Experimental strategy
We chose an in vivo approach for investigating the impact of chitin synthesis in mollusc larval shell formation. Therefore, we reared mollusc larvae in the presence of NikkomycinZ, a small-molecule drug that is well-known to inhibit enzymatic chitin synthesis in a competitive manner in vivo and in vitro. We used polarized light video microscopy for the in vivo investigations and scanning electron microscopy imaging of extracted shell material prepared from NikkomycinZ treated Mytilus galloprovincialis larvae of various age.
Shell development under the conditions of the test (control experiments)
The metamorphosis of the mollusc larvae into the pediveliger and subsequently into the adult stage within the expected time frames (Fig. 1a, Additional Files 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) was one of the indications that our established culture conditions meet suitable quality standards for in vivo experiments. For control purposes, larvae of Mytilus galloprovincialis (Mollusca: Bivalvia) were reared under conditions matching the ones of the test series as closely as possible with respect to culture size, water reservoir, animal density, feeding, light, aeration, water exchange, and temperature, however in the absence of the competitive inhibitor of chitin synthase, NikkomycinZ. The viability as well as the behaviour of the larvae and the development of the larval shell during the treatment was monitored (see Additional Files 5, 6, 7, 8, 9, 10). We found that larvae can be reared in small-scale cultures (1 ml) without any significant loss of viability as compared to 10 l-scale cultures, as long as appropriate concentrations of algae are supplied, and if the culture medium is frequently replaced in order to keep the salt concentration constant and to remove contaminants, excess algae, and larval debris. A scanning electron microscopy image of a well developed, 19-day shell reared under the conditions of the test without NikkomycinZ is shown in Fig. 1b.
Additional File 1: Video clip of spawning female. Demonstration of spawning female and eggs prior to fertilization. (MPG 2 MB)
Additional File 2: Video clip of spawning male. Demonstration of spawning male and sperm prior to fertilization. (MPG 4 MB)
Additional File 3: Video clip of fertilization procedure. Demonstration of fertilization procedure and collection of fertilized eggs. (MPG 2 MB)
Additional File 4: Video clip of large scale larvae culture (10 l). Demonstration of the culture conditions of larvae in artificial seawater at 10 l-scale. (MPG 365 KB)
Additional File 5: Video microscopy of 2-day larvae. Video microscopic (32× objective) demonstration of the behaviour of 2-day larvae. Note that the larvae are already in the early veliger stage and have a D-shaped shell. (MPG 3 MB)
Additional File 6: Video microscopy of 5-day larvae. Video microscopic (32× objective) demonstration of the behaviour of 5-day larvae. The size of the velum and the motility of the larvae increased. Note that the hinge of the larval shell became straight. (MPG 3 MB)
Additional File 7: Video microscopy of 8-day larvae. Video microscopic (32× objective) demonstration of the behaviour of 8-day larvae. The motility of the larvae is similar to 5-day larvae. The size of the larval shells slightly increased. Most of the shells are still in D-shape. (MPG 3 MB)
Additional File 8: Video microscopy of 12-day larvae. Video microscopic (20× objective) demonstration of the behaviour of 12-day larvae. Note that the size of the shell increased, and the larvae switched from the D-shape stage to the umbo stage of shell formation. (MPG 2 MB)
Additional File 9: Video microscopy of 31-day larvae. Video microscopic (20× objective) demonstration of the behaviour of 31-day larvae. Larvae are in different developmental stages, such as veliger larvae and pediveliger larvae. The latter are indicated by the developing foot and a degenerate, vanishing velum that is retracted into the shell. (MPG 3 MB)
Additional File 10: Video microscopy of 36-day larvae. Video microscopic (20× objective) demonstration of the behaviour of 36-day larvae. At this age, the velum disappeared, and metamorphosis into the adult was completed as indicated by the functional foot. (MPG 5 MB)
Statistical effects of NikkomycinZ treatment observed in larvae cultures
The effects of NikkomycinZ on the cultivation of larvae were evaluated by estimating the percentage of affected individuals per culture well. It was not possible to determine accurate numbers due to the high motility of the individuals. Based on values obtained from different experimenters, we estimated a deviation of ~20%. This deviation includes also effects of variable numbers of individuals per culture well. We defined test conditions to be appropriate when larvae in the control culture without NikkomycinZ represented viability and motility comparable to a regular 10 l scale culture, which allowed us to grow larvae until metamorphosis into the adult stage occurred. When NikkomycinZ was applied in concentrations of > 25 μM, most of the individuals did not survive for the duration of the test. When NikkomycinZ was applied in concentrations of less than 5 μM, no significant effects on viability or increased morphological changes were observed. We found NikkomycinZ suitable to be applied in concentrations of 5 μM and 10 μM in order to compromise between the detrimental effects on viability and inducing significant effects on shell formation.
