DOI: 10.5061/dryad.vq83bk46p
title: Morphological and biomechanical adaptations of larval mandibles in Trichoptera (Insecta)

description: This study examines the larval mandibles of five caddisfly species,
 documenting their morphological and biomechanical adaptations to different
 feeding strategies. Three predatory species (Molanna angustata,
 Plectrocnemia conspersa, Rhyacophila fasciata) and two algae-grazing
 species (Silo nigricornis, Tinodes pallidulus) were investigated using
 scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy
 (EDX), confocal laser scanning microscopy (CLSM), and nanoindentation. SEM
 analysis revealed distinct mandible structures: grazing species possessed
 various collecting setae and sharp mandible cutting edges, while predatory
 species exhibited more pointed incisors. By EDX, low concentrations of Ca,
 Cl, Cu, Fe, K, Mg, Mn, P, S, Si, and Zn were detected with no relationship
 to mechanical properties, suggesting limited elemental reinforcement in
 larval Trichoptera mandibles. CLSM imaging, however, revealed regional
 material heterogeneities related to the mechanical properties, indicating
 that the mechanical parameters depend on the degree of cuticle tanning. We
 detected harder and stiffer mandible cuticle in predatory species, likely
 enhancing the mechanical resistance of the material during prey capture.
 Nanoindentation analysis identified two functional mandible types in
 grazers, adapted for scraping and collecting, with differential regional
 cuticle hardness and stiffness. Predatory species exhibited three mandible
 types, likely specialized for grabbing and crushing, piercing and cutting,
 or grabbing, piercing, and cutting. Decreased mechanical properties of
 condyles in predators suggested enhanced flexibility for prey handling,
 whereas grazers, showing higher Young’s moduli and hardness values in
 condyles, likely required higher pressure for scraping. In some species
 (Silo nigricornis and Rhyacophila fasciata), the heterogeneity between
 lateral and medial mandible cuticle indicated that self-sharpening
 mechanisms could be present. These findings provide insight into the
 functional morphology and material adaptations of mandibles of
 trichopteran larvae in relation to their feeding ecology.
description: <em>Studied species</em> Five
 species were investigated and compared to the published data on
 <em>Glossosoma boltoni</em> (Spicipalpia) [42]:
 <em>Plectrocnemia conspersa</em> (Curtis, 1834) and
 <em>Tinodes pallidulus</em> McLachlan, 1878 (both
 Annulipalpia), <em>Silo nigricornis</em> (Pictet, 1834),
 <em>Rhyacophila fasciata</em> Hagen, 1859, and
 <em>Molanna angustata</em> Curtis, 1834 (all Integripalpia)
 (Supplementary Table 1). Two species primarily feeding through “grazing”
 (i.e., scraping algae from solid surfaces) (<em>Tinodes pallidulus,
 Silo nigricornis</em>) and three species primarily predatory
 (<em>Plectrocnemia conspersa, Rhyacophila fasciata, Molanna
 angustata</em>) were selected. Data regarding trophic specialization
 were obtained from the literature [36]. For this study,
 eight specimens of the ultimate larval instar per species were examined
 (Supplementary Table 2). The specimens of <em>P.
 conspersa</em>, <em>S. nigricornis</em>, <em>M.
 angustata</em>, and <em>R. fasciata</em> were obtained
 from the Georg Ulmer collection, curated at the Zoological Museum Hamburg
 (ZMH), now part of the Leibniz Institute for the Analysis of Biodiversity
 Change Hamburg (LIB). Most specimens were collected by August Thienemann
 around the turn of the 20th century in northern Germany. <em>T.
 pallidulus</em> was collected by Martin Kubiak in 2013 in
 Saxony-Anhalt. Specimens were preserved in 70% EtOH.
 Mandibles were first documented using scanning electron
 microscopy (SEM) to identify specific areas affected by wear.
 Subsequently, elemental analysis on the mandibular cuticle was conducted
 using energy-dispersive X-ray spectroscopy (EDX). Nanoindentation was
 performed to test for mechanical property gradients within each mandible.
