Anchored by Chemistry: The Adaptations of Barnacles and Limpets 

Laetitia Abdallah, Mohammad Jibran Budullah, Lucas Chan, Ralph El Hajj

Abstract

The chemical properties of barnacles and limpets were analyzed in this paper. These organisms use many chemical and biochemical phenomena to strive in their respective ecological niches. First, barnacles and limpets show complex mating behavior with different reproduction mechanisms, each governed by different chemical interactions. Furthermore, barnacle larvae use different chemical cues to find a suitable settling location. On the other hand, limpets use chemical techniques to defend themselves against predators. Moreover, adult barnacles use complex proteins to secrete a cement-like material, which they use to adhere to numerous substrates permanently. Similarly, limpets secrete mucus, called pedal mucus, with the interaction of multiple glands. Finally, to keep their radula robust, limpets undergo complex biomineralization processes to embed iron oxide into their radula. Hence, it has been clearly shown that chemical processes and phenomena govern the very fundamentals of barnacles and limpets. 

Introduction

The intertidal zone is characterized by rocky shores, rough currents, and drastically varying water temperatures (Adey & Loveland, 2007). Being sessile organisms living in this region, barnacles and limpets must devise solutions to various challenges that stem from this unique environment. Since the former are completely immobile upon reaching adulthood, and the latter have very limited locomotion, both must ensure that they can feed and reproduce within the area immediately around them. They must ensure that they do not fall prey and that they can adequately protect their colonies from the prying eyes of predators. Moreover, they must cope with the harsh environmental conditions of the intertidal zone to ensure that they are not swept away by the flow of the current or desiccated under the UV radiation from the sun. These organisms possess unique chemical adaptations that have allowed them to flourish in a rather difficult environment, making them particularly interesting to study. This paper will present a review of some of the most interesting chemical adaptations that make barnacles and limpets suitable for life in the intertidal zone. 

Reproduction

Barnacle Sexual Diversity

Barnacles exhibit a fascinating diversity of sexual systems which rise due to environmental stresses. The main drivers for the latter diversity are barnacles’ sessile lifestyle and the limited mating opportunities.  As a result, three types of sexualities are known to barnacles: hermaphroditism, the presence of both male and female gametes within one organism; androdioecy, the coexistence of dwarf males and hermaphrodites; and dioecy, the coexistence of dwarf males and females. In dense populations, hermaphroditism is prevalent since individuals can function as both males and females, maximizing reproductive success through both self-fertilization and cross-fertilization. Barnacles achieve the latter through cross-fertilizing with nearby individuals using their relatively long penises, as seen in Fig. 1 (Yusa et al., 2011).  

Fig. 1 External body form and sperm leakage of the hermaphrodite stalked barnacle Pollicipes polymerus. (a) relaxed penis (arrow) and feeding legs. (b) sperm leakage to cross-fertilize nearby barnacles (Barazandeh et al., 2013) 

In lower-density populations, barnacles develop into dwarf barnacles, which are smaller in size and mature faster, allowing for earlier reproduction and minimizing resource expenditure on growth. In androdioecious systems, dwarf barnacles attach to hermaphroditic individuals to enable cross-fertilization. They are especially advantageous in low-density populations, where greater distance between hermaphrodites reduces the chances of successful mating. By attaching themselves directly to the hermaphrodites, the dwarf males ensure greater reproductive success as they are positioned closer to fertilization sites. In addition, lower populations also drive hermaphrodites to mature and develop into female barnacles due to extremely limited mating opportunities. Once matured, the female barnacles allocate all their resources to producing eggs, maximizing reproductive output. In this case, dwarf males attach to larger female barnacles, as seen in Fig. 2, to ensure fertilization without competing for egg production, often referred to as dioecy (Yusa et al., 2011).  

Fig. 2 Dwarf male located inside a larger female representing dioecy in Ibla cumingi . Adapted from (Lin et al., 2015) 

Biochemical transformations  

After fertilization, the embryo forms in the mantle cavity of hermaphroditic or female barnacles; as seen in Fig. 3. Once the embryo forms, it undergoes development into different stages before ultimately becoming a nauplius larva, which can swim and feed on its own. During the development of the embryo, significant biochemical transformations occur, which are key for the embryo’s growth. Some of these transformations include changes in carbohydrate and protein metabolism and the associated oxygen consumption (Barnes, 1965). 