Four independent test series were included in the evaluation. NikkomycinZ was applied in concentrations of 0 μM (control), 5 μM, and 10 μM to 2-day (2d), 5-day, 8-day, and 12-day larvae. The cultures were grown to a final larval age of 15 days (15d). The estimated values for viability and morphological abnormalities of living individuals in each culture are summarized in Fig. 2 and Fig. 3 (see also Additional File 11). Morphological abnormality (Fig. 3) was defined as 100% for a particular culture when no individual survived the treatment. The average percentage of abnormally developed individuals or an increase in mortality in cultures without NikkomycinZ during the observed time span was slightly higher than compared to 10 l cultures due to the higher population density.
As demonstrated in Fig. 2, the early larval stages are more affected by NikkomycinZ treatment than the older larvae. If NikkomycinZ is added to a 2-day culture, the survival rate is below 20% on the 8th day (Fig. 2a). A comparable decrease in the survival rate is obtained when 5-day larvae are treated with NikkomycinZ for seven days (Fig. 2b). 8-day larvae were not affected comparably as much on the average (Fig. 2c). About 50% more individuals than in the control culture died within seven days in the presence of NikkomycinZ. As shown in Fig. 2d, no significant decrease in survival rate was observed during the first three days of NikkomycinZ incubation in 12d old cultures, whereas all younger larval stages did show an effect after three days.
The following criteria were defined in order to classify shell phenotypes that were observed in NikkomycinZ treated cultures: undulated shell edge, bilaterally asymmetrical valves, lack of umbo stage after 12 days, extraordinary small shell relative to the size of the organism, no straight hinge line, increased transparency of the shell, fractured shell. A larval shell was considered affected by NikkomycinZ treatment if one or more of the described phenotypes were applicable. Only motile individuals were taken into account. Fig. 3 shows the percentages of individuals with abnormally developed shells grown in the presence of NikkomycinZ with respect to the criteria described above. With progressive incubation time, the NikkomycinZ treatment from day 2 on induced a steady increase of abnormally developed individuals in the range of three times higher than observed in the control culture (Fig. 3a). Similar results were obtained from cultures incubated from day 5 on. Three days of a treatment with 10 μM NikkomycinZ slightly increased the number of observed shell defects in the population (Fig. 3b). No significant effects of the NikkomycinZ treatment with respect to shell development were observed at the binocular microscope level at 80 × magnification for the older stages of 8–15 days and 12–15 days (Fig. 3c,d).
Morphological effects of NikkomycinZ treatment on the organism scale (> 100 μm)
It was observed in Mytilus galloprovincialis populations grown in the presence of NikkomycinZ that the growth rate of the larval shell is reduced relative to the growth rate of the body tissue. Despite the fact that shells were not adequate in size and thus not suitable to envelope the organism, the animals were still alive and motile as observed by video light microscopy (see Additional Files 11, 12, 13, 14, 15). Fig. 4a shows one representative example (compare also between control and NikkomycinZ treated populations in the video data supplied as Additional Files 5, 6, 7, 8, and 12, 13, 14, 15, respectively). The second main feature observed in NikkomycinZ treated individuals was the asymmetry of the two shell valves. Scanning electron microscopy of shells extracted from NikkomycinZ treated individuals confirmed this impression gained from video microscopy in more detail: the two valves differ significantly in size, thus forming an asymmetric shell where the rims do not fit each other (Fig. 4b). Summarizing the effects on the 100 μm – 1000 μm scale, the asymmetry of the valves, a reduced size of one or both valves relative to the organism's body size, and also a slightly reduced size of the whole individual were among the phenomena observed comparably more often in NikkomycinZ treated cultures.
