Fine Structural Characteristics of the Chorionic Microspheres on the Egg Surface of the Orb Web Spider Trichonephila clavata

doi:10.21203/rs.3.rs-2857404/v1

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Fine Structural Characteristics of the Chorionic Microspheres on the Egg Surface of the Orb Web Spider Trichonephila clavata

https://doi.org/10.21203/rs.3.rs-2857404/v1

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published 18 Jul, 2023

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The eggs laid by the orb web spider Trichonephila clavata must overwinter in very harsh weather conditions before hatching, but there does not seem to be any protection like a compact silk case covering the entire eggmass. Instead, the surface of the eggmass is completely coated with a milky coating called chorionic microspheres (CM). Therefore, we investigated the fine structural characteristics of CM to demonstrate the role of their ecological importance. Although the CM exhibits an uneven size distribution in outer eggmass, the chorionic surface is evenly covered with a single layer with a diameter of 2.3 µm approximately. The surface structure of aggregated CM shows short papillary projections demonstrating segmental adhesion of mucous components. CM is insoluble in water but partially soluble in absolute ethanol, and its spherical structure is completely decomposed by HFIP, a strong organic solvent. Since our fine structural observations clearly show that CM is not derived from vitellogenic or choriogenetic processes, the CM adhesive coatings during ovipositional process appears to be equivalent to cocoon silk for various protective functions in silken eggcase.

Egg

Chorion

Fine structure

Microspheres

Spider

Maternal secretions can be observed on the egg surface of arthropods. The secretions have been identified in arthropods such as dragonflies (Family: Libellulidae) (Miller 1987) and mites (Family: Acarididae) (Witaliński 1993). Microspherical secretions have been observed on the eggs of 41 spider species (Humphrey 1987).

The eggs of spiders, unlike that of other terrestrial arthropods, do not have a shell structure that could protect them during embryonic development (Schultz 1987). Instead, the outer membrane of their eggs is surrounded by two layers: the inner vitelline membrane and the outer chorion (Foelix 2011). The chorion contains chorionic microspheres (CM) of different diameters and densities that vary based on the species type (Grim and Slobodchikoff 1982). Nevertheless, the spherical morphology has no significant correlation with the arachnid taxa (Humphrey 1983, 1987).

Grim and Slobodchikoff (1978) argued that CM reduce the fungal infection by delaying water absorption. Austin and Anderson (1978) suggested that the CM was hydrophobic and therefore provide protection against moisture. Hadley (1978) confirmed the presence of a lipid layer impermeable to water on the egg surface. Witaliński and Žuwała (1981) suggested that the mucus-containing oviposition fluid coats the chorion, causing aggregation of eggs. Conti et al. (2015) suggested that the aggregation of the eggs restricts their dehydration, as they become tightly attached to each other. Despite the various functionalities proposed for CM, the observation of its fine morphology remains severely limited.

Most spiders wrap their eggs in cocoon silk derived from the tubuliform silk gland (Foelix 2011). The cocoon silk is known to protect the eggs from predators and parasites (Austin 1985), reduce the predator's perception of the background (Barrantes 2013), decrease desiccation during a long overwintering period (Hieber 1992), and regulate the temperature (Hieber 1985). Some species, such as Theridiidae and Lycosidae, build their cocoon as a compact “eggcase”, while others like T. edulis use a loose “eggpad” (Grim and Slobodchikoff 1982; Humphrey 1995).

In this context, the density of silk is determined by whether the internal eggs are visible through it. When examining the CM of various spider species based on the density of their silken cocoon, we observed a negative correlation between the density of silk and the abundance of CM. Therefore, we suggest considering the possibility of a complementarity between silk and CM.

We herein describe the fine structure of the chorion, attached with a massive amount of CM, of the orb-web spider, T. clavata, with aid of an electron microscope. To understand its role on the spider ecophysiology, solubility CM in water and organic solvents was evaluated. This study further examined the abdomen of T. clavata, a sexually mature individual to determine the origin or source of the CM.

Adult females of the golden orb web spider, Trichonephila clavata were collected in a local area near the Dankook University, Chungnam, Korea. This species was moved to Trichonephila from the genus Nephila along with 10 other species (Kuntner et al. 2018).

All spiders were maintained under ambient conditions with natural lighting in enclosures comprising a wooden frame (height × length × width, 50 cm × 50 cm × 10 cm) with glass panels on the front and back, and fed insect larvae and water daily. After spawning, the spiders were released, and the eggmasses were stored in room temperature.