Fig. 3 Internal anatomy of a Northern Rock Barnacle. Note the mantle cavity where the embryo develops (Department, 2012) 

Initially, the embryo exhibits a moderate amount of carbohydrates, in the form of glucose, soluble and insoluble polymers. As the embryo develops, carbohydrate reserves are being metabolized to fuel the growth and differentiation of embryonic tissue. For example, in Table 1 below, the B. balanus embryos started with an amount of 93.5 mg of glucose per 10⁶  eggs. In the span of approximately fifty days, the concentration of carbohydrates depleted to 18.9 mg per 10⁶ eggs, using almost 80% of their carbohydrate reserves to fuel growth and development of the embryo. A similar trend can be observed for protein nitrogen reserves for these the B. balanus barnacles. Continuous reduction in the amount of protein nitrogen suggests protein catabolism for energy as well. In addition, the catabolism of carbohydrate and protein reserves is accompanied by oxygen consumption. Oxygen uptake increases with increasing metabolic activity, providing adequate resources to oxidize substrates like carbohydrates and proteins. Thus, the intricate balance of biochemical energy required for optimal embryonic development is highlighted by the synchronization of oxygen consumption with carbohydrate and protein metabolism (Barnes, 1965). 

Table 1 Biochemical composition of eggs during development in Balanus balanus Barnacles (Barnes, 1965) 

Limpet reproductive strategies  

Similar to barnacles, various species of limpets exhibit different reproductive strategies. For example, limpets commonly exhibit hermaphroditism, where individuals possess both male and female reproductive organs simultaneously, and dioecy, in which individuals are exclusively male or female. An intriguing reproductive system is that of P. rustica, which is characterized by protandric hermaphroditism. Individuals exhibiting a protandric hermaphroditic reproductive system initially mature into males, then develop into larger females. Generally, for all limpets, the reproductive cycle is characterized by multiple developmental stages to their gonads, the reproductive organs. Gonadal development is called spermatogenesis in males and oogenesis in females. In male gonads, the gonadal tubules contain spermatogonia, spermatocytes, spermatids, and spermatozoa. Each type represents a different developmental stage of the gonad respectively, with spermatozoa being the fully developed; these stages of the gonad can be observed in Fig. 4. Similar to males, female gonads undergo development, where oocytes mature to prepare for fertilization. Once gonadal development is complete, fertilization happens externally. Sperms and eggs are released into the water and fertilize externally, developing into free swimming larvae. These larvae swim and develop until they find a suitable substrate to settle on (Prusina et al., 2014).  

Fig. 4 Spermatogenesis of Patella rustica limpets. A) earliest stage through D) later stage. SPG, spermatogonia; TW, tubular wall; SPC, spermatocytes; SPD, spermatids; SPZ, spermatozoa [Adapted from Prusina et al., 2014] 

Environmental influence on reproduction 

The reproduction of limpets is influenced by environmental cues such as temperature and chemicals. Temperature plays a crucial role in limpet reproductive cycles as it affects gonadal development and release of gametes, called spawning time. Limpets tend to reproduce depending on seasonal change, where warmer water is more favorable. Higher temperatures are favored since they accelerate metabolic processes, which include gonadal development. In addition, temperature determines the time of release of the gametes. When the temperature is optimal, limpets release their gametes to fertilize externally. This synchronization of sperm and egg release results in a greater success rate for fertilization. For example, in species like the Patella rustica limpets, reproduction occurs in late summer or early autumn when there is optimal temperature for gonadal development and spawning (Prusina et al., 2014). Furthermore, it has been found that chemicals like hydrogen peroxide (H₂O₂), and potassium chloride (KCl), influence the reproductive cycle of limpets. A study done on Patella caerulea limpets tested the spawning induction of the limpets under different chemical treatments. A higher concentration of H₂O₂, combined with the presence of KCl, was found to result in increased spawning. Therefore,  both, KCl and H₂O_2, contribute to the reproduction cycle of limpets, as they are naturally found in low concentrations in marine environments (Ferranti et al., 2018).  

Search and Settle 

Settlement-Inducing Protein Complex in Barnacles 

Barnacle larvae are challenged with the difficult task of finding a suitable spot to settle for the rest of their lives. Once chosen, they will not move from their initial settlement location. This presents multiple challenges: vulnerability to attack by predators, limited ability to reproduce, and challenges in finding food. Thus, selecting an appropriate place to call home is extremely critical to ensure the organism’s survival, and nature has provided barnacles with a remarkable way to achieve this (Aldred et al., 2018). 

