Small Bodies, Big Potential: Chemically Bonding Salps and their Environment

Keywords: Salps; Tunic; Biogeochemical cycling; Innate immunology; Sensory cells; Carbon cycling

Abstract

Salps are gelatinous invertebrate marine organisms belonging to the phylum Chordata. The chemical and cellular processes that occur within their fragile bodies contain fascinating and novel information in various subjects varying from organic chemistry to immunology to environmental science. Salps are encased in a transparent tunic that does not absorb visible light, making them nearly invisible to sighted predators. This tunic is mainly composed of a network of crystalline cellulose molecules, leading to a durable, yet lightweight exoskeleton. The high tensile strength of cellulose fibers is due to the hydrogen bonding and crystalline structure that protects them from environmental debris. The preference of possessing the cellulose Iβ allomorph provides better flexibility for the salp’s movement compared to cellulose Iα or cellulose II. The innate immune system of salps functions with a combination of reactive enzymes such as hydrolytic enzymes and phenoloxidase, proteins such as specialized anti-microbial proteins, signal factors such as tumor necrosis factor-α and carbohydrate-binding lectins. Communication between salps occurs within chains and relies on synaptic activity between epithelial cells and specialized sensory and button cells. Finally, salps function as an efficient cleaner condensing particulate organic matter and moving carbon along the biogeochemical cycle in marine ecosystems. These studies reveal the important information behind the salps that can be utilized in bioengineering fields.

Introduction

The loss of many genes and structures in salps is the main cause for the difficulty in deducing the ancestors and evolutionary path of salps. Tunicata is a diverse subphylum whose members have evolved to efficiently survive in all depths of marine habitats. Tunicate species genomes can range from 70-190 Mb in their genomes, compared to the human 3 Gb. This is due to the loss of intergenic regions, the loss of certain developmental Hox genes, and high density of genes in operons. Phylogenic analysis of genes found in the nucleus shows that tunicates have evolved at rapid rates, thus the relationship between salps and other tunicates is difficult to pin down. Salps have undergone secondary simplification at some point during their evolution, meaning complex structures and features present in their ancestors are completely absent in salps, further obscuring evolutionary relationships (Holland, 2016).

What allowed these tunicates to display more evolutionary adaptations than their relatives? One hypothesis is that early determination of cell fates, or determinate development, has led to divergence of traits at later developmental stages (Holland, 2016). However, their evolution and ancestry has yet to be proven. One example of successful tunicate evolution is seen in members of the class Thaliacea such as salps, with selective advantage from asexual reproduction since it allows them to rapidly grow in population in favorable conditions such as high blooming of phytoplankton (Holland, 2016). Tunicates include three classes, where all the species in Ascidiacea and Thaliacea (exception with Appendicularia that do not have a tunic, but still secrete cellulose) possess tunics, an adaptation important for protection and predator evasion (Zhao & Li, 2014). Salps have also evolved to function as a superorganism and have developed mechanisms to communicate along the chains or aggregates. As individuals or as a group, salps may appear simple to the untrained eye, but possess many adaptations to their environment and contribute to the biogeochemical carbon cycling of the oceanic environments.

Salps employ innate immune systems to combat infection along chains. Innate immune systems consist of generalized immune functions that allow for quick response to all manners of immunological threats, in contrast to pathogen-specialized adaptive immune function. Upon infection, molecules in the tunic and circulatory network identify foreign material like invading bacteria and viruses, such as lectin tagging bacteria, by binding to specific sugars on its surface. Additional signaling factors are released by granular cells (immune cells that carry proteins in granules) that recruit phagocytes (cells that ingest or encapsulate foreign material) and cytotoxic cells (cells that release materials toxic to pathogens) to travel to the site of infection. The defense mechanisms can be categorized as cellular response, involving the destruction of non-self molecules and cells by phagocytes, or humoral response, involving the secretion of chemicals or proteins into extracellular fluids to destroy foreign cells (Franchi & Ballarin, 2017). The close evolutional relationship between tunicates and vertebrates emphasize the importance of studying their immunological system, as it contains information of the formation and development of innate immune systems in our body (Cima et al., 2018).