After physically disintegrating the eggmass, the outer eggs and the inner eggs were separated. The eggs with white coating layer and chorion surface were classified. The specimens were attached to a flexible cover glass (0.14 mm × 18 mm × 18 mm, Marienfeld, Germany) and were treated for various solvent (distilled water, EtOH, HFIP) conditions under different times (20 seconds, 1 hours, 24 hours).

Microscopic images were photographed using Motic digital imaging system (Motic Instruments Inc., Richmond, Canada). and Nikon Eclipse 80i microscope.

For scanning electron microscopic examination, the samples were mounted on stubs using double sided tape and were coated with platinum-palladium with a thickness of 20 nm, using a Hitachi E-1030 ion sputter coater (Hitachi Co., Tokyo, Japan). Coated samples were observed with a Hitachi S-4300 (Hitachi Co., Tokyo, Japan) field emission scanning electron microscopy (FESEM) with an accelerating voltage of 5–20 kV (Sun et al. 2021).

To analyze egg development in the maternal ovary, specimens were slowly cooled down to 4℃ and dissected under light microscope in a drop of spider Ringer’s solution consisting of 160 mM NaCl, 7.5 mM KCl, 4 mM CaCl₂, 1 mM MgCl₂, 4 mM NaHCO₃, 20 mM glucose, pH 7.4 (Moon and Tillinghast 2020). The ovary was gently removed. For transmission electron microscopy examination, the tissues were fixed in a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde buffer with phosphate buffer.

Post-fixation was performed with 1% osmium tetroxide in the same buffer solution. They were dehydrated in graded concentrations of ethanol and propylene oxide, and embedded in EM-Bed 812 medium (Sun et al. 2023). Semithin sections stained with toluidine blue were used to examine the histology of the tissues. Ultrathin sections were obtained from a LKB ultramicrotome, and were double stained with uranyl acetate and lead citrate. The sections were examined with a JEM 100 CX-II (JEOL, Japan) electron microscopy at 80 kV.

Statistical analyses were performed using SPSS 21 Statistics (IBM Korea, Korea) to determine frequencies of CM size variation by location. A p value of < 0.05 was considered statistically significant.

The eggs are clustered in an ellipsoidal shape to form a mass (Fig. 1A). Following spawning, the eggmass is randomly wrapped with eggcase silk. The wrapping silk is characterized by an extensive enveloping of the eggmass. The eggpad is attached radially to the wooden frame by the pyriform gland silk.

A thin coating comprising a milky substance surrounds the outer edge of the eggmass (Fig. 1B). This coating is also vulnerable to physical impact and can be shredded into particles. Eggcase silk passes through the inside of the coated surface. The egg is generally crumpled spherical and attached to each other (Fig. 1C). The empty space between the eggs is filled with the CM lump. Unlike the outer coating surface, the inside of the eggmass has a low coating distribution, which allows the observation of the egg chorion (Fig. 1D).

The coating substance on the eggs were photographed with FESEM (Fig. 2A). A color effect was applied to distinguish the outer surface of the eggmass from the inner surface of the egg; green indicates the eggmass surface and brown indicates the inner egg surface (Fig. 2B). The inner eggs inside the mass are characterized by a pressed surface by the attached surface with adjacent eggs (Fig. 2C). The adhesive surface has a coating with CM, forming a single homogeneous layer of the CM. Microsphere lumps (Lu) are observed (Fig. 2D). Depending on the amount of CM, the CM may form a multilayer in the form of a lump.

Treatment with EtOH for 48 h confirm that the CM-coated chorion peeled off on the cover-glass bottom (Fig. 3A). The partially peeled-off chorion shows that CM is attached only above the surface (Fig. 3B). No spherical structure is found under the CM-coated layer. Adhesiveness decreases in the old chorion (over 6 months) surface and CM can fall off (Fig. 3C). Adhesion traces are clearly observed at the location where the CM has fallen off. The CM was found to have a uniform size distribution, with a diameter of approximately 2.3 µm. The mean diameter of 80 adhesion traces was measured to be 2 µm.

Egg coating can be deposited on the tubuliform silk (Fig. 4A). The solid platform is completely closed and hence does not form an CM layer (Fig. 4B). Meanwhile, a non-porous CM layer is also observed, which maintained its spherical shape (Fig. 4C). Additionally, pyriform gland-derived glue proteins (Py) adhere to CM.