To tackle the challenges that arise from a sessile lifestyle, barnacles are gregarious: organisms of the same species aggregate together and form large colonies. Not only does this afford a notable increase in reproductive success, but being present in such large numbers reduces the risk of predation (Clare, 2011). Yet, there can be downsides to living in a large group, as neighbouring barnacles will be competing against one another for space and food. Research has shown, however, that one species—Semibalanus balanoides—in fact has had increased success in foraging for food by being in a group. After a predator passes, neighbouring barnacles send an all-clear signal to one another so they can emerge from their shell and resume filter-feeding. It has been found that these signals allow gregarious barnacles to emerge faster than solitary ones (Clare, 2011). 

But how exactly do barnacle larvae detect the presence of other conspecific individuals? Evidence is accumulating that one or more chemical cues, secreted by adult individuals, are detected by the larvae (or cyprids), signaling the presence of an agglomeration. Upon detection of these chemical cues, the cyprids temporarily bind to the substrate and attempt to locate the colony. If found, they will analyse the substrate using chemo- and neuro-receptors and determine if its mechanical and chemical properties are suitable for settlement (Clare, 2011). Fig. 5 below illustrates this search-and-settle behaviour. 

Fig. 5 Settlement process of cyprids. a) Cyprids approach the substrate, perhaps after detecting a waterborne cue. b) Initial contact with the substrate. c) Searching behaviour and analysis of the substrate. d) The cyprids locate the colony. (Clare, 2011) 

This settlement cue is believed to be a water-soluble glycoprotein, referred to by researchers as the settlement-inducing protein complex (SIPC). Using a nitrocellulose membrane assay, the SIPC was isolated in one barnacle species, Balanus amphitrite, and proteins of similar structure were found in other examined species. It is believed that small differences in the settlement-inducing proteins of different species allow cyprids to distinguish between con- and hetero- specific individuals (Clare, 2011; Matsumura et al., 1998). 

Another protein, unrelated to the SIPC, has recently been isolated from B. amphitrite. It is believed that this protein may induce cyprids to approach the substrate and begin searching for a colony, as seen in Fig. 5 (a). However, the precise nature of this protein remains unknown, and researchers note that further evidence confirming its secretion by adults is needed to validate the hypothesis (Endo et al., 2009). 

Cyprids possess specialized antennules (sensory appendages) that allow them to detect these chemical cues. These antennules are further subdivided into four segments, as shown in Fig. 6 below (Abramova et al., 2019). The SIPC receptors are believed to be located in segments 3 and 4, but further research is needed to support this hypothesis. However, what is known is that the hair-like structures at the distal ends of these antennules, called setae, are highly innervated, and remarkable similarities have been found with other crustacean olfactory systems (Clare, 2011). As such, it is believed that the setae play a crucial role in sensory detection. Though researchers have yet to uncover the specifics of the chemical sensory cues related to settlement, and the ways in which they are perceived by cyprids, it is nonetheless remarkable that barnacles have developed such an elaborate system to finding the perfect home even before their physical development is complete. 

Fig. 6 a) Barnacle cyprid and its antennules, viewed under a stereomicroscope. b) Close-up structure of an antennule. Segments 1-4, and the setae, are visible (Abramova et al., 2019). 

Trail-Following in Limpets 

It is a well-documented fact that most limpet species will return to their home scar after a period of grazing and foraging (Cook et al., 1969). However, very little research has been done so far to investigate the process by which this homing behaviour takes place. Nonetheless, it is known that limpets do follow a specific path home, and this path may differ from its outward path. It is believed that the limpet orients itself by sensing topographic or chemical cues on the substrate, and that once arrived at its home scar, it detects an individualized chemical signal which triggers it to settle (Cook et al., 1969; Santina, 1994).  

The chemistry behind barnacle and limpet adhesion 

Adult barnacle underwater adhesion 

In acorn barnacles, the cement glands are grouped around the mantle chamber next to the basal portion of their body (Fig. 7A). Their cytoplasm is heterogeneous with two distinct regions: the synthetic region which is rich in RNA, and the secretory or storage region where synthesized barnacle cement proteins (BCPs) are transported and packed in large vacuoles with an acidic internal environment  (Liang et al., 2019). The acidic environment is beneficial for protein condensation to form high-density granules and for the self-assembly of nanofibrous structures. 

When the cement is secreted, the stored cement is delivered through an extracellular canal that includes collective canals (CCs), secondary canals (SCs), and primary canals (PCs). It then reaches the adhesive joint where the cement is secreted (Fig. 7B). Stalked barnacles have an almost identical cement apparatus with minor differences seen at the location of the gland cell, the heterogeneity of their cytoplasm, and they possess intracellular canals for cement secretion. This can be seen in Fig. 7 C and D (Liang et al., 2019). 

Fig. 7 The cement apparatus of adult barnacles. A, B) Schematics of cement glands and components for acorn barnacles. C, D) Schematics of cement glands and components for stalked barnacles (Liang et al., 2019). 

