Porous substrate affects a subsequent attachment ability of the beetle Harmonia axyridis (Coleoptera, Coccinellidae)

Abstract

According to literature data, porous substrates can cause a reduction of insect attachment ability. We carried out traction experiments with adult ladybird beetles Harmonia axyridis on the smooth solid glass sample and rough porous Al₂O₃ membrane to prove the primary effect of absorption of the insect pad secretion by porous media, rather than surface roughness, on the attachment force on the porous sample. With each insect individual, a set of five experiments was conducted: (1) on glass; (2) on the porous membrane; (3–5) on glass immediately after the test on the porous surface, then after 30 min and 1 h of recovery time. On the porous substrate, the forces, being similar in females and males, were greatly reduced compared to those measured on glass. A significant difference between the force values obtained in the first (before the test on the porous sample) and second (immediately after the experiment on the porous sample) tests on glass was observed. After 30 min recovery time, beetles completely regained their attachment ability. Females produced significantly lower forces than males in all experiments on glass: the differences are probably caused by the sexual dimorphism in the microstructure of their adhesive pads. The obtained results are of fundamental importance for further application in biomimetics of novel insect-repelling surfaces and in plant protection by using porous materials.

1. Introduction

According to the recent literature, insects equipped with hairy attachment devices (adhesive pads) are able to establish a highly reliable contact and attach successfully to a substrate [1–3]. However, plant surfaces bearing three-dimensional epicuticular wax coverage usually minimize insect attachment (see review [4]). Such anti-adhesive properties of waxy plant surfaces were explained by the effects of the microroughness created by microscopic wax projections covering the plant cuticle (roughness hypothesis), contamination of insect adhesive pads by wax material detached from the waxy plant surface during the contact (contamination hypothesis), absorbing of the fluid from the pad surface due to the high capillarity of the wax coverage (fluid absorption hypothesis) and hydroplaning caused by the plant wax dissolving in the insect pad secretion (wax dissolving hypothesis) [5]. Also, it was recently suggested that detached wax particles can serve as a separation layer between insect attachment organs and plant surfaces (separation layer hypothesis) [6].

To date, only the first two hypotheses have been experimentally tested and supported. The impact of the surface microroughness on insect attachment has been proven in several studies using mostly artificial substrates with different roughness parameters [1,7–12]. These experiments have demonstrated much higher attachment forces on either smooth or relatively coarse microrough surfaces (with an asperity size exceeding 3.0 μm), whereas test insects completely failed on substrates with a roughness of 0.3 and 1.0 µm because of the strongly reduced real contact area there.

The contaminating ability of plant waxes has been verified for many plants [13–20]. It has been found that plants differ in their contaminating effects on insect pads and the degree of contamination is determined by the micromorphology (primarily size and shape) of wax projections [20].

As for the fluid absorption hypothesis, very recently, the fluid absorption capability of the plant wax coverage in Nepenthes alata has been revealed for oil [21] that represents an important, in beetles possibly the main [22–26], component of the pad secretion. Previous experiments have demonstrated heavily reduced insect attachment caused by treatment of pads with lipid solvent in the case of the assassin bug Rhodnius prolixus or after walking on silica gel in the case of the aphid Aphis fabae [27,28]. Also smooth polyimide substrates that selectively absorbed the watery component of the pad fluid significantly lessened the attachment forces in the stick insect Carausius morosus [29].

Our previous study was performed with three porous substrates (anodisc membranes) having the same pore diameter (ca 200 nm) but different porosity (28%, 42% and 51%), which are able to absorb both polar (water) and non-polar (oil) fluids [30]. On all these substrates, attachment forces of the ladybird beetle Coccinella septempunctata were highly reduced if compared to those on reference smooth solid substrates. The reduction in insect attachment was explained by (i) absorption of the fluid from insect adhesive pads by porous media and/or (ii) the effect of surface roughness. The present study was carried out in order to discriminate between these two effects by comparing the initial attachment ability of beetles with the subsequent one after they had walked on the porous substrate.

Here, we measured traction forces of the ladybird beetle Harmonia axyridis (Pallas) (Coleoptera, Coccinellidae) on a rough porous substrate and a reference smooth solid glass surface. The traction experiments were designed to test whether there is an effect of the porous substrate on the ability of insects to subsequently attach to the smooth surface or not. If there is such an effect, tarsal fluid absorption has a primary effect on the force reduction, whereas if not, it is the surface roughness that matters. Contamination of pads was excluded, as the porous substrate was synthesized from very stiff material by etching and not by particle sintering. We also tested differences in performance between female and male beetles.

