Snustorr Defines Cuticle Elasticity in Drosophila melanogaster

TL;DR

This study shows that the ABC transporter Snustorr is essential for lipid incorporation into the cuticle, supporting a decades-old hypothesis about how insect cuticles harden.

Contribution

The study provides molecular evidence that Snustorr is required for lipid incorporation into the cuticle, confirming a historical hypothesis.

Findings

Larvae lacking Snustorr have a 10-fold reduction in cuticle hardness compared to wild-type larvae.

Snustorr is necessary for the incorporation of lipids into the cuticulin layer of the cuticle.

The findings support Sir Vincent Wigglesworth's hypothesis that lipids contribute to insect cuticle hardening.

Abstract

Sir Vincent Wigglesworth proposed in his seminal work on insect cuticle lipids that lipids are involved in cuticle hardening. In brief, in histological experiments, he demonstrated the presence of lipids as a component of cuticulin, a “hard amber material of the insect cuticle.” The molecular evidence for this postulate awaits demonstration. In the present work, we examined the cuticle of larvae lacking the function of the cuticle lipid transporter Snustorr (Snu) by nanoindentation with atomic force microscopy in order to analyze the role of lipids on cuticle hardness in the fruit fly Drosophila melnoagaster. Compared to wild‐type larvae, we found that the Young's modulus is 10 times reduced in snu mutant larvae that lack cuticulin at their surface. Thus, this simple result supports the finding of Sir Vincent Wigglesworth that lipids contribute to cuticle hardness in insects.

Based on histological experiments, Sir V. Wigglesworth proposed that lipids may interact with catecholamines and proteins to contribute to insect cuticle hardening. In this work, we show that the ABC transporter Snu is involved in the incorporation of lipids for this process.

Based on histological experiments, Sir V. Wigglesworth proposed that lipids may interact with catecholamines and proteins to contribute to insect cuticle hardening. In this work, we show that the ABC transporter Snu is involved in the incorporation of lipids for this process.

Sir Wigglesworth postulated that insect cuticle hardening depended on the incorporation of lipids into the upper parts of the cuticle. We show that the delivery of these lipids needed for cuticle hardening requires the ABC transporter Snu in the fruit fly Drosophila melanogaster larva.

Figures

Mechanical stress ruptures the cuticle of snu mutant larvae. As seen on electron‐micrographs, at the muscle attachment site in the ready to hatch wild‐type larvae, the cuticle (epi, epicuticle; pro, procuticle) follows the pull of the muscles (left image). At the muscle attachments site in snuDf(98E2) mutant larvae the procuticle (pro) detaches from the epicuticle (right image). The adhesion of the epidermal surface (dotted line) and the procuticle is, by contrast, stable. The scale bar in the left micrograph represents 500 nm and applies also to the micrograph on the right.

The Young's modulus of wild‐type and snuDf(98E2) mutant larval cuticle measured using AFM nanoindentation. Box endings correspond to the 25th and 75th percentiles; the line within shows the median; error bars define the 10th and 90th percentiles; outliers are marked by circles. The values of cuticle Young's modulus for the two embyo types are statistically significantly different (Mann−Whitney U test, p < 0.005). The indented regions in the abdominal of the larvae are marked on cryo‐SEM images by yellow triangles.

Full text

Introduction

1

The insect cuticle is a trade‐off physical structure serving as a barrier against free inward and outward water flux at the same time as an exoskeleton needed for body shape and locomotion (Moussian 2013; Neville 1975; Hepburn and Chandler 1976). The physical properties of the cuticle are set the way to accommodate these requirements, and they vary between species, lifecycle stages and body parts (Vincent and Wegst 2004). Hard regions, sclerites, are connected to each other by flexible regions called arthrodial membrane to ensure movability of the body (Wigglesworth 1957).

Commonly, the stereotype of the cuticle consists of three composite horizontal layers (Moussian 2010; Locke 2001). The outermost layer is called the envelope that contains proteins and lipids. Underneath lies the bipartirte epicuticle with an electron‐lucid apical half and an electron dense basal half. This layer is formed proteins such as the Tweedle proteins (Zuber et al. 2020). The innermost procuticle is a stack of helicoidally arranged sheets of parallel running chitin microfibrils (lamina) (Moussian 2010).

Cuticle hardening (sclerotization) occurs mainly in the upper half of the procuticle and involves especially dopamine and derivatives. The underlying biochemical reactions are very well studied. Dopamine is produced in the cytoplasm by the tyrosine hydroxylase (TH) and dopa‐decaboxylase (Ddc); Dopamine is modified to N‐acetyldopamine (NADA) or N‐βalanyldopamine (NBAD) that are subsequently secreted to the extracellular space, where they may be further modified and covalently bind to specific proteins thereby reducing the water content of the cuticle and promoting its hardening (Vincent and Wegst 2004; Andersen 2010; Noh et al. 2016). Besides this canonical pathway, Sir Wigglesworth proposed the involvement of lipids in cuticle hardeining (Wigglesworth 1990). According to him, the cuticle surface molecule cuticulin consists of sclerotin, that is composed of proteins linked to polyphenols, and lipids. There is simple evidence that the dopamine‐ and the lipid‐based hardening pathways are acting independently. Indeed, they do not seem to occur at the same site within the cuticle. Consistently, envelope and epicuticle formation is not affected in the larval cuticle of the fruit fly Drosophila melanogaster that are compromised in dopamine production (Moussian et al. 2006).