 Finally, mandibles were examined by confocal laser scanning microscopy
 (CLSM) to reveal material composition. Using this combination of methods,
 the morphologies and material properties of the mandibles were compared
 and analyzed in the context of feeding strategies (grazing and
 predatory). <em>Light
 microscopy</em> Initially, the specimens were
 documented using a Keyence Digital Microscope VHX-7000 (KEYENCE,
 Neu-Isenburg, Germany) equipped with automatic stacking software in
 dorsal, ventral, and lateral views (Supplementary Figures 1–5). Images of
 individuals were also used to determine the ratio of body and head length
 to mandibular length. Measurements of the individuals were taken by
 drawing a line from anterior to posterior along the larva in Adobe
 Photoshop, because most specimens were curled. Then, the length of the
 line, given in pixels, was converted into mm, taking the number of pixels
 of the scale bar as a conversion factor. Additionally, all extracted
 mandibles were documented using light microscopy before further
 analysis. <em>Scanning electron microscopy
 (SEM)</em> For documenting head morphology, seven
 specimens per species were cut along with the first abdominal segment,
 briefly cleaned in 70% EtOH using an ultrasonic bath, mounted on SEM
 sample holders using double-sided carbon adhesive, and air dried. The
 specimens were coated with a 5 nm thick layer of platinum (Polaron SC7640
 Sputter Coater) and visualized with a Zeiss LEO 1525 (One Zeiss Drive,
 Thornwood, USA) at an acceleration voltage of 15kV. Subsequently, the
 specimens were rehydrated with 70% EtOH, detached from the sample holders,
 and the mandibles were carefully dissected from the heads. The mandibles
 were cleaned again in the ultrasonic bath and documented using SEM. The
 images were used to describe morphology and measure the length of the
 mandible. Afterwards, mandibles were detached from the sample holders with
 70% EtOH, cleaned in a short ultrasonic bath, and used for either EDX or
 nanoindentation. <em>Energy-dispersive X-ray
 spectroscopy (EDX)</em> Two specimens per species
 were used for this analysis (one left mandible of one specimen and one
 right mandible of another specimen to compare for differences between
 individuals and between the right and left sides). The same localities of
 the countermandibles from the same specimens were later tested by
 nanoindentation.  The mandibles were initially mounted
 on glass slides using double-sided carbon adhesive. Each sample was
 surrounded by a small metal ring, which was then filled with epoxy resin
 (<em>Reckli Epoxi WST,</em> RECKLI GmbH, Herne, Germany)
 following the protocol described by [43,44,45]. Polymerization lasted
 three days at room temperature. Afterwards, the glass slides and carbon
 adhesive were removed. Samples were polished using sandpapers of varying
 grit sizes until the first layers of the cuticle became visible.
 Subsequently, the exposed surfaces were polished with a 0.3 µm aluminum
 oxide polishing powder suspension (PRESI GmbH, Hagen, Germany) on a
 polishing machine (<em>Minitech 233/333</em>, PRESI GmbH,
 Hagen, Germany) to achieve a smooth surface. Since the various regions of
 interest were located at different depths within the sample, each sample
 was sequentially polished to expose one target region at a time. Once a
 target region was revealed, EDX analyses were conducted. The sample was
 then further polished to expose the next target region, after which the
 same protocol was repeated (see below) to test the subsequent
 regions. After smoothing, the samples (Figure 1) were
 cleaned in an ultrasonic bath for five minutes, mounted onto SEM sample
 holders, air-dried, and coated with a 5 nm layer of platinum. The
 elemental composition was determined using the SEM (<em>Zeiss LEO
 1525</em>), equipped with an Octane silicon drift detector (SDD)
 (TEAM microanalysis system, EDAX Inc., New Jersey, USA). Consistent
 settings were applied for all samples (e.g., acceleration voltage of 20
 kV, working distance, lens aperture, etc.). Prior to analyzing each
 sample, the detector was calibrated using copper. Small areas of the
 cuticle sections (1–2 x 1–2 µm) were tested. The
 detected elements included aluminum (Al), carbon (C),
 calcium (Ca), chlorine (Cl), copper (Cu), hydrogen (H), iron (Fe),