2. Material and methods

2.1. Insects

The harlequin, multicoloured Asian ladybird beetle H. axyridis was used as a model insect species because of its appropriate size (body length: 6–9 mm) and because it occurred in great numbers at the study site. This coccinellid originated from eastern Asia, but has been introduced into North America and Europe for biological control of aphids and scale insects [31]. Now, it is common and spreading in these regions.

Adult beetles were collected near Niendorf (surroundings of Timmendorfer Strand, SH, Germany) and kept in small ventilated cages (15 × 8 × 10 cm³) at a temperature of 10°C and 40% relative humidity for three weeks. Insects were fed with aphids collected from different plants, e.g. apple trees and roses. Water in the cages was changed every second day and the cages were sprayed with water daily. Before starting the experiments, the cages were transferred to the room conditions (temperature of 24–26°C and 52–57% relative humidity) and kept there for ca 30 min until insects were acclimatized.

2.2. Substrates

As a porous substrate, we selected a disc-shaped Al₂O₃ membrane (Anodiscs, Whatman, Schleicher and Schuell, Whatman International Ltd, Maidstone, UK) having columnar pores of 200–250 nm in diameter and 51% porosity [30]. The thickness of the walls separating the pores equals 74.87 ± 52.15 nm (n = 94) [30]. A hydrophilic soda-lime glass plate was used as a reference surface.

2.3. Microscopy

Morphology of the attachment devices in female and male beetles was studied using scanning electron microscopy (SEM). Insects were air-dried, mounted dorsally on holders, sputter-coated with gold-palladium (6 nm) and examined in a SEM Hitachi S-4800 (Hitachi High-Technologies Corporation, Tokyo, Japan) at 3 kV accelerating voltage.

2.4. Traction force tests

The traction experiments with tethered walking beetles were performed using a load cell force transducer (10 g capacity, Biopac Systems Ltd, Santa Barbara, CA, USA). Beetles were narcotized with CO₂ for 30–40 s in order to glue together their elytra and to attach a human hair (length: 15–20 cm) to the insect with a small droplet of a molten bee wax. After recovery for ca 15 min, the beetles were used in the experiments. The experimental set-up is described in detail by Gorb et al. [30]. With each insect individual, a set of five measurements was carried out: (1) on the glass plate (glass1), where individual beetles showed their maximum attachment performance; (2) on the porous disc (porous); (3–5) on glass immediately after the test on the porous surface (glass2), after 30 min (glass3) and 1 h recovery time (glass4). The force produced by the insect moving on the horizontal test substrate was recorded. Since the beetles were constrained to pulling parallel (not at an angle) to the measurement axis of the transducer, the registered force corresponded to the total traction force. Obtained force–time curves of the beetles stretching the hair for 10–30 s were used to calculate the maximal traction force (electronic supplementary material, figure S1). The tests were performed at a temperature of 24–26°C and 52–57% relative humidity. Twenty females and 20 males were tested. In all, 200 force measurements were conducted.

3. Results

3.1. Morphology of the attachment system in Harmonia axyridis beetles

The tarsus of H. axyridis bears two ventrally curved claws and adhesive pads on the first and second proximal tarsomeres (figure 1a–d). The adhesive pads are of the hairy type [1]: the ventral surface of the tarsomeres is covered by microscopic tenent setae (figure 1). There are several types of setae: (1) with a pointed sharp tip (figure 1m); (2) with a flattened and widened endplate called the spatula (figure 1o); (3) transitional type, often with a pointed tip and relatively narrow elongated endplate (figure 1n) and (4) mushroom-like setae with a flat discoid terminal element (figure 1p). The structure of adhesive pads shows a distinct sexual dimorphism: only males have mushroom-like setae.

Figure 1.

SEM micrographs of attachment devices in the beetle Harmonia axyridis. (a) Tarsus of the female foreleg. (b–d) Tarsi of fore- (b), mid- (c) and hindlegs (d) in a male. (e,f) The second (e) and first proximal (f) tarsomeres in a female. (g,i,k) The second proximal tarsomere of fore- (g), mid- (i) and hindlegs (k) in males. (h,j,l) The first proximal tarsomere of fore- (h), mid- (j) and hindlegs (l) in males. (m–p) Different types of tenent setae. Arrows in (a)–(l) show the distal direction. CW, claw; T1, the first proximal tarsomere; T2, the second proximal tarsomere. Scale bars: 200 µm in (a–d); 100 µm in (e–l); 20 µm in (m); 10 µm in (n–p).