The outside plane of the eggmass has CM of various diameters (Fig. 4D). The CM has diameters that may vary by up to about 39 fold. The CM in the multilayer are predominantly aggregated together, which appears to enable adhesion between CM (Fig. 4E). On the surface of the chorion, a fine papillary projection, along with a broken strand, is mainly observed rather than aggregated form (Fig. 4F). Two or more strands and “papillae” can be observed in one CM unit,

Next, 500 CM were photographed and their diameters were determined using the freeware imageJ. The diameter distributions are plotted in box plots for each location (chorionic surface, outer surface, and silk) (Fig. 5). Their averages diameters are determined to be 2329, 1692, and 1594 nm, respectively (standard deviation: 438.83, 444.67, and 601.79). The boxplot shows that the CM size can vary significantly by location (P < 0.005). In particular, the size of the CM inside is significantly larger than that of the outside.

To confirm the affinity of the CM for various solvents, the eggs were treated with water and 100% EtOH and hexafluorisopropanol (HFIP) for 48 h (Fig. 6). The white structure (CM) on the eggs was hardly soluble in water (Fig. 6A) and hence no change is observed on the electron microscope (Fig. 6B). In contrast, the CM partially dissolved in EtOH (Fig. 6C) with considerable amount of white coating detached on the cover glass. The partially dissolved surface of CM appeared as cracked structures aligned on a single “plane” (Fig. 6D). HFIP, a highly volatile organic solvent, can melt the CM layer (Fig. 6E). Most of the spherical structures are lost when treated with HFIP, while some barely retained their shapes (Fig. 6F). The egg surface is found to have a surface that ranges from smooth to a densely CM-coated one.

To confirm the intermediate state of the dramatic structural change caused by HFIP, HFIP was applied to the surface for only about 20 seconds. Outside plane not treated with HFIP comprises a large portion of the multilayer surrounding the egg (Fig. 7A). Under the multilayers, a single layer is directly attached to the chorionic surface (Fig. 7B). In contrast, eggs treated with HFIP lost most of the multilayers (Fig. 7C). The CM on the chorionic surface is arranged in a hexagonal pattern (Fig. 7D). Rather than aggregate, strand and papilae structures dominate the CM.

The CM HFIP-treated was immediately dried and melted (Fig. 8A). The spherical, but severely distorted CM and a sticky CM mass are mainly observed. The shrinkage of the spherical shape suggested that the CM dissolved in HFIP (Fig. 8B). Fine structure of the eggs not treated with HFIP showed that the CM has a severely uneven surface with agglomerated fine particles (Fig. 8C). The adhesive strand has the same texture as the CM surface, which contains nanoparticles. The CM of the HFIP-treated eggs immediately dried and were relatively less granular. In addition, the surface of the merged strand has a softer texture than that of the control (Fig. 8D).

Each mature oocyte is composed of glycogen and yolks of various sizes and types (Fig. 9A). Transmission electron microscopy images shows that the yolk comprised two types of granules: large sized and highly electron-dense yolk protein and low electron-dense yolk lipid. In addition, fine glycogen particles are found to aggregate together and form a high-electron component. The vitelline membrane is formed after the completion of vitellogenesis. Before choriogenesis, the vitelline membrane has an inner homogeneous electron-lucent component, a denser middle layer, and a less dense outer layer (Fig. 9B, C). Unlike the layers in which the vitelline membranes face each other in oocytes (Fig. 9B), the vitelline membrane facing outward has a low electron density (Fig. 9C). Each oocyte has well-developed microvilli along the surface of the egg envelope. CM or its precursors are not identified until chorions are formed.

The orb-web spider, T. clavata secretes a large amount of the oviposition fluid while laying eggs. Its eggs are first reddish, which then gradually turn brown. The oviposition fluid surrounds whole eggmass, soon dries to a white CM. This milky coating consists of a high-density CM surrounding the chorion. In the genus Trichonephila, the CM coating is thought to be significantly thicker than that in other reported spider taxa (Humphrey 1987, 1995). Therefore, CM exhibits minor morphological differences depending on their location. This study describes the structural variations and features of CM in T. clavata.