Additional File 12: Video microscopy of 8-day larvae after 6 days of NikkomycinZ treatment. Video microscopic (32× objective) demonstration of the behaviour and phenotype of 8-day larvae after 6 days of treatment with the chitin synthase inhibitor NikkomycinZ. The overall motility of the larvae decreased as compared to untreated control cultures. Note that NikkomycinZ appears to have a dramatic effect on shell development. The size of the shells is comparable to 2-day larvae. The hinge is not a straight line, and shell edges appear irregular or undulated. (MPG 4 MB)
Additional File 13: Video microscopy of 12-day larvae after 7 days of NikkomycinZ treatment. Video microscopic (32× objective) demonstration of the behaviour and phenotype of 12-day larvae after 7 days of treatment with the chitin synthase inhibitor NikkomycinZ. The overall motility of the larvae decreased as compared to untreated control cultures. Note that NikkomycinZ appears to have a dramatic effect on shell development. The formation of the umbo was prevented (see additional file 8). The size of the shells is comparable to 5-day larvae. The most prominent features were curved hinges and malformed shell edges. Shells of some individuals were too small to host the organism completely. (MPG 2 MB)
Additional File 14: Video microscopy of 12-day larvae after 4 days of NikkomycinZ treatment. Video microscopic (32× objective) demonstration of the behaviour and phenotype of 12-day larvae treated from the 8th day on with the chitin synthase inhibitor NikkomycinZ. Even four days of treatment with NikkomycinZ have similar effects on shell development of individuals as described in additional file 13. Note that less affected individuals were highly motile and therefore out of focus in this data set. (MPG 2 MB)
Additional File 15: Video microscopy of 15-day larvae after 7 days of NikkomycinZ treatment. Video microscopic (32× objective) demonstration of the behaviour and phenotype of 15-day larvae treated from the 8th day on with the chitin synthase inhibitor NikkomycinZ. Even in later developmental stages, NikkomycinZ induced characteristic effects on the shell development of living individuals. Note that the previously straight hinge (see additional files 7 &8) appears now curved. This indicates that NikkomycinZ interferes with the remodelling of the hinge region. The fact that shells are much smaller than in the untreated control cultures (additional file 8) suggests that either the solubility of the newly formed shell is increased, or lateral shell growth is blocked by the chitin synthase inhibitor drug. Note that also shell remnants of larvae that died at undefined age are present in this data set. (MPG 5 MB)
Structural effects of NikkomycinZ treatment on the tissue scale (10 μm – 100 μm)
Larval shell valves grown in the presence of NikkomycinZ exhibited drastic abnormalities. As demonstrated in Fig. 5a, the rims (edges) of most NikkomycinZ treated Mytilus galloprovincialis shells exhibited undulations. Such undulations were not observed in the control populations cultured in the absence of NikkomycinZ. Especially at the growth front, the rims of the valves appeared deformed or squeezed. In particular cases, the growth lines were curving or appeared rolled-up (Fig. 5b). The hinge line of NikkomycinZ treated animals was not straight but curved or undulated along the provinculum connecting the two valves (Fig. 5c). The development of the umbo stage was usually inhibited or delayed in NikkomycinZ treated larvae; in other words, the shell valves appeared comparably "flat" rather than cone-shaped. The outer surfaces of NikkomycinZ treated larvae showed unusual undulations on the length scale of square microns (Fig. 5d, circles). Growth lines were missing completely or were irregular in shape (Fig. 5d, arrowheads; compare control specimen in Fig. 1b).