1. Tunic and Body Composition

1.1 Body adaptations

The name of the subphylum Tunicata comes from the protective outer layer formed from proteins and cellulose called a tunic that covers their entire epidermis, the outermost layer of the skin. The tunic and other anatomical features of a salp are illustrated in Fig.1. The cellulose that composes the framework of the tunics is proposed to be acquired through horizontal gene transfer deriving from bacteria (Holland, 2016). The tunic has many functions, including phagocytosis, impulse conduction and contraction, bioluminescence, and allorecognition. The skeletal structure of the tunic is composed of cellulose (Zhao & Li, 2014). For salps, the tunic is the protection mechanism against pathogens, parasites, and physical damage. A marine habitat can contain 10⁵–10⁶ microbes/ml in a water column without including the sediments (Franchi & Ballarin, 2017). The tunic immune cells will be further discussed in section 2.

Fig. 1. Schematic anatomical diagram of a female Thalia democratica blastozooid isolated from the chain. The tunic surrounds the entire body in a protective cellulose and protein covering (Pascual, 2016).

Salp tunics are specialized for defense against predators with sight by having a lightweight and transparent tunic. Salps, as a pelagic tunicate, need tunics with neutral buoyancy for easier movement, in contrast with benthic tunicates that require heavy and rigid tunics for protection and competition for space (Hirose et al., 1999). With no other defense than swimming away, salps rely on the transparency of their tunics as their main predator avoidance strategy. Two species of salps, Thetys vagina and Thalia rhomboides, were found to have no specific absorption spectra in the visible range of 280-800 nm, leaving them transparent to visual predators, including those with eyesight in the UV spectrum. However, their body contouring can sometimes catch the reflection of light. Some salps choose to combat this by living in darker regions during the day and only move to shallower regions at night, known as diel vertical migration (Kakiuchida et al., 2017). Other species of salps have nano-sized nipple-like arrays on the cuticle surface of their tunic that is used to reduce the reflection of light, and is a similar technique seen with the eyes of moths (Kakiuchida et al., 2017). This nipple-like array is seen in the salp species Thetys vagina, which is represented by Fig.2a and can be compared to a flat tunic structure seen in the ascidiacea species Rhopalaea seen by Fig.2c.

Fig. 2. a) The tunic structure of Thetys vagina taken by Transmission Electron Microscopy (TEM) showing the nipple-like structures, b) the model of the nipple array and c) tunic of Rhopalaea species that has no repetitive structures, but not completely flat due to damage, image taken by TEM (Kakiuchida et al., 2017).

1.2 Cellulose

By using electron diffractograms, salps were discovered to possess cellulose I microfibrils with high crystallinity. In salp species, such as Salpa fusiformis, the tunic is primarily composed of monoclinic crystalline cellulose (Iβ) with no triclinic crystalline cellulose (Iα) (Hirose et al., 1999). Iβ cellulose has a lower degree of polymerization and crystallinity compared to its Iα counterpart, where the main structural difference is found in the symmetry of the packing of their chains (Poma et al., 2016). The Iβ-cellulose allomorph is favorable for structures that require more flexibility rather than stronger durability. Salps generally had fewer tunic cells since tunic cells evolved to protect the more sessile tunicates and the Salpidae species already have the jet propulsion escape mechanism. Salps also presented tunic cells with an amoeboid shape, suggesting an adaptation for cell motility. This suggests tunic cells can play a role in tissue renewal or in the immune system, like phagocytosis (Hirose et al., 1999). A visualization of the cellulose I microfibrils can be seen by Fig.3 from an ascidian tunic.

Fun Fact! Tunicates possess almost 100% of the Iβ-cellulose, while the other extreme can be found in the cell wall of algae Glaucocystis with nearly 100% of the Iα cellulose (Poma et al., 2016).

