usually be doomed in such situations due to their limited ability to walk on water surfaces. Consequently, they may drown or, in turn, would be an easy prey for other predators.3 Water striders for instance feed to a large extent from nonaquatic insects accidentally trapped at the water surface.4 In order to escape on such occasions, the Stenus beetle is pointing its abdomen tip toward the water surface and releases a multicomponent secretion, which propels the animal to the safety of the shore.2 Thereby the beetle can reach maximum velocities of 0.4−0.75 m s−1 and distances up to 15 m.5 The underlying physicochemical process is called Marangoni propulsion.6 By excretion of surface active substances a gradient in surface tension γ along the contact line between the animal body and the fluid is established, providing a force pushing the beetle forward. The active ingredients in the Stenus comma secretion are two piperidine alkaloids. Schildknecht et al.7 found the mixture’s main component to be N-ethyl-3-(2-methylbutyl)piperidine, also referred to as stenusine. The structurally similar molecule norstenusine, differing by one fewer methylene group, was detected later in smaller relative amounts.8 Further constituents such as 1,8-cineole appear only in lower quantities and are therefore regarded to be of minor importance for the skimming activity but serve other purposes.2 Presumably the generation of locomotion was not the pygidial glands’ excretions main © XXXX American Chemical Society
Received: May 13, 2016 Revised: June 24, 2016
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DOI: 10.1021/acs.jpcb.6b04871 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry B properties of the liquid. A viscoelastic behavior of the adsorbed layer would imply that for each propulsion stroke an additional amount of energy is required due to dissipative losses within the adsorption layer. Thus, it would be tremendously more difficult for predators to pursue the beetle upon skimming. To clarify the existence of this effect, the surface dilatational properties of interfacial layers formed from the spreading alkaloids stenusine and norstenusine were measured by means of the oscillating bubble technique in this study. Our instrumental design gave access to a frequency range from 2 to 500 Hz, which allows capturing the rapid surface deformation of walking striders.
Table 1. Best Values of Parameters for Matching the Model Described in Eq 2 to Experimental Data Obtained from the Oscillating Bubble Device
THEORY The surface dilatational modulus E describes the ability of a system to restore its surface tension in an instant of stress. When applying periodic expansion−compression cycles to an interface, the associated dilatational viscoelasticity of the adsorption layer is characterized by the surface dilatational modulus E. It is defined as the change in surface tension γ upon a relative change in surface area A and given by
1 + ζ + iζ + i 2πfκ 1 + 2ζ + 2ζ 2
κ [N s m−1]
stenusine norstenusine
20.6 20.6
54.8 53.0
1.245 1.051
1.305 × 10−6 1.919 × 10−6
Both oily compounds were diluted to stock solutions with a concentration of 20.6 mmol L−1, which is marginally lower than the visually observable solubility limit. An aqueous phosphate buffer, containing KH2PO4 (26 mmol L−1) and Na2HPO4 (41 mmol L−1), was used to constantly maintain the pH at 7. Since alkali phosphates act as bulk electrolytes, no noteworthy difference in the equilibrium surface tension of the buffer solutions compared to pure deionized water was observed. Solutions of lower alkaloid concentrations were produced by successive dilution of the purified stock solutions with untreated buffer solution by a dilution factor of 2. Water used in the course of the experiments was taken from a Millipore purification system (>18 MΩcm) and the glassware was cleaned by soaking in KOH and subsequently HCl for at least 24 h followed by exhaustive rinsing with deionized water. Prior to oscillating bubble experiments, the aqueous solutions were degassed by ultrasoniaction for 10 min. Stock Solution Purification. To remove residual trace impurities originating from the synthesis, both stock solutions were brought to a surface chemically pure state using a protocol and an apparatus developed by Lunkenheimer and coworkers.14 The applied purification scheme ensures a complete removal of any surface active impurities by repeated cycles of (a) compression of the surface layer, (b) its removal with the aid of a capillary, and (c) dilation to an increased surface area. These cycles are repeated until the equilibrium surface tension between subsequent cycles remains constant. Solutions at lower concentrations were prepared as described above. Surface Tension. Equilibrium surface tension isotherms were obtained by a profile analysis tensiometer (PAT-1M, SINTERFACE Technologies, Berlin) using the pendant drop configuration. The shape of a drop is determined by the interplay between gravity and surface tension. An analysis of the drop shape according to the Gauss−Laplace equation yields the surface tension. All measurements were carried out in a temperature-controlled room at 23 ± 1 °C. Drops were formed at the tip of a stainless steel capillary of 3 mm in diameter. Equilibrium surface tension values were determined by
(2)
fdiff 2f
fdiff [Hz]
(1)
for the surface dilatational modulus E, whereas ζ is related to both the characteristic diffusion frequency fdiff and the applied perturbation frequency f and defined as ζ=
ϵm [mN m−1]
Figure 1. Molecular structures of the spreading alkaloids (a) stenusine and (b) norstenusine in their neutral forms.