Ultrastructural effects of NikkomycinZ treatment on the subcellular to cellular scale (<1 μm – 10 μm)
Inner surface layer – lateral growth and thickening of the larval shell
The shell edge of Mytilus galloprovincialis larvae usually appears smooth and compact. As demonstrated in Fig. 6a, the inner shell surface appears to consist of submicron flakes, covered by a different kind of fine dispersed granular material on the surface. The inner surface of shells extracted from larvae grown in the presence of NikkomycinZ appeared irregular and hackly shaped (Fig. 6b). Additional irregular agglomerates (Fig. 6b, dotted lines) were associated with the inner shell surface. These phenomena were predominantly observed at the newly formed shell edge. In principle, the same features as described for 5 day old larval shells (Fig. 6a,b) are present, and even more pronounced in 22 day old shells (Fig. 6c,d). The edge of shells extracted from an untreated control population (Fig. 6c) is smoothly fringed, and the material appears compact from the shell's centre to the rim. Again, the material consists of flakes that fit to each other like pieces of a puzzle. The surface is still covered by a fine dispersed material. NikkomycinZ treated 22 day old larvae formed valves with irregular rough margins (Fig. 6d). The inner surface was covered with square-edged flakes. It was observed that the size of these flakes decreases towards the shell margin, which indicates either the interference of NikkomycinZ with at least two different growth mechanisms (lateral growth, thickening), or a variation in the effective concentration of NikkomycinZ (see discussion for details).
These shells (Fig. 6d) also lack the granular layer, which usually covers the flake-like material on the inner shell surface (also compare Fig. 6c). The uncovered flakes (Fig. 6d) have evident interspaces between them, which also decrease towards the shell edge.
Fig. 7a–d show the inner surface of central parts of shells of 5 day old (Fig. 7a,b) and 19 day old (Fig. 7c,d) larvae, respectively. In these areas, the NikkomycinZ treatment (Fig. 7b,d) affected predominantly the surface of the material. The overall structure of the inner shell's surface did not differ significantly from the control. However, the fine structure of the individual flake's surface did: As demonstrated for a 5 day old larval shell in Fig. 7a, the surface texture of the flakes formed without NikkomycinZ appears fine-dispersed granular and compact. When the larvae were grown in the presence of NikkomycinZ from day 2 on, the inner surface of the 5 day old larval shells showed irregular agglomerates (Fig. 7b dotted lines) that were associated with the shell's surface. The shell flakes (several hundreds of nm in diameter) were interspersed with numerous slits of a few nm in diameter (Fig. 7b, arrowheads). In later developmental stages the inner surface of larval shells is usually smooth, homogeneous, compact, and the shell flakes are more confluent than in the younger developmental stage. This is demonstrated in Fig. 7c for a 19 day old individual. Once larvae were grown in the presence of NikkomycinZ from day 8 on until the same age (Fig. 7d), their shells exhibited a porous and flake-like structure on their inner surface, which is structurally comparable to the one described for the 5 day old larvae treated with NikkomycinZ from day 2 on (Fig. 7b). It can be concluded from these results that a partial inhibition of chitin synthesis by NikkomycinZ influences not only the lateral growth and the thickening of the larval shell, but also the fine-tuning of interfaces of macromolecular components that aggregate with the mineral phase into the final shell.
Outer surface layer – interference of NikkomycinZ with periostracum formation
The outer surface of Mytilus galloprovincialis larval shells appears smooth in the prodissoconch I stage, whereas the prodissoconch II exhibits regular growth lines, and a well-defined, smooth edge (Fig. 8a). If larvae are exposed to NikkomycinZ, the appearance of the growth lines of the outer surface of prodissoconch II changes. This is shown in Fig. 8b. At higher magnification, the material deposited in the presence of NikkomycinZ at the shell's edge by an 8 day old larva reveals irregular growth lines and a porous fine structure (Fig. 8b, inset). In older stages of shell development, the influence of chitin synthase inhibition by NikkomycinZ is predominantly apparent at the shell's edge. The edge of untreated shells consists of globular elements that are regularly deposited on the outer shell surface on top of an inner layer. The particles closest to the shell edge appear embedded into the inner, homogeneous layer. As demonstrated in Fig. 8c, the globular particles are covered by a fine-dispersed homogeneous layer in the previously formed shell parts (Fig. 8c, arrowheads). Fig. 8d demonstrates that this fine-dispersed layer is not completely homogeneous, but porous in the shells of NikkomycinZ treated larvae cultures. The respective close-up views show more clearly the differences in the fine structure of this layer formed in the absence (Fig. 8e) and presence (Fig. 8f) of NikkomycinZ. It has to be taken into account that the appearance of these layers is influenced to a certain extent by partial etching, due to an exposure to deionised water during the preparation for SEM imaging. There is also no protection against etching by the periostracum, which has been removed during the previous sodium hypochlorite extraction of larval shells. These observations suggest that even the formation of the layer next to the periostracum is in some way influenced by the NikkomycinZ inhibited chitin synthesis. When NikkomycinZ was applied to larvae cultures in the very early veliger stage, the structure of the outer surfaces of both, prodissoconch I and II were affected.