 potassium (K), magnesium (Mg), manganese (Mn), nitrogen (N), sodium (Na),
 oxygen (O), phosphorus (P), platinum (Pt), sulfur (S), silicon (Si), and
 zinc (Zn). However, some elements were excluded from
 discussion as they are either fundamental components of chitin and
 proteins (H, C, N, O), part of the coating (Pt), or derived from the
 polishing powder (Al, O). The remaining elements were collectively
 categorized as "all elements, Ae". Since Pt and P share a
 spectral peak and the software cannot distinguish between the two, their
 values were combined as (P + Pt). To refine the estimation of P content,
 the elemental composition of 20 areas of the epoxy resin was analyzed,
 yielding a mean ± SD Pt content of 0.15 ± 0.02 atomic %. The analysis
 confirmed that the resin contained none of the other discussed
 elements. The measurements were sorted into the
 following mandibular zones: (A) medial cuticle of the mandible tip, (B)
 lateral cuticle of the mandible tip, (C) medial surface of the mandible,
 (D) lateral surface of the mandible, (E) dorsal condyle, and (F) ventral
 condyle (Figure 1). The mandible surfaces were then divided into the
 sections a to k (Figure 1B). Since mandibles were not of the same size, we
 assorted these sections by proportions.  A total of
 1006 localities from 10 mandibles were tested via EDX (Figure 1). For
 analysis of elemental content of the suborders, the data from <em>G.
 boltoni</em> was included from [42].
 <em>Nanoindentation</em>
 Nanoindentation was conducted on the two opposing mandibles from
 the same specimens analyzed by EDX. To expand the sample size, six
 additional mandibles from three other specimens per species were included
 for mechanical property testing, resulting in a total of four individuals
 per species studied. The preparation of samples for nanoindentation
 followed the same protocol as for EDX: mandibles were embedded in epoxy,
 polished, and smoothened [for detailed protocol, see 42].
 The prepared samples were mounted onto the nanoindenter sample
 holder. Indentations were performed using a nanoindenter SA2 (MTS Nano
 Instruments, Oak Ridge, USA) equipped with a Berkovich indenter tip and a
 dynamic contact module (DCM) head. The mechanical properties — hardness
 (<em>H</em>) and Young’s modulus (<em>E</em>) —
 were calculated from force-displacement curves using the continuous
 stiffness mode. <em>H</em> measures a material's
 resistance to localized plastic deformation caused by indentation.
 <em>E</em> represents the stiffness of a solid material,
 describing the relationship between stress and axial strain. 
 These properties were determined at penetration depths of
 800–1000 nm. For each indentation site, approximately 30 measurements were
 recorded at varying depths and then averaged to produce one mean value for
 <em>H</em> and one for <em>E</em> per indent. All
 tests were carried out under standard room conditions (relative humidity
 28–30%, temperature 22–24 °C), with each indent and its corresponding
 curve manually inspected. After testing, each sample was further polished
 to reveal the next target region for analysis. A total
 of 1040 localities from 40 mandibles were tested using nanoindentation
 (Figure 1). For analysis of the mechanical properties of the suborders,
 the data from <em>G. boltoni</em> was included from
 [42].
description: # Morphological and biomechanical adaptations of larval mandibles in
 Trichoptera (Insecta) Dataset DOI:
 [10.5061/dryad.vq83bk46p](10.5061/dryad.vq83bk46p) ## Description of the
 data and file structure Regions were tested by nanoindentation and
 afterwards by elemental analysis. ### Files and variables #### File:
 Nano_and_EDX_for_Dryad.xlsx **Description:** Mechanical properties, tested
 by nanoindentation, and elemental content, tested by EDX ##### Variables *
 Sheet one: Young's modulus E, in GPa, and Hardness H, in GPa. The
 hardness values can be found in column F, and the Young's modulus
 data in column G. * Sheet two: Elements, given in atomic % (atom %). The
 results can be found in columns F-S. Column T is the sum of Pt and P
 content. Column U is the sum of all listed elements. * The areas tested
 can be identified from the paper, where you can find figures with the
 localities. ## Code/software Excel ## Access information NA