Proteins in barnacle cement

Underwater adhesion can be classified based on the different functions fulfilled by its bioadhesive components. The components can be further classified as surface and bulk components. Surface components are located at the boundary of the bulk glue and the substratum, while the bulk components are found in the innermost of the glue and are responsible for the cross-linking of different glue components. To achieve proper bioadhesion, these surface components need to form interfacial adhesion, and bulk components must develop bulk cohesion (Liang et al., 2019).  

Different barnacle species possess different surface and bulk components of the class of proteins called “barnacle cement proteins”. For example, M. rosa showed that its cp20k, cp19k, and cp68k proteins fulfilled the roles of surface components through non-covalent interactions, while cp100k and cp52k acted as bulk components by self-assembling into amyloid fibrils. However, in other barnacle species, such as A. amphitrite, the cp19k and cp68k proteins fulfilled the functions of both surface and bulk components (Liang et al., 2019). 

The amino acid composition of many barnacle cement proteins 

The primary structure of the barnacle cement proteins (BCPs)—their amino acid composition—will dictate most of their properties. Although the exact composition of each protein differs from species to species, it is possible to generalize some patterns in important proteins. In most barnacle species, cp100k and cp52k possess a high quantity of hydrophobic amino acids, with a high amount of Phe and Tyr (9.3% in cp100k and 15.7% in cp52k), a relatively high amount of Arg and Lys (11.5% in cp100k and 15.7% in cp52k), but a very small amount of Cys. Their average GRAVY (grand average of hydropathicity)—which describes how hydrophilic or hydrophobic a protein is on average— was of 0.089 and −0.081. This indicates their relative tendency to be hydrophobic, as their value are close to zero. On the other hand, cp20k do not possess as many hydrophobic amino acids, but rather is characterized in its very high composition of Cys. For example, in Mrcp20k and Balcp20k, their Cys content was of 17.5% and 17.1%. They also showed a high percentage of other charged amino acids, such as His (10.4%), Asp (11.5%), and Glu (10.4%) in Mrcp20k, and His (20.0%), Asp (11.5%) and Lys (9.5%) in Balcp20k. Finally, cp19k and cp68k show a high concentration of Ser, Thr, Gly, Ala, Lys, and Val, making up about 70% of the protein. Moreover, they have a very low amount of aromatic amino acids, with a composition of 1.8% of Phe in cp19k, and no Tyr or Trp, much alike cp68k (Liang et al., 2019). 

More onto surface components 

Studies have shown that BCPs such as cp20k specifically adhere to calcite, while cp19k could bind to many different substrata with diverse properties. Both were deemed as surface components. It has been further shown that the function of different proteins was correlated to their location in the barnacles. Supporting this empirical principle, it was shown that cp20k was produced between the barnacle calcite base and bulk cement, while cp19k was found between the bulk cement and the external surface (Liang et al., 2019). 

As barnacles can adhere to almost any surface, their surface components are capable of adhesion to many substrata. The cp19k and cp68k proteins contain many amino acids with side-chain amine or hydroxyl group, such as Cys, and it is believed they help the removal of water on the substratum. Moreover, it was shown that the presence of multiple positively charged Lys amino acid was able to displace the adsorbed cations on minerals to ease the binding of the proteins (Liang et al., 2019). 

The role of amyloid fibrils in barnacle cement 

With the use of microscale and nanoscale imaging techniques, the structure of barnacle cement on different surfaces has been greatly studied. For instance, it has been shown that the protein content of their cement is about 90%. Moreover, barnacle cement comprises nanoscale globules which self-assemble into microscale structures, such as dense meshes on certain metal surfaces or fibril meshes on soft surfaces as polydimethylsilane (PDMS). Such discoveries have pointed to the fibrillar morphology of barnacle cement.  

In the past few years, the view that amyloid fibrils form a vital component of barnacle cement has become prominent for three reasons. First, it has been shown that β-sheet, the structural basis of amyloid fibrils, is the primary secondary basis of barnacle-cured cement. Furthermore, it has been revealed through atomic force microscopy that the cured cement is composed of smooth, straight, and unbranched fibrils of 10 to 40 nm in diameter. Finally, the barnacle cement can be stained with an amyloid fibril-specific dye, Thioflavin T (Liu et al., 2017). 