The histological characteriation of cuticulin is not paralleled by its molecular identification. Especially, the impact of lipids in this context remains enigmatic. Lipid transport to the cuticle depends on the function of two ATP‐binding cassette (ABC) type H transporters that are specific to arthropods (Popovic et al. 2010). In D. melanogaster, two of these ATP transporters, named Oskkydad (Osy) and Snustorr (Snu), are responsible for the deposition of the cuticular hydrocarbons (CHC) that prevent water‐loss and xenobiotics penetration in larvae and adults (Zuber et al. 2018; Wang et al. 2020). Snu function is also needed for complete envelope formation and the correct localization of the extracellular protein Snustorr‐snarlik (Snsl) that contributes to the barrier function of the cuticle (Zuber et al. 2018). Similarly, in the migratory locust (Locusta migratoria), the Snu orthologue LmABCH‐9C delivers lipids to the cuticle surface thereby protecting the animal against dehydration (Yu et al. 2017). Likewise, the function of Snsl is exerted by its orthologue in L. migratoria (Liu et al. 2025), suggesting an evolutionary conserved mechanism in insect cuticle barrier formation. The contribution of these proteins to cuticle sclerotization has not been reported, yet. Hypothetically, both Snu and Snsl may contribute to cuticulin formation and/or function.

In the present work, we addressed the question whether Snu might be implicated in cuticle hardening or sclerotization. To this end, we measured their cuticle elasticity by atomic force microscopy. In brief, our results indicate that Snu contributes to the correct organization of the apical portions of the cuticle and to cuticle elasticity.

Materials and Methods

2

Fly Husbandary

2.1

For egg collection, fly populations were kept in cages on apple juice agar plates garnished with fresh baker's yeast. Embryos were staged according to the time point after egg laying and gut morphology (Moussian et al. 2006). Over 22 h post egg laying, highly meandering gut tubes marked the end of cuticle formation; movements of the ready to hatch larvae indicated that the embryos/larvae were not dead when measurments were done. Eggs were dechorionated for 3 min in commercially available chlorine bleach diluted 1:1 in undestilled water. Homozygous mutant embryos were manually collected based on the presence (heterozygous sibling) or absence (mutant) of a balancer chromosome expressing GFP (Dfd > YFP).

Nanoindentation With Atomic Force Microscopy

2.2

Mature wild‐type and snu ^ Df(98E2) ^ mature embryos (larvae) were manually freed from the egg case in PBS, mounted in cyanoacrylate glue with the lateral surface up and immediately covered by PBS. A NanoWizard scanning probe microscope (JPK Instruments, Berlin, Germany), mounted on an inverted light microscope (Zeiss Axiovert 135, Carl Zeiss MicroImaging GmbH, Göttingen, Germany) operating in phase contrast mode was used for imaging the embryos prior to indentation to select the topmost position on the lateral surface for nanoindentation. NanoWizard SPM software 4.0.31 (JPK Instruments, Berlin, Germany) was used to obtain the force distance curves. To prevent desiccation during the experiment the measurements were performed in PBS.

Indentation depth was set to 50 nm to exclude influence of the underlying tissues on the Young's modulus measurements. The spring constant of the cantilever (1.0 N ∙ m^–1^) and the tip radius (10 nm) were ascertained before the experiments using the thermal noise method and calibration grids, respectively. The cantilever cone half‐angle was 12° (according to NCH, NanoSensors, Neuchatel, Switzerland). Force—distance curves were recorded for 100 sites distributed over a 10 × 10 µm grid (distance between sites was approximately 1 µm). The curves demonstrated irregularities were excluded from the following analysis.

Overall, indentation on 10 wild‐type and 10 snu ^ Df(98E2) ^ embryos was performed. Scanning probe data processing software was applied to fit the data (SPIP ver. 5.1.2, Image Metrology A/S, Hørsholm, Denmark). The retraction part of the force—distance curves were fitted with Sneddon modelfor estimation of the Young's modulus, E, from the following equation (Snaddon 1965):

$$[eqn]$$

where F is the force, ν is the assumed Poisson ratio (set to 0.5 here), θ is the cantilever cone half‐angle (12°), d is the indentation depth. Mann‐Whitney U test within SigmaStat (ver. 3.5, Systat Software Inc., San Jose, CA, USA) was used for evaluation of statistical differences between the two types of embryos.