Fig. 3. Electron micrographs from a scanning electron microscope (SEM) showing cellulose I microfibrils of the ascidian tunic (a) of a Metandrocarpa uedai outer tunic and (b) of Polyandrocarpa misakiensis. The inset in (b) is a diffraction pattern showing high crystallinity – visible as intense spotty reflections (Kimura & Itoh, 2007).

While cellulose can be found mostly in cell walls of plants and of some algae and bacteria species, the only known animal source of cellulose is found in tunicates. According to Zhao & Li (Zhao & Li., 2014), the cellulose found in tunics is also chemically identical to the cellulose produced by plants but is formed by a cellulose-synthesizing enzyme in the membrane of the epidermis. Tunics typically contain 60% cellulose, with cellulose microfibrils ranging from 100 nm to several micrometers in length and 15–30 nm in diameter (Zhao & Li, 2014). Cellulose consists of D-glucopyranose linked ring units in the lowest energy state of C1-chair conformation represented by Fig.4. Of note are the linking β-1,4-glycosidic bonds that allow for a 180-degree rotation around the center axis and the three reaction hydroxyl groups in the cellulose chain. Its strong hydrogen bonding system reveals multiple properties about the molecule: limited solubility in most solvents, the hydroxyl reactivity. Its long-range repetitive molecular structure leads to its crystalline form (Heinze, 2015).

Fig. 4. Cellulose molecule (C₆H₁₀O₅) in its chair conformation linked by the β-1,4-glycosidic bonds, and the multiple reactive hydroxyl groups (Heinze, 2015).

The reactive hydroxyl groups and the oxygen molecules create both intermolecular and intramolecular hydrogen bonds, that along with their free rotation create crystalline or fibrillar structures. The native form of cellulose is named cellulose I (with two subclasses Iα and Iβ) and is formed by a bond between the oxygen on carbon-3 and the OH on carbon-6. This hydrogen bond, along with weak C-H-O bonds and hydrophobic interactions, creates the layering and crystal assembly seen in cellulose. Cellulose II is another form with a different hydrogen bonding system that leads to it being more closely packed and therefore less reactive (Heinze, 2015). Both forms can be seen in Fig.5, however, only cellulose I can be found in salps.

Fig. 5. a) cellulose I and b) cellulose II molecular structures (Heinze, 2015).

Cellulose nanocrystals are known for being insoluble in natural environments and for their strong tensile strength. Both ramie fibers from the plant (cellulose I) and fortisan fibers from yarn (cellulose II) were tested to have a specific breaking stress value of 0.59 Pa mm³/g, which is comparable to a steel wire with a value of 0.26 Pa mm³/g. Along with their density, the cellulose breaking stress values are converted to values of 0.9 and 2.0 GPa respectively (French et al., 2002). The insolubility and strength combined allow for a strong and stable structure that is suitable for an outer layer in salps. The cellulose tunic therefore protects salps from their sea environment, like predators or floating debris that can cause physical damage.

Cellulose is found in many applications, like ethanol production, medical devices and pulp and paper production, and has become highly researched for its high crystallinity, processability and specific strength (Zhao & Li, 2014). Dunlop et al. (Dunlop et al., 2018) also state that this nanocrystalline cellulose is useful due to its optical transparency, low thermal expansion, biodegradability, low toxicity, and low cost. Tunicates also produce a high ratio of pure I-β nanocrystalline cellulose compared to their biomass with a ratio of 148:23 (Dunlop et al., 2018). Therefore, tunicates are a useful source of cellulose and could be farmed for large-scale production.

2. Immunology

Salps and other invertebrates lack adaptive immune systems present in vertebrates, meaning they only use non-pathogen specific methods to combat infection or innate immunity (Litman et al., 2010). Like vertebrates, salp immune cells travel throughout the body by the circulatory system of the body. However, salp circulatory systems are distinct from vertebrates due to their open circulatory system lacking blood vessels and a heart that pumps hemolymph, a fluid that serves a similar function as blood, at lower pressures than vertebrates. Instead, hemolymph in salps is pumped through cavities between epidermal cells called hemocoels and diffuse in the space around body cells (Cima et al., 2018).