Amyloid fibrils are made of nanofibers that self-assemble from non-covalent interactions between components rich in β-sheet secondary structures. They exhibit great mechanical stability and are resistant to enzymatic degradation (Liang et al., 2019). Studies have shown that proteins such as cp52k and cp100k could indeed self-assemble into amyloid-like fibrils in given conditions, and thus play stabilizing roles during adhesion. It has been demonstrated that hydrogen bonding between the backbones, and π-π stacking between aromatic amino acids are crucial for the self-assembly of those proteins into amyloid fibrils (Liang et al., 2019).  

Furthermore, Liu et al., researched a full-length 19kDa cement protein from a barnacle species called Balanus albicostatus, which is referred to as Balcp19k (2017). They isolated the protein and expressed it in Escherichia coli to investigate its self-assembly properties. By doing so, they showed that the time for the protein to fully self-assemble into amyloid fibrils, which was about 24 hours, was almost identical to that of a liquid barnacle cement to cure. Moreover, the solubility of the Balcp19k in 8 M urea and 6 M GdnHCl (guanidine hydrochloride) was the same as that of the barnacle-cured cement. Thus, those findings show the self-assembly tendency of the Balcp19k protein, implying how the self-assembly of amyloid fibrils plays an essential role in the curing process of barnacle cement and contributes to its insolubility (Liu et al., 2017).  

Limpets’ mucus 

Limpets alternate between suction and mucus for their adhesion to different substrata. It has been shown that they use different gels during locomotion and gluing. In the genus Lottia, the gel used for gluing has been studied, and it has been found that it presents a particular 118 kDa protein that is absent in the gel used for locomotion and suction. Further studies have then shown that this protein was needed for the stiffening of the gel, which may also help for the cross-link of the gel (Smith, 2010). 

Limpets’ mucus also shows peculiarities compared to other gels. Indeed, most other mucous gels depend on the tangling of protein-carbohydrates complexes. However, limpets’ mucus is predominantly made with proteins—making up about 3% of the mucus composition—while carbohydrates only make up about 1% of the mucus (Smith, 2010). 

Limpets possess specialized glands, each producing different secretory products. In the limpet P. vulgata, nine distinct glands have been identified, six of which secrete directly onto the limpet’s sole (Fig. 9). 

Fig. 9 Schematic of P. vulgata with its different glands identified (Smith, 2010). 

The glands identified in Fig. 9 as P2, P5, P8, and P9 are the only ones found on the surface of the sole. They secrete acid mucopolysaccharides, with a mixture of non-sulfated and sulfated sugar, along with some neutral mucopolysaccharides. The P9 gland was identified as a general epithelial mucocyte, also seen in many other gastropods—they are usually used for lubrification. The other glands, P2, P5, and P8, are believed to be used for adhesion. The secreted acidic mucopolysaccharides and their location at the surface of the sole are used to adhere to the substratum. Although acidic mucopolysaccharides do not inherently have adhesive properties, the acidic side favors cross-linking, which helps the adhesion of the limpet on the surface. Furthermore, as seen in Fig. 9, these three glands are subepithelial, a common feature in gastropod adhesive glands. The P8 cells are club-shaped and extend into the subepithelial region with a neck about 60 μm long. P2 and P5 glands are much thinner and are situated deeper into the subepithelial region. It has been noticed that the P5 glands were much more common around the foot’s edge, forming a sealing ring, as adhesive failure most often begins at the edge. It has also been shown that magnesium and calcium form almost two-thirds of the total inorganic composition of those cells’ secretion, which is important as calcium may greatly impact the mechanics of the gel as well as its adhesive properties. These glands are hence believed to be the main source of limpet adhesion (Smith, 2010). 

Limpets on Guard 

Unlike barnacles, which use gregarious settlement to deter predatory attacks and increase feeding efficacy, limpets use chemical techniques as a means of defense. This phenomenon has been exhibited in Siphonaria capensis, a limpet species off the coast of South Africa. This species coexists with another, Patella granularis, but it has been shown that P. granularis occurs disproportionately in the diets of most predators in the area (McQuaid et al., 1999). How is it then that predators seem to prefer one species over another? Generally, this is a result of one species being especially desirable, or others being difficult to catch or undesirable in some other way. In the case of S. capensis, the undesirability is believed to be achieved through chemical means of defence (McQuaid et al., 1999).  

Chemical defence can be expressed as a toxin present within the soft tissues of an organism. In general, this is accompanied by a bright coloration to warn predators of the presence of this toxin, thus deterring them (Halpin et al., 2008). Sometimes, the bright coloration is not present to deter possible predators, despite the presence of a toxin. This is the case for S. capensis. As the predator makes contact with the prey, the toxic or distasteful chemicals cause it to reject its prey. In some extreme cases, when the toxins are strong enough, the predator can be killed on the spot (Clucas, 2010; McQuaid et al., 1999).  