In females, the distribution of setal types (figure 1e,f) is rather similar in all legs. In males, mushroom-like setae occur on both tarsomeres of the fore- and midlegs (figure 1b,c,g–j), but they are absent on the hindlegs (figure 1d,k,l). These setae are situated in the central region of the first tarsomere (figure 1h,j) and more laterally in the second tarsomere (figure 1g,i).

3.2. Attachment ability of Harmonia axyridis beetles

The traction force of insects ranged from 0.37 to 12.44 mN in females and from 0.43 to 15.49 mN in males (figure 2a,b). The forces obtained in different experiments showed significant differences in both females and males (females: H = 57.088; males: H = 60.211; for both d.f. = 4, p < 0.001, Kruskal–Wallis one-way ANOVA on ranks). In both samplings (figure 2a,b, table 1), beetles produced the lowest force values on the porous substrate and the highest force in the first experiment on glass (p < 0.05, Tukey test). In other experiments on glass, the force increased gradually, but non-significantly (p > 0.05, Tukey test) in the following order: glass2 – glass3 – glass4. The force values obtained in the first, third and fourth experiments on glass were similar (p > 0.05, Tukey test).

Figure 2.

Traction forces of females (a), males (b) and females versus males (c) obtained in the experiments on the porous substrate (porous) and in the first, second, third and fourth experiments on the glass surface (glass1, glass2, glass3 and glass4, respectively). f, female; m, male. Medians with different letters in (a) and (b) differ significantly from one another (p < 0.05, Tukey test, see table 1). In (c), differences between sexes are indicated: NS, not significant (p = 0.25); *p < 0.05; ***p < 0.001 (t-test and Mann–Whitney rank sum test, see table 2).

Table 1.

Results of multiple pairwise comparisons (Tukey test: p, probability value; q, test statistics) of the force values obtained in the experiments on the porous substrate (porous) and in the first, second, third and fourth experiments on the glass surface (glass1, glass2, glass3 and glass4, respectively) with the beetle Harmonia axyridis.

experiment	glass1	porous	glass2	glass3
females
porous	q = 9.950 p < 0.05	—	—	—
glass2	q = 4.540 p < 0.05	q = 5.411 p < 0.05	—	—
glass3	q = 3.014 p > 0.05	q = 6.936 p < 0.05	q = 1.526 p > 0.05	—
glass4	q = 1.881 p > 0.05	q = 8.070 p < 0.05	q = 2.269 p > 0.05	q = 1.113 p > 0.05
males
porous	q = 10.197 p < 0.05	—	—	—
glass2	q = 4.933 p < 0.05	q = 5.264 p < 0.05	—	—
glass3	q = 3.021 p > 0.05	q = 7.176 p < 0.05	q = 1.191 p > 0.05	—
glass4	q = 2.004 p > 0.05	q = 8.193 p < 0.05	q = 2.929 p > 0.05	q = 1.017 p > 0.05

Comparison between females and males (figure 2c, table 2) showed that both sexes performed equally bad on the porous substrate (p < 0.05, Mann–Whitney rank sum test), whereas males produced significantly stronger forces than females in all experiments on glass (p > 0.05, t-test and Mann–Whitney rank sum test).

Table 2.

Results of the comparisons (t-test and Mann–Whitney rank sum test: p, probability value; t and T, test statistics, respectively) between the force values obtained with females versus males of the beetle Harmonia axyridis in the experiments on the porous substrate (porous) and in the first, second, third and fourth experiments on the glass surface (glass1, glass2, glass3 and glass4, respectively).

experiment	test statistics	p-value
glass1	t = −4.143	<0.001
porous	T = 450.000	0.250
glass2	T = 324.000	0.021
glass3	t = −3.658	<0.001
glass4	t = −4.129	<0.001

4. Discussion

Sexual dimorphism of adhesive pads in H. axyridis beetles (along with four other coccinellids studied) has been mentioned almost one decade before [32]. More recent study on the microstructure of attachment organs in this species, being very detailed and extensive, dealt with only male beetles, although did not indicate this fact [33]. Our results on the micromorphology of female and male adhesive pads are in line with the previously published data.