The complex nature of the modulus manifests as a phase shift between the induced change in surface area and the measured variation in surface tension. Assuming a purely diffusional relaxation mechanism between the surfactant adsorption layer and the adjacent bulk phase, the surface dilatational modulus E is given by an expression derived by Lucassen and van den Tempel.11 Therein, the high frequency limiting elasticity ϵm and characteristic diffusion frequency fdiff are used as tunable parameters. In an extension of the model, the intrinsic surface viscosity κ leads to a significant modification at higher frequencies and captures dissipative losses within the surface region.12 A combination of the latter two contributions leads to the frequency-dependent expression E(f ) = ϵm
c [mmol L−1]
hyde under acidic conditions and 20 bar of hydrogen pressure. Distillation yielded stenusine and norstenusine, as verified by means of NMR spectroscopy (1H and 13C). The corresponding molecular structures of both compounds are shown in Figure 1.
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dγ E= d ln A
solution
(3)
In the latter expression, i represents the imaginary unit with i2 = −1. The experimental oscillating bubble data, i.e., amplitude and phase angle values, were simultaneously fitted to eq 2 using the three model characteristics high frequency limiting elasticity ϵm, characteristic diffusion frequency fdiff, and intrinsic surface viscosity κ as fitting parameters. Best agreement with experimental data is achieved using the parameter given in Table 1.
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EXPERIMENTAL SECTION Materials. The spreading alkaloids stenusine and norstenusine were synthesized in a two-step procedure starting from 3picoline.13 After alkylation with 2-bromobutane and 2bromopropane, respectively, the intermediate products were N-ethylated and hydrogenated simultaneously with acetaldeB
DOI: 10.1021/acs.jpcb.6b04871 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry B averaging over a period of 100 s after allowing the drop to age for 200 s. Oscillating Bubble. Surface dilatational properties of adsorption layers are determined by the oscillating bubble technique in a frequency range from 2 to 500 Hz. An air bubble is formed at the tip of a capillary, which is immersed in the sample solution. The bubble is monitored by a CCD camera and forced to sinusoidal oscillations by a piezo driver. The system response is detected by a pressure sensor and evaluated by a lock-in scheme. A cross-sectional view of the apparatus is given in Figure 2.15,16
Figure 3. Experimental equilibrium adsorption isotherms of stenusine, norstenusine, and the respective best fits (dotted lines) according to the Frumkin adsorption isotherm. Published literature data extracted from dynamic surface tension measurements for comparison.13
in particular considering the pKa, a comparison of the latter with our data (cf. Figure 3) is justified due to a similar protonation state. However, some discrepancies to our measurements for both compounds were observed. That is a significantly lower surface tension, especially for the less concentrated alkaloid solutions. We ascribe these differences to the previous paper to trace impurities originating from the synthesis, which come more into effect for lower bulk concentrations, due to higher surface activity of the contaminants. By applying the previously described purification procedure, surface tension values are typically shifted upward with increasing number of cycles until surface chemical purity is accomplished.18 Oscillating Bubble. The frequency-dependent values of the amplitude and phase shift of the surface dilatational modulus E for aqueous solutions of stenusine and norstenusine at concentrations of 20.6 mmol L−1 are shown in Figures 4 and 5, respectively. For comparison, the theoretical curves of a surface elastic reference system are shown as dotted lines. In this case, the amplitude approaches a saturation value and the phase angle approaches zero. For the studied solutions of stenusine and norstenusine, the amplitude of the modulus is characterized by a moderate increase at frequencies above 100 Hz and a rising phase angle between the induced surface area perturbation and the measured pressure response in the same frequency range. This behavior is referred to as surface viscoelastic. The experimental data can be appropriately fitted to the extended Lucassen−van den Tempel theory (eq 2) by tuning the parameters to simultaneously match the amplitude and phase angle to the latter equation. The