Hinge formation and hinge ultrastructure
A straight hinge line is characteristic for healthy bivalve larvae. We observed that a big proportion of individuals that grew in the presence of NikkomycinZ revealed a curved hinge (Fig. 5c). The formation of the hinge teeth was affected by NikkomycinZ as demonstrated in Fig. 9. The hinge of a larval shell grown in the absence of NikkomycinZ contains up to 24 teeth periodically arranged in two rows, one per valve, on the 8th day after fertilization (Fig. 9a). Each hinge tooth has a compact cubic shape with a smooth, granular surface structure (Fig. 9b). The size of the hinge teeth varies from the centre of the hinge line towards the edge. When NikkomycinZ was added to the culture medium on day 5, the hinge of 8 day old larvae did not develop well-shaped hinge teeth. An extreme example of one specimen without any visible hinge teeth is shown in Fig. 9c. The hinge teeth of another 8 day old larvae grown in the presence of NikkomycinZ are shown in detail in Fig. 9d. They appear like the fragments of hinge teeth, probably due to either an increased dissolution of shell material or limited deposition of chitin, and thus limited deposition of other shell material.
The influence of NikkomycinZ on the development of hinge teeth is strongly dependent on the time frame of NikkomycinZ application. In older developmental stages, hinge tooth formation is not completely inhibited by NikkomycinZ. However, we observed structural alterations that might predominantly affect the functionality of the youngest formed hinge teeth. For comparison, the hinge of a larval shell from the control culture without NikkomycinZ is shown in Fig. 9e. Even the youngest teeth in the centre of the hinge represented at least small protuberances. The transition region between the smaller (Fig. 9e, arrow) and the bigger hinge teeth is shown in detail in Fig. 9f. All teeth exhibited smoothly curved edges. Each tooth spans the whole cross-section (thickness) of the shell's hinge throughout the complete line. The picture changes, once NikkomycinZ was present in the culture medium during shell growth (Fig. 9g,h). It is obvious that especially the smaller hinge teeth (Fig. 9g) were not well formed. As observed more clearly at higher magnification (Fig. 9h, arrow indicates the zoom-in position in Fig. 9g), such hinge teeth did not span the whole thickness of the shell. The edges of the hinge teeth did not confine cubic elements. The same structural features applied to the bigger hinge teeth. The close-up view of the small hinge teeth (Fig. 9h) revealed that the building blocks of the hinge region in larvae grown in the presence of NikkomycinZ consisted of small, flat, and sharp-edged prismatic building blocks with a planar upper surface. Such sharp-edged building blocks were never observed in hinges of control larvae, which were smoothly curved-edged, and which were equipped with a fine-dispersed globular surface cover (Fig. 9b,f). These results indicate a direct interference of NikkomycinZ with the formation and remodelling of hinge teeth in the bivalve larvae of Mytilus galloprovincialis throughout development.
