FEG-SEM and TEM imaging combined with EDXS analyses of cuticle differentiation during early ontogenetic development

in terrestrial crustaceaPolona Mrak1, Nada Žnidaršič1, Kristina Žagar2, Miran Čeh2, Jasna Štrus1

polona.mrak@bf.uni-lj.si; nada.znidarsic@bf.uni-lj.si; jasna.strus@bf.uni-lj.si1. Department of Biology, Biotechnical faculty, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana, Slovenia

2. Department for Nanostructured Materials, Jožef Stefan Institute, Jamova cesta 39, SI-1000 Ljubljana, Slovenia

Participation in International Microscopy Congress 2014 in Prague, Czech Republic, is financially supported by the IMC European scholarship and the scholarship ofSlovene Society for Microscopy (SDM) and FEI company.

Exoskeletal cuticle of arthropods is an extracellular matrix based on chitin-protein fibers and secreted apically by epidermal cells. Crustaceans additionally harden their exoskeleton by calcification. Fully formed cuticle of adults comprises the outermost thin lipo-protein epicuticle and the inner chitin-protein procuticle, divided into exocuticle and endocuticle, which are mineralized by calcite and by amorphous calcium carbonate and calcium phosphate [1]. In terrestrial isopod crustaceans, cuticle is formed during ontogenetic development in the female brood pouch - marsupium [2, 3] and is renewed periodically during molting. To follow dynamic and complex processes of cuticle differentiation, a study of cuticle structure and composition in progressive developmental stages is required. Application of different complementary microscopic techniques, including correlative approaches, is valuable for description of cuticle formation.

Different preparative, imaging and analytical microscopic approaches were applied to investigate the exoskeletal cuticle differentiation in intramarsupial development of isopod Porcellio scaber, compared to adult cuticle.Aldehyde fixation was performed to preserve organic components, required for analysing detailed ultrastructure of surface matrices and epidermal cells and for localization of N-acetyl-glucosamine oligomers, including chitin, that was performed by labelling with lectin WGA-gold in ultrathin sections of tergite cuticle.Preservation of the mineral phases, including amorphous polymorphs in the cuticle, is achieved by methanol-fixation and no exposure to water during samplepreparation, as suggested in the literature [4]. Elemental composition and ultrastructure of tergite cuticle were revealed in methanol-fixed intact samples by FEG-SEMimaging in LEI mode, supplemented by EDXS analyses. Next, sample block faces of methanol-fixed and resin embedded specimens were analysed by correlative LEIimaging and EDXS analyses of tergite cuticle, combining with imaging and specific histochemistry of the corresponding ultrathin and semithin sections, respectively.

Figure 1: Opened marsupium (fluid-filled brood pouch) with marsupial larvae mancae on the ventral side of Porcellio scaber female. Intramarsupial development, from fertilized egg to embryo and marsupial larva manca, lasts approximatelly 35 days.

CUTICLE OF ADULTS

CUTICLE OF ADVANCED EARLY MARSUPIAL MANCA

e. FEG-SEM imaging of the intact methanol-fixed manca and depicted locations of the obtained EDXS spectra 1 and 2 in the manca surface. The spectra show presence of calcium, phosphorus and magnesium, with Ca peaks always lower than P peaks.

f. FEG-SEM imaging of the intact methanol-fixed manca and EDXS spectrum 5 obtained in the manca surface. The spectrum shows similar elemental composition as EDXS analyses of the cuticle in the block face (spectra 3 and 4).

Figure 2: Precuticular matrix(PCM), consisting of electron dense lamina (L) and subjacent loose material.a. The epidermis of the mid-stage embryo secretes at least two precuticular matrices (L1, L2).b. In late embryo, hatched from the chorion, a substantial precuticular matrix covers the epidermis. The previous precuticular matrix is shed.

SURFACE MATRICES DURING EMBRYONIC DEVELOPMENTFigure 3: Cuticle covers the epidermal cells.a. The first cuticle formation is observed underneath the precuticular matrix (PCM) in the prehatching embryo of stage 18. b, c. In the last stage of embryonic development, stage 19, the cuticle is differentiated into epicuticle (EPI) and exo-(EXO) and endocuticle (ENDO) with sublayers. Morphological features of cuticle renewal, namely cuticle detachment (*) and degradation (d) and pore canals (pc) are already observed in this stage.

CONCLUSIONS• Our results reveal that several epidermal extracellular matrices are formed during intramarsupial

development of isopod Porcellio scaber, with early signs of matrix deposition in mid-stage embryo and the first cuticle deposition in prehatching embryo.

• Newly hatched manca secretes a new cuticle that displays differentiated structure of epi- and procuticle, a conspicuous labelling with the lectin WGA-gold probes and calcium contents.

• In sequential marsupial manca development the cuticle architecture, thickness, labelling with WGA-gold complexes, histochemical localization of calcified tissue and EDXS analyses show that the exoskeleton structure and composition are already similar to those in adults.