best values of the model parameters are summarized in Table 1, and the corresponding theoretical curves of magnitude and phase angle are shown in Figures 4 and 5. The values of the high frequency limiting elasticity ϵm agree within experimental error for the studied solutions, even though the frequency-dependent values of the amplitude are not indicative for this behavior. However, the different best fit parameters for the characteristic diffusion frequency fdiff can be rationalized in terms of the polarity of the alkaloids. A higher degree of hydrophobicity due to the additional methylene group in stenusine as compared to norstenusine manifests as an increased characteristic diffusion frequency fdiff. The widely differing values of intrinsic surface viscosity κ are hard to be interpreted in terms of molecular structure, as the increased
Figure 2. Schematic representation of the oscillating bubble device (not to scale). The surface area of an air bubble formed at the tip of a capillary is perturbed in a sinusoidal fashion in a frequency range from 2 to 500 Hz, and the change in pressure across the curved interface is measured by a sensitive transducer. Reprinted with permission from ref 16.
For each studied solution, three independent measurements were carried out; i.e., the sample chamber was opened and refilled with the solution to be studied. The frequencydependent results shown in Figures 4 and 5 represent the averaged values obtained from these measurements. Within one measurement, the automated frequency scanning mode was used. This protocol provides for measuring the amplitude and phase angle of the pressure response at a certain frequency before changing the perturbation frequency. Between changes of the frequency, the bubble size was corrected to half-sphere geometry by an implemented control loop. All measurements were carried out at room temperature at a fixed relative deformation amplitude of 5%.
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RESULTS AND DISCUSSION Surface Tension. Figure 3 displays the equilibrium adsorption isotherms of stenusine and norstenusine. Starting from low alkaloid concentrations, a considerable reduction of the equilibrium surface tension of water sets in around 1 mmol L−1 for stenusine and at about the 5-fold molar concentration for norstenusine. Increasing the concentrations further induces a dropoff in γ to values of 58 or 64 mN m−1 for the respective stock solutions (20.6 mmol L−1). This behavior seems reasonable considering that norstenusine bears one fewer methylene group, thus resulting in a lower hydrophobicity of norstenusine in comparison to stenusine. A Frumkin type adsorption isotherm was chosen in order to fit the experimental data. For compounds of the N-alkylpiperidine type pKa values around 10 are reported.17 Therefore, one can assume quantitative protonation of the alkaloids in the present case of pH 7. Gedig et al. performed similar experiments in buffer solutions of pH 6.13 Since the difference in pH is rather small, C
DOI: 10.1021/acs.jpcb.6b04871 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry B
norstenusine are characterized by a nonzero value of the intrinsic surface viscosity κ; i.e., the expansion−compression deformation type is associated with a loss in energy, which makes it increasingly difficult for other water walking arthropods to follow the Stenus beetle. To conclude, the spreading alkaloids stenusine and norstenusine excreted by the Stenus beetle in the course of escaping from a water surface have a twofold purpose in terms of their physicochemical properties at concentrations close to the solubility limit at room temperature: inducing a surface pressure driving the beetle to the water’s edge and rendering the adsorption layer surface viscoelastic, which additionally favors the beetle’s escape. Semiaquatic bugs are supported by the surface tension. Their weight distorts the surface thereby forming characteristic dimples at the legs. Water striders propel themselves by driving their central pair of legs in a sculling motion; spiders take advantage of the galloping technique. The locomotion of water walking arthropods leads to deformations of the water surface and the formation of capillary waves. The decisive frequency range for our studies is the range of 50−500 Hz matching the characteristic time of the surface deformations caused by the legs of water striders and spiders.19 The use of the oscillating bubble technique enabled to study the highfrequency dilatational rheology of adsorption layers at the air− water interface up to 500 Hz.