Previous traction experiments carried out with Coccinella septempunctata ladybird beetles have demonstrated a great force reduction on three porous anodisc membranes compared to both reference smooth solid samples (hydrophilic glass and Al₂O₃ (sapphire)) [30]. Also in our tests with H. axyridis beetles, the force on glass sample was nearly one order of magnitude higher than that on the porous substrate (comparison glass1 versus porous). The significant difference in the force values obtained in the first (before the test on the porous sample) and second (immediately after the test on the porous sample) tests on glass clearly indicated the primary effect of the sample's porosity, not of the surface roughness, on the force reduction on the porous sample. Here, the absorption of the insect pad secretion by the porous media led to a drastical decrease of the fluid thickness between the tips of insect pads and the substrate and resulted in adhesion force reduction (fluid absorption hypothesis according to [5]). Previously, we have demonstrated that nanoporous media strongly absorb oil-based fluids [21,30]. Using a cryo scanning electron microscopy approach and high-speed video recordings of the behaviour of fluid drops, followed by numerical modelling of experimental data, we showed that nanoporous materials, such as plant epicuticular wax coverages [21] and anodisc membranes [30], readily adsorb oil: strong changes in the contact angle, base, height, and volume of the oil drops within the first few seconds have been detected. The absorption phenomenon found in these previous studies explains the effect of the porous substrate on wet adhesive system of the beetle observed in the present study. However, after 30 min of recovery time, the attachment ability of H. axyridis was completely regained (comparison glass1 versus glass3).

Our data showing the reduction of the attachment force on the glass surface after the pad fluid was adsorbed while walking on the porous substrate are in line with results obtained in attachment experiments with Rhodnius prolixus, Aphis fabae and Carausius morosus [27–29], but contrast the findings of the previous authors showing a negative correlation between the adhesion force and the amount of fluid on the smooth surface for a single, cut-off pad of Carausius morosus [34]. In the latter study, the amount of the pad secretion was gradually depleted, whereas we confirm strong reduction of the amount of the fluid from the adhesive pads during our experiments on the porous sample.

Our experiments clearly showed that the sex of beetle individuals influenced the attachment force. In all tests on smooth glass, we obtained significantly higher forces with males compared to those with females. These results are congruent with data previously obtained for the beetles Leptinotarsa decemlineata [8], Gastrophysa viridula [10], Coccinella septempunctata [30], Chrysolina polita [35] and Ch. americana [36] on smooth surfaces. The difference in attachment ability between females and males could be explained by the need for males to hold during long-term copulation and mate guarding on the smooth elytra of females [8,10,35,36]. This difference presumably resulted from the sexual dimorphism in types and distribution of tenent setae: females possess spatula-like and lanceolate setae, whereas males additionally possess mushroom-like ones. Spatula-shaped and lanceolate setae can generate adhesion only if a shear force is applied [37], and shear movement can be generated only if a counterforce is applied in the opposite direction. The contact elements with mushroom-shaped geometry are independent of applied shear and, due to the geometry that maintain homogeneous stress distribution in contact, can remain passively adhered without the external support of muscular force. This is also the reason why this latter type of contact element geometry can also be used in synthetic patterned dry adhesives [37].

Interestingly, on the rough porous surface, similar force values were obtained for H. axyridis females and males. This result contradicts the previous data obtained for other beetle species on microrough substrates, where females performed better than males [8,10,30], however, types of substrates and conditions differ between the present and previous studies. The fact that we observed no difference between sexes on the porous surface can be also an indication that substrate porosity causing pad fluid absorption led to the attachment force reduction rather than surface microroughness, which usually results in higher attachment forces in females compared to males.

As the obtained results uncover the mechanism of the insect attachment reduction on porous substrates and can be generalized to artificial and natural nano- and microporous substrates and insects with oil-based pad fluids (e.g. beetles), they are of fundamental importance for further application in biomimetics for technological development of novel insect-repelling surfaces and anti-adhesive surfaces in general. As some plant surfaces demonstrate nanoscale porosity due to the presence of 3D waxes, the results might be also used in plant protection either by applying porous materials or by breeding plants with specific 3D coverages.

Ethics

The work was undertaken with ethical approval of the Kiel University, Germany.

Data accessibility

Data supporting this article are available in electronic supplementary material, table S1.

Authors' contributions

S.N.G. and E.V.G. conceived the work, analysed the data and prepared figures. S.N.G. performed the microscopy study; W.L. collected experimental data. E.V.G. wrote the draft manuscript; all authors approved the final version.

Competing interests

We declare we have no competing interests.

Funding

This study was partly funded by the Human Frontier Science Program (research grant ‘A dung beetle's life: how miniature creatures perform extraordinary feats with limited resources’ to S.N.G.).