The CM of Araneus (Araneidae), was observed to have an average diameter of 4.6 µm or higher, while most families of Araneae exhibit a CM of around 1 µm (Grim and Slobodchikoff 1982; Humphrey 1983). At the level of Araneidae, the average size of the CM ranges from 1.16 µm (Argiope aetherea) to 3.55 µm (Archemorus simsoni) (Humphrey 1987). In Trichonephila, Trichonephila edulis was found to have a uniform CM diameter with an average of 1.6 µm (Humphrey 1995).

This study demonstrates that the CM of T. clavata exhibits a consistent size distribution of 2.3 µm, limited to the chorionic surface within the eggmass. However, average diameter of the CM exposed on the outer eggmass was approximately 1.7 µm. The reason for the existence of such spherical size variation is not yet known, but it seems to be a common phenomenon during the production and secretion of granules. This is because mature granules stored within secretory cells are first deposited on the chorionic surface, and immature granules subsequently accumulate on the outer surface.

Alternatively, the size difference of CM may have been caused by the difference in exposure to external environment due to the thick CM layer. A diverse range of CM with varying diameters was found on the silk covering the eggmass, but their varied forms make it difficult to determine when they were attached to the silk.

Adhesive structures formed between CM can be categorized into three stages: aggregates, strands, and papillae. Aggregates refer to clusters of CM, while strands connect individual CM unit, and papillae are short projectional structure. The “papilla” was also firstly reported by Humphrey (1995). These three structures are believed to form aggregated CM clumps that elongate because of external factors (e.g., diffusion of secretions or airflow) and form a strand, which eventually breaks off to become papillae.

These segmental adhesions are composed of a substance, which is different from the spherical scaffolds that maintains its shape for a long time. According to Witaliński and Žuwała (1981) and Makover et al. (2019), the oviposition fluid contains mucus and low-molecular-weight proteins. The low-molecular-weight proteins indicates the basic spherical units of CM, while mucus forms the adhesion between the units. Makover et al. (2019) reported that the CM in the Latrodectus geometricus egg is insoluble, although it is superhydrophilic with a water contact angle of < 10°. Because CM itself is found to be insoluble, the superhydrophilic layer on the CM is probably made of mucus secretions.

If CM acquires adhesive ability through the mucus components, it can explain the stronger adhesion on the chorionic surface than between CM. The chorion exposed to air for a long period (over 3 months) shows poor adhesion. The CM that do not adhere directly to the surface are more likely to fall off. Nevertheless, the diameter of the fall-off trace is comparable to 87% of the CM diameter. Therefore, approximately 34% of the CM is in contact with the chorionic surface. These quantitative measures could potentially serve as indicators for explaining the differences in adhesive properties. What is certain is that CM themselves do not possess any adhesive properties.

CM or its precursors is not observed in the vitelline membrane during oogenesis. In addition, the egg does not contain any tissues or cells secreting a microgranular structure. Therefore, this result suggests that the CM does not derived from both vitellogenenic and choriogenetic processes. Morishita et al. (2003) also reported that the eggs of Loxosceles intermedia become granular during the transition of the oocytes through the oviduct and the uterus. Therefore, it is presumed that the CM components are separately secreted from the exocrine ducts or secretory cells present in the oviduct and the uterus.

Regarding CM as an ecophysiological parental investment, it is necessary to compare the function of eggcase silk, as same means. Spiders that overwinter in the cocoon protect the larva from desiccation, while those that do not overwinter provide no protection to the eggs from transpiration (Hieber 1992). According to Austin (1984), the cocoons of sac spiders Clubiona robusta (Clubionidae) have a higher humidity than the surrounding air and prevent the eggs from drying out. Schaefer (1976) experimentally demonstrated that the eggs of Floronia bucculenta (Linyphiidae) survive in the cocoon for 68 days at 32% relative humidity and 5°C. Without the eggcase, the eggs dry out in 37 days.

Austin and Anderson (1978) showed that the cocoons of orb-web spider, Trichonephila edulis, which is a non-overwintering species, offer no protection to the eggs from desiccation. However, since T. clavata makes loosely silken eggpad, more precise humidity control is required to overwinter. It is expected that this function will be complemented by thick CM layers. Alternatively, a closed solid platform, presumed to be a mixture of silk proteins and non-silk CM, may be associated with protection from desiccation.