Figure 4. Amplitude of the frequency-dependent surface dilatational modulus E of stenusine and norstenusine and the respective best fits (colored dotted lines) compared to an elastic reference substance.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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Figure 5. Frequency-dependent phase angle ϕ of stenusine and norstenusine and the respective best fits (colored dotted lines) compared to an elastic reference substance.
REFERENCES
(1) Jenkins, M. F. On the Method by Which Stenus And Dianous (Coleoptera: Staphylinidae) Return to the Banks of a Pool. Trans. Entomol. Soc. London 1960, 112, 1−14. (2) Schildknecht, H.; Krauss, D.; Connert, J.; Essenbreis, H.; Orfanides, N. Das Spreitungsalkaloid Stenusin aus dem Kurzflügler Stenus comma (Coleoptera: Staphylinidae). Angew. Chem. 1975, 87, 421−422. (3) Lang, C.; Seifert, K.; Dettner, K. Skimming Behaviour and Spreading Potential of Stenus Species and Dianous Coerulescens (Coleoptera: Staphylinidae). Naturwissenschaften 2012, 99, 937−947. (4) Møller Andersen, N. The Semiaquatic Bugs (Hemiptera, Gerromorpha): Phylogeny, Adaptations, Biogeography and Classification; Entomonograph; Scandinavian Science Press: Klampenborg, 1982; Vol. 3. (5) Linsenmair, K. E.; Jander, R. Das Entspannungsschwimmen von Velia und Stenus. Naturwissenschaften 1963, 50, 231. (6) Bush, J. W. M.; Hu, D. L. Walking on Water: Biolocomotion at the Interface. Annu. Rev. Fluid Mech. 2006, 38, 339−369. (7) Schildknecht, H.; Berger, D.; Krauss, D.; Connert, J.; Gehlhaus, J.; Essenbreis, H. Defense Chemistry of Stenus comma (Coleoptera: Staphylinidae). J. Chem. Ecol. 1976, 2, 1−11. (8) Kohler, P. Die absolute Konfiguration des Stenusins und die Aufklärung weiterer Inhaltsstoffe des Spreitungsschwimmers Stenus comma, 1979. (9) Baudoin, R. P. A. La Physico-chimie des surfaces dans la vie des Arthropodes aériens, des miroirs d’eau des rivages marins et lacustres de la zone intercotidale, 1955. (10) Scriven, L. E.; Sternling, C. V. The Marangoni Effects. Nature 1960, 187, 186−188. (11) Lucassen, J.; van den Tempel, M. Dynamic Measurements of Dilational Properties of a Liquid Interface. Chem. Eng. Sci. 1972, 27, 1283−1291.
energetic loss upon dilatational deformation at higher frequencies is apparently not associated with a larger number of degrees of conformational freedom in norstenusine.
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SUMMARY The natural spreading alkaloids stenusine and norstenusine occurring in the Stenus beetle were synthesized in a two-step reaction and purified to reach the state of surface chemical purity. For both molecules, the induced surface pressure is rather low as compared to classical long-chain surfactants at comparable concentrations. This moderate reduction of surface tension of pure water is sufficient, however, to induce a surface pressure driving the beetle toward the water’s edge, but at the same time sufficiently low to avoid a drowning of the beetle. The Baudoin number is especially important for skimming beetles since reduction of γ below a critical value would cause the animal to sink immediately. The spreading alkaloids modify the dynamics of the respective adsorption layers as evidenced by the oscillating bubble device as well. In both cases, a shift toward surface viscoelastic behavior characterized by an increasing value of the amplitude of the surface dilatational modulus E and a nonzero phase angle ϕ at perturbation frequencies above 100 Hz was observed. The experimental values of amplitude and phase angle could be successfully modeled by the extended Lucassen−van den Tempel theory giving an expression for the frequency-dependent surface dilatational modulus. Adsorption layers of stenusine and D
DOI: 10.1021/acs.jpcb.6b04871 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.jpcb.6b04871 J. Phys. Chem. B XXXX, XXX, XXX−XXX