Defensive chemicals can either be sequenced de novo by the prey—that is, the prey synthesizes the chemicals internally, from scratch. They can also be sequestered from the diet by consuming other organisms that contain either the desired chemical itself or some precursor to it. Or, if it occurs naturally, the prey can extract the chemical by rubbing or rolling in it (Clucas, 2010). 

It is believed that S. capensis synthesizes the compounds needed for chemical defence de novo. Since its diet is chemically bland,  these limpets are unable to remove large algae from the shore and seafloor. They can only consume small algae on the surface of hard substrates. These algae do not contain complex chemicals, nor do they contain the desired defensive substances (McQuaid et al., 1999). 

Researchers tested the preference of various limpet-eating predators towards either S. capensis, a species which is believed to manifest a form of chemical defence, versus P. granularis, which does not. As the results show in Table 3, all four predators tested showed a significant preference for P. granularis. Furthermore, it was observed that the tube-feet of the starfish Marthasterias retracted upon contact with either limpet. However, with P. granularis they were re-extended within seconds, whereas it took minutes with S. capensis. This delay in recovery resulted in each individual taking significantly longer to return for a second prey after contact with S. capensis, and this is believed to be caused by a chemical present in the mucus secreted by the limpet. What is interesting to note is that Chorisochismus and Marthasterias systematically avoided P. granularis when the limpets’ shells were coated with S. capensis extract. As such, it is concluded that this preferential selection and avoidance is not caused by a behavioural or structural adaptation; rather, one or more chemicals secreted by S. capensis are believed to be responsible. Further studies are needed to determine the exact nature of these chemicals. 

Table 3: Number of live limpets eaten per predator (McQuaid et al., 1999) 

Strength in Nature 

The Chemistry of Limpet Radula Strength 

When feeding, limpets must constantly grind against rocky, uneven surfaces, which challenges their ability to maintain a strong, wear-resistant tool. This is why they have developed an ingenious approach whereby they naturally embed iron oxide minerals into their radula through a complex biomineralization process. 

Limpets’ mineralized teeth, shown in Fig. 10, are known to be among the strongest and hardest biological materials (Wang et al., 2022). 

Fig. 10 Scanning electron microscope image of limpet teeth (Viegas, 2015). 

The mechanical characteristics of limpet teeth arise from an exclusive, well-organized composite structure made of flexible chitin nanofibers separated by filamentous iron oxide crystals in the form of goethite (whose chemical structure is shown in Fig. 11), which is the biomaterial with the highest known tensile strength. In particular, the existence of a chitin scaffold was discovered in 1907; goethite was recognized as a crucial component in the 1960s; and in the 1980s, it was reported that each tooth is composed of highly organized chitin matrix that contains directionally oriented nanofibrous crystals of goethite (Sone et al., 2007). 

Fig. 11 Crystal structure of goethite, α-Fe(III)O(OH) (Icenhower et al., 2010). 

Limpet teeth begin as chitin scaffolding that accumulates iron oxide minerals throughout each developmental stage of the radula. Radula cells produce extracellular components that play an important role in chitin mineralization, resulting in a high-strength bio composite of interlaced chitin fibres and iron oxide crystals in a completely acellular process. The chitin fibers seem to be the site of nucleation for these linear deposits of goethite, which in turn control the crystals’ orientation (Rumney et al., 2022). 

The radula, a ribbon-like structure that supports several transverse rows of mineralized teeth, is used by these marine animals to scrape algae and other microbes off the rocks, where they are then consumed. The last ten or so rows of teeth are actually only used for scraping; when these teeth wear down and fall out, the radula, which is always expanding, advances new teeth into position (Sone et al., 2007). 

Along the whole length of the radula, teeth can be discovered at different stages of development. The α-chitin matrix and related proteins make up the newly created teeth inside the animal’s cavity. At least one iron and one non-iron biomineral may be found in mature teeth, which prompts intriguing issues about ion transport as well as the temporal regulation of mineralization (Rumney et al., 2022). 

The process of mineralization in limpet teeth starts with the elongated goethite crystals that are tightly linked to the organic matrix. Later, when the process moves closer to the mouth, opal—a hydrated amorphous silica phase (SiO₂·nH₂0)—implores the space between goethite crystals. The fracture and wear properties of the functioning tooth are largely determined by the arrangement of the goethite crystals. The orientations of goethite crystals in teeth are thought to be controlled by the chitin fibers of the organic matrix. This leads to teeth that are resistant to fracture and self-sharpening (Rumney et al., 2022). 