The CM of Licosidae, which builds compact eggcase, are attached to chorionic surface without forming any distinct CM layers (Grim and Slobodchikoff 1982). The microparticle structure of the exochorion can be found in several insects. Witaliński (1993) suggested that the exochorion granular structure in mites (Acaridida) such as Aleuroglyphus ovatus, Tyrophagus perniciosus, and Notoedres cati functions as an adhesive layer that fixes the eggs to the host epidermis. Dragonflies such as Brachythemis lacustris and Tholymis tillarga contain a trabeculate endochorion and an exochorion perforated by aeropyles (Miller 1987).

Wetting the exochorion at the oviposition causes it to become sticky and to attach firmly to the substrate. In the case of T. clavata, the abundant outer layer of CM in the eggmass serves as a barrier that prevents the eggs from scattering, much like a compact eggcase. Conversely, if an "eggcase" is capable of sufficiently preventing the dispersion of eggs, there may be no need for a large secretion of oviposition fluid.

Makover et al. (2019) also reported that the CM of eggs in Latrodectus geometricus had antibacterial effects. Esteves et al. (2009) described an antimicrobial microplus structure on the surface of the eggs of the Rhipicephalus tick (Boophilus). Similar findings were reported by Yu et al. (2012) and Zimmer et al. (2013) for microplus of ticks in Amblyomma hebraeum and Rhipicephalus, respectively. Nelson et al. (1999, 2000) described a waxy, fluffy material produced by whitefly females into which they lay eggs.

The material, according to the authors, camouflages the eggs, thus protecting it against predators. In vertebrates, D'Alba et al. (2013) showed that the surface topography of brush-turkey Alectura lathami eggs, determined by the presence of nanoscale spheres composed of calcium phosphate, renders the eggs hydrophobic, decreases bacterial attachment, and is most likely the major component preventing trans-shell penetration. Although the morphology is different, these egg surface microstructures are confirmed to be the result of convergent evolution that have protecting eggs.

The egg-coating function of the CM in spiders has been anticipated to be similar to that of cocoon silk. However, the morphology and characteristics of CM have remained understudied for a long time. The results of this study strengthen the premise that the CM of T. clavata is formed by the combination of spherical insoluble proteineous units and adhesive mucus component. On the other hand, it is proposed that CM and eggcase silk have a high complementarity in their functions, suggesting that they have evolved to have close interactions. This idea could also be applicable to further biological studies on the egg secretion of silk-producing arthropods. It would be interesting to investigate whether there is a correlation between the amount of CM secretion and the average winter climate in the area, in relation to the proliferation and northward expansion of T. clavata (Chuang et al. 2023), which has been introduced into the United States.

We examined the fine structural characteristics of the CM coating chorionic surface of T. clavata.
Morphological differences of CM were observed using scanning electron microscope.
To confirm the affinity of the CM for various solvents, the eggs were treated with water and absolute EtOH and hexafluorisopropanol (HFIP).
Although the CM exhibits an uneven size distribution in outer egg mass, the chorionic surface is evenly covered with a single layer with a diameter of 2.3 µm approximately.
CM is insoluble in water but partially soluble in absolute ethanol, and its spherical structure is completely decomposed by HFIP, a strong organic solvent.
It was found that CM units consist of insoluble proteineous sphere and mucous components that provide adhesion in CM.
Transmission electron microscopic observation suggests that the CM does not derived from both vitellogenenic and choriogenetic processes.
The CM acquires adhesiveness by mucus in oviposition fluid. CM can be lost by drying or exposure to organic solvents.

HFIP, hexafluorisopropanol; CM, chorionic microspheres.

Acknowledgements

This research was supported by the National Research Foundation (NRF) of Korea funded by the Ministry of Education (NRF-2014R1A1A2056398) and the Korea government (MSIT) (NRF-2019R1I1A3A01062105)

Authors' contributions

Myung-Jin MOON (Corresponding Author): contributions to the conception, design of the work as well as contribution to the acquisition, analysis, and interpretation of data. Seung-Min LEE (First Author): contribution to analysis, and interpretation of data.

Funding

This research was supported by the National Research Foundation (NRF) of Korea funded by the Korea government (MSIT) (NRF-2019R1I1A3A01062105)

Availability of data and materials

Materials described in the manuscript, including all relevant raw data, will be freely available to any scientist wishing to use them for non-commercial purposes

Competing interests

Not applicable

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Fine Structural Characteristics of the Chorionic Microspheres on the Egg Surface of the Orb Web Spider Trichonephila clavata

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