• Our results show gradual formation and calcification of the exoskeletal cuticle during P. scaber larval development within the marsupium, indicating involvement of exoskeleton in animal mobility which was observed to progress in developing marsupial larva [2].

• Combination of different microscopic techniques is beneficial for parallel examination of cuticle structure and composition. Examination of the same sample is achieved by correlative FEG-SEM imaging and EDXS analyses of the sample block face in combination with specific histochemistry and imaging of the corresponding semithin and ultrathin sections.

REFERENCES[1] Dillaman R., Roer R., Shafer T., Modla S. 2013. The crustacean integument: Structure and function. In: Watling L., Thiel M. (Eds), Functional Morphology and Diversity, vol. 1. Oxford University Press, New York, pp. 140-166 (5).[2] Mrak P., Žnidaršič N., Tušek-Žnidarič M., Klepal W., Gruber D., Štrus J. 2012. Egg envelopes and cuticle renewal in Porcellioembryos and marsupial mancas. Zookeys 176: 55-72.[3] Mrak P., Žnidaršič N., Žagar K., Čeh M., Štrus J. 2014. Exoskeletal cuticle differentiation during intramarsupial development of Porcellio scaber (Crustacea: Isopoda). Arthropod Structure and Development 43: 423-439.[4] Becker A., Bismayer U., Epple M., Fabritius H., Hasse B.,Shi J., Ziegler A. 2003. Structural characterisation of X-rayamorphous calcium carbonate (ACC) in sternal deposits of the crustacea Porcellio scaber. Dalton Transactions: 551-555.

a. TEM image of glutaraldehyde-fixed andosmicated specimen. Forming cuticle displayselaborated epicuticle and no distinctivestructural division of the procuticle. Theprocuticle is invaded by broad cytoplasmicprojections of the epidermal cell. b. Localization of N-acetyl-glucosamine oligomers by lectin WGA-gold labelling in ultrathin section. The exoskeletal cuticle is conspicuously labelled in comparison to the underlying epidermal cell.

c, d. Corresponding semi- and ultrathin sections of methanol-fixed and resin embedded manca. c. Alizarin red S staining for calcified tissue localization on the semithin section shows no differential staining of the exoskeletal cuticle.d. TEM imaging of the cuticular matrix reveals typical chitin-protein fibers arrangement in the procuticle.

CUTICLE OF NEWLY HATCHED MARSUPIAL MANCA

Figure 4: Cuticle of newly hatched marsupial manca of P. scaber Figure 5: Advanced early marsupial manca of P. scaber.

a. TEM image of glutaraldehyde-fixed and osmicated specimen. Exoskeletal cuticle is thicker than in the earlier stage and displays all three principal layers: epicuticle, exocuticle and endocuticle with lamellar sublayers. b. Localization of N-acetyl-glucosamine oligomers by lectin WGA-gold labelling in ultrathin section shows intense binding of WGA-gold in the exoskeletal cuticle, resembling the labelling of the adult cuticle (Fig. 6b).

c. FEG-SEM imaging in LEI mode of the sample block face reveals transversely cut cuticle and nuclei of underlying cells (white arrow). EDXS spectra 3 and 4, recorded from the depicted locations in the cuticle show conspicuous calcium peaks, that are evidentely higher than phosphorus peaks, and magnesium peaks, similarly as in the cuticle of adults (spectrum 6). d. Alizarin red S staining in the semithin section: calcified tissue is differentially demonstrated, although the integrity of the cuticle is not complete due to the cutting. e. TEM image of the cuticle reveals lamellae of chitin-protein fibers in endocuticle. Empty spaces presumablyrepresent lost material due to the exposure to water during cutting. Figure 6:

a. Ultrastructure of intermolt adult cuticle, composed of distinct horizontal layers: thin epicuticle with scales, exocuticle with characteristically arranged chitin-protein fibers and endocuticle with lamellar chitin-protein sublayers. b. Localization of N-acetyl-glucosamine oligomers by lectin WGA-gold complexes: labelling of different regions of the cuticle.

c, d, e. Methanol-fixed and resin embedded manca. Correlative FEG-SEM imaging and EDXS analyses on the block face and the corresponding semithin and ultrathin sections.

c, d, e. Methanol-fixed resin embedded cuticle of postmolt adult.c. FEG-SEM imaging in LEI mode of the sample block face displaysmatrix subregions and structure. At the selected spot in the matrix, EDXS spectrum 6 was recorded, showing prominent contents of calcium and presence of phosphorus and magnesium.d. Alizarin red S histochemical staining for calcified tissue localization in the corresponding semithin section shows differences of staining intensity within cuticular matrix. e. Ultrastructure of the cuticle in the corresponding ultrathin section. Exocuticle and endocuticle with typical chitin-protein fibers arrangement and basal newly forming layers of cuticular matrix.