As shown in Fig. 13, sections of unmineralized limpet teeth, mostly made of α-chitin (whose chemical structure is shown in Fig. 12), are very thin and exhibit parallel chitin fibers that fluctuate in width and spacing on a regular basis. The preparation methods used for the Epon-embedded, methyl cellulose, and cryo- Transmission Electron Microscopy (TEM) sections resulted in differences in fiber size and organization, with the Epon sections displaying larger strands as a result of dehydration (Sone et al., 2007). 

Fig. 12 Structure of different chitin conformations (Roy et al., 2017). 

Fig. 13 TEM micrographs of unmineralized limpet teeth show fiber structures using (a) Cryo-TEM, (b) Methyl cellulose, and (c) The insets are electron diffraction patterns obtained by Fast Fourier Transformation (FFT) [Adapted from (Sone et al., 2007)]. 

The formation zone, a soft tissue structure shaped like a bulb, is where the tooth growth process starts, and this is where the limpet radula is arranged like a conveyor belt (Fig. 14 a). It has been demonstrated in other gastropods that teeth arise from this area in their ultimate form. The formation zone must constantly create teeth throughout the limpet lifecycle. As seen in Fig. 14, the four unique phases of teeth in the limpet are immature (Stage I, the first 15–20 rows without any indications of mineralization), early maturing (Stage II, the following 12 rows), late maturing (Stage III, the next 30 rows), and mature (Stage IV, the remaining rows). The degree of mineralization and tooth formation varies between these stages (Rumney et al., 2022). 

Fig. 14 The formation zone of the limpet radula [Adapted from (Rumney et al., 2022)]. 

Furthermore, proteins coat the chitin matrix. It has been shown that the α-chitin matrix is composed of organized, tightly spaced chitin fibers with just a few nanometers separating neighboring fibers, particularly in the limpet species Parella vulgata, P. athletica, and P. caerulae. These very hard minerals in limpet teeth are used to scrape rocks in search of algae (Wang et al., 2022).  

It was discovered that the cusp is made up of needle-like goethite crystals, but the tooth base is composed of microcrystalline (superparamagnetic) and disordered goethite (Wang et al., 2022). The organic matrix is in charge of the organization of the shell mineral and controls the polymorphic type, size, and orientation of shell-forming crystals (Rodríguez-Navarro et al., 2006a). This control results in needle goethites with smaller crystals in the anterior region and bigger crystals in the posterior region, and they are oriented along the direction of the fibers (Wang et al., 2022).  

The radula is first released as a mixture of tyrosine-rich proteins and chitin that may undergo quinine tanning to crosslink. Then, as it moves toward the front, the cusps and bases of the radula’s teeth absorb inorganic salts (Fe, Si, P, and Ca) that settle inside the organic matrix and give them a particularly hard texture. The substrate is compressed and dragged over by the recurved, hook-like teeth. Therefore, denticles need to be more durable than substrates made of calcium carbonate. This is accomplished by impregnating the tooth with goethite (FeOOH) (shown in Fig. 11) and opal (SiO₂. nH₂O), which together comprise around 10% silica and 12% ferric oxide by mass of the tooth (Rinkevich, 1993). 

Ultimately, limpets’ constant struggle when feeding involves clenching their teeth against rough surfaces. A precise biomineralization process that embeds iron oxide crystals like goethite in chitin strands is their clever, natural answer. Because of this, its radula becomes a very durable and self-sharpening instrument that can function well in the harshest environments. 

Building Barnacle Armour 

Sessile barnacles’ mineralized exoskeleton is essential for shielding the organism from predators and the tide’s fluctuating hydration levels. The barnacle grows rapidly in its base region in the 6–37 hours that follow metamorphosis. While there is remarkable development during the first few hours after transformation, mineralization is noticeably lacking. The beginning of exoskeleton mineralization takes many hours to days, despite its critical role in defense (Metzler et al., 2020). 

As shown in Fig. 15, calcified basal plate is topped by six parietal (lateral) shell plates that shield barnacles. Calcite is found in shell plates, which are formed of a matrix of chitin, acidic proteins, and sulfate-rich polymers. These components combine hardness (from calcite) with toughness and flexibility (from chitin) to contribute to the barnacle’s defensive qualities (Nardone et al., 2018). 

Fig. 15  Main plates of Pollicipes (Barnacle species) : (A) Right side, (B) posterior side, (C) left side, (D) anterior side (Álvarez Fernandez et al., 2010). 

In addition to intracrystalline organics, the calcite component of barnacles includes trace amounts of magnesium (1-2 mol%) and strontium (about 0.5 mol%). Compared to its geological counterpart, the calcite in the exoskeleton of barnacles is more disordered at the atomic level. Because the crystals in the barnacle shell vary in size, shape, and orientation, the calcite material within is more resilient to fracture (Metzler et al., 2020). 