Effects of NikkomycinZ treatment on the crystallization and molecular self-assembly scale (Å – 100 nm)
Several observations indicated that the effects of the chitin synthase inhibitor NikkomycinZ did not only cover the length scales of the organism, organ, tissue, cells, and subcellular compartments, but as well the mineralization process. Several prominent features have been discussed in the previous section, such as the sharp-edged appearance of building blocks of the hinge teeth (Fig. 9h). The presence of flat prisms in NikkomycinZ treated animals (Fig. 6d, Fig. 9h) instead of granular material indicates a strong interaction between the NikkomycinZ sensitive biochemical synthesis pathways such as chitin synthesis and the way of mineral deposition and crystallization. In the following, two additional key observations with respect to shell mineralization are presented: The first one refers to structures that were obtained presumably due to partial dissolution of the mineral phase in distilled water, to which the samples were exposed during the sample preparation for scanning electron microscopy. The dissolution process apparently affected larval shell preparations from NikkomycinZ treated and non-treated cultures, each in a different manner. An example for a non-treated 19 day old shell is given in Fig. 10a. The outer surface of the shell edge consists of columnar depositions that are arranged in a regular manner. The surface of each column is smooth, and each column or stalk is terminated by a globular structure (~50 nm in diameter). The columns or stalks are bridged between each other (Fig. 10a, arrowheads) by elongated structures that appear to exist separately without any stalk. The hollow space between the columnar structures indicates that the original shell texture was altered by extended etching in deionised water (for comparison, see Fig. 8e). Comparably dissolved edges of shells extracted from NikkomycinZ treated cultures appeared differently (Fig. 10b). No regular columns were observed, and neither smooth globular edges, nor elongated bridges were present. The overall appearance of the dissolved shell edge was irregular and porous. The porosity of each particular columnar equivalent in Fig. 10b to the smooth globular edged columns in Fig. 10a appears self-evident. Conceivably, the dissolution process seems to affect the mineral composite structures obtained in the presence and absence of NikkomycinZ each in its own way. The formation process and, conceivably, the materials properties of the two mineral composites are therefore different. As shown in Fig. 9d, even the hinge region is probably more sensitive to dissolution due to NikkomycinZ treatment during its formation.
The second key observation refers to polarized light microscopic analyses of NikkomycinZ treated larvae (Fig. 11). Almost no birefringence is observed in 2 day old larval shells (Fig. 11a) due to the fact that the shell consists mainly of amorphous calcium carbonate. The increased birefringence of a 5 day old larval shell that was grown in the absence of NikkomycinZ is shown in Fig. 11b. Three major shell phenotypes with respect to birefringence were observed in larvae that were grown in the presence of NikkomycinZ (Fig. 11c–e). As demonstrated in the polarized light microscopy image (Fig. 11c), larval shells that were too small compared to the size of the organism ("half-naked" bivalve) exhibited the characteristic dark cross (Fig. 11c, arrowheads), which indicates a more or less radial arrangement of crystals in the larval shell [51]. The overall birefringence, and thus the crystallinity of such shells was comparably high, taking into account that this shell part represents to a large extent the first formed shell (prodissoconch I) which is supposed to consist of a large fraction of stable amorphous calcium carbonate (ACC) (see Fig. 11a for comparison with a 2 day old control larva, and the central shell part (prodissoconch I) of the 5 day old larva in Fig. 11b). With respect to the highly irregular shape of such shells, this result suggests that the inhibition of chitin synthesis by NikkomycinZ does not necessarily interfere with the initial deposition (radial arrangement) of crystals, but rather with the local stabilization or controlled transformation of ACC into aragonite. A second characteristic birefringence phenotype of an otherwise "healthy" looking shell was the split-up appearance of the dark cross (Fig. 11d, arrowheads), which could either be an artefact due to NikkomycinZ induced shell undulations, or indicate that the undulations are caused by NikkomycinZ induced alterations in the crystallization process. Both, the increased birefringence and the splitting of the dark cross were observed as well in shells exhibiting a curved hinge due to NikkomycinZ treatment (Fig. 11e). In general, the shell size of 5 day old NikkomycinZ treated larvae (Fig. 11c–e) was comparable to 2 day old untreated larvae (Fig. 11a) and thus smaller than the regular size of 5 day old untreated larvae (Fig. 11b). Although we currently lack a detailed comparison of cross-sections, our visual microscopic impression was that shells of NikkomycinZ treated larvae appeared more transparent, or thinner than shells of control animals.
Effects of NikkomycinZ treatment on the physical properties and functionality
From all the previously described structural phenomena it can be deduced that NikkomycinZ affects shell formation and thus also the functionality of the larval shell. One of the key properties of mollusc shells is their remarkable fracture resistance compared to the quantity of material used. Even though chitin is not the only component responsible for the integrity of this complex composite ceramic material, it appears reasonable that the synthesis of chitin is coordinated with the secretion of other organic and inorganic shell precursors. Therefore, it is unsurprising that the fragility of larval shells grown in the presence of NikkomycinZ is increased, as shown in Fig. 12. The break lines of such shells follow the borders of the puzzle flakes as demonstrated in Fig. 12a. Furthermore, the outer shell surface appears brittle. This brittleness may also induce the formation of holes that were observed in some shell regions (Fig. 12b).