Light and Electron Microscopy

2.3

For transmission electron microscopy (TEM), embryos and larvae were treated as described in our previous works (Moussian et al. 2005; Moussian and Schwarz 2010). A Philips CM‐10 was used at 60 kV for data recording.

Cryo scanning electron microscopy (cryo‐SEM) was performed according to Gorb (Gorb 2006). Shortly, the devitellinized embryos were mounted on aluminium stubs, cryo‐fixed in liquid nitrogen (−196°C), and immediately transferred to the microscope stage. Then the samples were sublimated and sputter coated with 6 nm Au‐Pd with a Bal‐tec SCD 500 Sputter Coater (Bal‐tec AG, Balzers, Liechtenstein). Cryo‐samples were observed using Hitachi S‐4800 (Hitachi High‐Technologies Corp., Japan) at 2 kV accelerating voltage.

Results and Discussion

3

In a previous work, we had shown that at muscle attachment sites, the epidermis with the procuticle detached from the above epicuticle in snu mutant larvae (Figure 1) (Zuber et al. 2018). This distinct phenotype indicates that the snu mutant cuticle is unable to withstand mechanical stress occurring when muscles pull on the cuticle. This defect is quite different from the one observed in larvae mutant for genes coding for proteins either acting on chitin organization such as obstructor‐A (obstA) and retroactive (rtv) or constituting the cuticle‐membrane anchor such as piopio (pio) (Göpfert et al. 2025). In contrast to the situation in snu mutant larvae, in these cases, the procuticle detaches from the apical surface of the epidermal cells because of the dysfunctional molecular anchor. Based on this particular phenotype, we hypothesized that Snu function is needed to establish cuticle Figure 2 stifness or elasticity.

Mechanical stress ruptures the cuticle of snu mutant larvae. As seen on electron‐micrographs, at the muscle attachment site in the ready to hatch wild‐type larvae, the cuticle (epi, epicuticle; pro, procuticle) follows the pull of the muscles (left image). At the muscle attachments site in snuDf(98E2) mutant larvae the procuticle (pro) detaches from the epicuticle (right image). The adhesion of the epidermal surface (dotted line) and the procuticle is, by contrast, stable. The scale bar in the left micrograph represents 500 nm and applies also to the micrograph on the right.

The Young's modulus of wild‐type and snuDf(98E2) mutant larval cuticle measured using AFM nanoindentation. Box endings correspond to the 25th and 75th percentiles; the line within shows the median; error bars define the 10th and 90th percentiles; outliers are marked by circles. The values of cuticle Young's modulus for the two embyo types are statistically significantly different (Mann−Whitney U test, p < 0.005). The indented regions in the abdominal of the larvae are marked on cryo‐SEM images by yellow triangles.

Cuticle Elasticity Depends on Snu Function

3.1

To evaluate cuticle elasticity in wild‐type and snu ^ Df(98E2) ^ mutant larvae, here, we measured the Young's modulus of the respective cuticle by atomic force microscopy. The indentation depth of 50 nm ensured that we analyzed the contribution of the relevant layers envelope and epicuticle, and minor contribution of endocuticle. The wild‐type larval cuticle has a median Young's modulus that equals to 1.70 MPa (90% confidence interval: 1.12–2.30 MPa). According to Vincent and Wegst (Vincent and Wegst 2004), the D. melanogaster larval cuticle is, thus, a soft type of cuticle with the Young's modulus ranging from 1 kPa to 50 MPa. Indeed, its elasticity corresponds to the one of Resilin containing cuticles with a Young's modulus of 1 MPa. In contrast to the normal cuticle, stiffness of the cuticle of snu mutant larvae is dramatically ten times smaller (median Young's modulus 0.16 MPa, 90% confidence interval: 0.12–0.22 MPa). Eleimination of Snu function, and by consequence, the removal of an Argentaffin‐positiv component from the epicuticle and envelope (Zuber et al. 2018), hence, enhances cuticle elasticity. This finding support the histological results of Sir Vincent Wigglesworth that lipids constitute an important component of cuticulin. In conclusion, Snu‐dependent delivery of lipids to the cuticle surface are not only contributing to the waterproof barrier function of the cuticle but also to its stiffness. Possibly, this second population of lipids is distinct from those responsible for cuticle waterproofness (CHCs). Due to experimental set ups, the cuticulin‐related lipids were not identified in the respective gas chromatography/mass spectrometry experiments and await, therefore, identification.

Author Contributions

Alexander Kovalev: formal analysis, validation, methodology, visualization, writing – review and original draft. Stanislav Gorb: supervision, formal analysis, validation, methodology, visualization, writing original draft, funding acquisition, project administration, resources. Bernard Moussian: supervision, formal analysis, validation, methodology, visualization, writing − review and editing, writing − original draft, funding acquisition, investigation, conceptualization, project administration, resources.

Conflicts of Interest

The authors declare no conflicts of interest.