The organic matrix’s concentric lamellae divide the mineralized layers that make up the barnacle shell. Chitin is the primary constituent of these lamellae; nevertheless, in order to get an infrared spectrum comparable to that of ordinary chitin (shown in Fig. 16), the organic matter has to be treated with NaOH, suggesting a tight association between chitin and proteins. It is anticipated that these chitin-associated proteins include Trp and Tyr residues, which may account for the significant autofluorescence seen prior to deproteination (Fernández et al., 2015). 

Fig. 16 Fourier-Transform Infrared Spectroscopy or FTIR spectra of various chitin-based materials (Kasprzak & Galiński, 2021). 

It’s interesting to note that from the inner to the outside shell surface, the crystallite size measured in the various structural components of the barnacle shell decreases. As shown in Fig. 17, the crystallinity of the shell mineral diminishes as the quantity of organic components rises (Rodríguez-Navarro et al., 2006b). 

Fig. 17 Evolution of crystallite size across the barnacle shell wall in relation to the organic matter content. A, B, C, and D correspond to the internal radii, internal pariete, external radii, and external pariete, respectively (Rodríguez-Navarro et al., 2006b). 

Fig. 18 Crystal structure of calcite at ambient conditions, which visualized using the software Vesta (Zhang et al., 2024). 

Upon examining longitudinal sections of a parietes using optical microscopy, it can be observed that this structural unit is composed of two laminas: an inner lamina consisting of mineral layers, approximately 20μm thick, separated by thin organic sheets, and an outer lamina with a massive microstructure composed of randomly oriented calcite microcrystals (Fig. 19 A and B) of equiaxial calcite (shown in Fig. 18) . Each mineral layer’s crystals extinguish synchronously under cross-polarized light, indicating a high degree of crystal orientation. Additionally, it is evident that both structural elements are rather porous than completely dense. A transverse slice of a parietes is seen in Fig. 19 C. It is composed of a mineral layer arranged like a bunch of grapes, and an outer organic-rich lamina with channels that run from the base to the cone tip. Every grape-like unit splits out from a central line that runs through the center of the structure and is rich in organic matrix. A central line of organic material also crosses each grape-like unit. The crystals in these units showed an undulating extinction under cross-polarized light (Rodríguez-Navarro et al., 2006b). 

Fig. 19 Various sections of the barnacle shell: photomicrographs of parietes layers viewed under cross- and parallel-polarized light. (A) longitudinal section of the pariete outer lamina (B) Longitudinal section of the pariete internal lamina (C) Transverse section (Rodríguez-Navarro et al., 2006b). 

By developing a mineralized shell made of calcite and organic molecules that solidifies with time, barnacles can withstand the severe conditions of their surroundings. 

Conclusion 

Barnacles and limpets exhibit fascinating design solutions which help them adapt to their severe and ever-changing environments. Regarding reproduction, barnacles face extreme obstacles due to their sessile lifestyles. Mating is deemed difficult due to lack of motility, which is the evolutionary cause of barnacles’ relatively large penises. In addition, less dense populations struggle even more to repopulate. A design solution for the latter struggle is the diverse sexual systems adopted by barnacles depending on the population density. Barnacles exhibit hermaphroditism, androdioecy, or dioecy, depending on the system that maximizes successful reproduction, rendering nature as the Master of Design solutions.  

A ubiquitous environmental stress for limpets is the threat of predators. To avoid predation, limpets were found to be using toxin-based defenses. Some limpets were found to produce these toxins internally, while others consumed these toxins or even rolled in the toxin to coat their exterior shell. These adaptations represent remarkable design solutions which help limpets to survive in the wild. 

One issue that barnacles must cope with is the harsh and constantly changing environment of the intertidal zone. The solution is to construct a robust and stable conical shell of polycrystalline calcite that provides shelter, allows for continuous filter feeding when the conditions permit, and accommodates growth. 

Limpets face a dual challenge, staying attached to surfaces in rough waters while avoiding infections. Their solution is a versatile mucus that acts as a strong adhesive, securing them to rocks, and provides antibacterial protection to ward off pathogens. This mucus serves as both, a physical and biological defence, ensuring limpet survival in the ever-changing circumstances of their habitat. 

Indeed, barnacles and limpets show nature’s ability to adapt to external circumstances. Their distinct adaptations offer insights that might stimulate creative solutions to engineering problems, especially in fields like environmental resilience, biomimetics, and materials science. 

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