A marine habitat can contain 10⁵–10⁶ microbes/ml in a water column and nearly 10 times as many viruses (Franchi & Ballarin, 2017). The dense tunics of salps serve as the primary barrier to prevent infection from occurring (Cima et al., 2018). However, if one salp in a chain loses a piece of their tunic or swallows a high load of bacteria, salps need to have strong immune systems to combat the spread of disease along the chain. Salps employ robust cellular and humoral (fluid-based) defenses around the anterior and posterior siphons, as well as in response to tunic damage. A population of phagocytes—immune cells that recognize and engulf foreign particles—adheres to the internal tunic at the anterior and posterior siphons, blocking access to the pharynx and atrium, respectively. Phagocytes assume two morphologies: spreading, also known as hyaline amoebocytes, which are cells that actively move towards and consume foreign materials as seen in Fig.6., and round, which are cells that do not actively move (Franchi et al., 2017).

Fig. 6. A spreading hyaline amoebocyte of a Thalia democratica salp observed using transmittion electron microscopy. In the blood lacuna (lac), a gap in the tunic matrix, the hyaline amoebocyte adheres to the basal lamina (bl), a thin sheet of extracellular proteins around epithelial cells. The arrowheads point to phagocytized bacteria in phagosomes. The scale bar is 1µm (Cima et al., 2018).

2.1 Inflammation

2.1.1 Cellular

The hyaline amoebocytes respond immediately after the first infection, in just 10 minutes (Cima et al., 2018). Phagocytosis occurs when phagocyte cells detect and engulf the pathogen into an organelle called the phagosome. The pathogen is then killed inside the phagocyte with hydrolytic enzymes for digestion of pathogen (Cima et al., 2014). The fragility of the salp tunic in laboratory conditions greatly limits researchers’ ability to study the specific mechanism of the innate immune response, but due to preservation of enzyme structure and functions across other tunicates, the same enzyme in different organisms shall have a similar function, allowing investigation of salp immune mechanisms with data from organisms in neighbour classes as shown in Fig.7. (Cima et al., 2018).

Fig. 7. Organisms with a close phylogenetic relationship to salps that can be used to help interpret enzyme functions. Adapted from (Franchi & Balla rin, 2017).

Three hydrolytic enzymes exist in amoebocytes: acid phosphatase, non-specific esterase, and tryptase, as shown in Fig.8 (Cima et al., 2014). Acid phosphatases are commonly observed in lysosomes (organelles specialized for breaking down cell parts) of phagocytes and hydrolyze phosphate esters, like those found in nucleic acids (Cohn & Wiener, 1963). The activity of acid phosphatases is in proportion to the ability of phagocytes to inhibit and kill pathogens (Black et al., 1985). Non-specific esterase also contributes to the digestion of pathogens by breaking down ester bonds, as the name implies (Cima et al., 2014). Although tryptase is classified as a hydrolytic enzyme, they are involved in the synthesis of oxidative enzymes and regulation of cytotoxic activities (Cima et al., 2014; Muhlia-Almazán et al., 2008).

Fig. 8. The highlighted circles in the picture indicate the existence of selected enzymes marked by immunofluorescence: (A) and (B) are Immunohistochemical assays for cell identification that are not relevant to this essay. (C). Acid phosphatase (D). Non-specific esterase (E). Peroxidase and (F). phenoloxidase. Adapted from (Cima et al., 2014).

2.1.2 Humoral

After the initial phagocytosis of the pathogen, the amoebocytes release stem cell factors (SCF), signal molecules that trigger the accumulation of granular cells (granulocytes) (Cima et al., 2018). Granulocytes are immune cells containing regulatory and cytotoxic molecules in vesicles within the cytosol (Cima et al., 2014). These cytotoxic materials include oxidative enzymes such as phenoloxidase (PO) and antimicrobial peptides (AMPs), which are small polypeptides that disrupt microbial functions. These cytotoxins are secreted into the tunic and other tissues to degrade large numbers of invading pathogens. These cells share a similar structure and function to mast cells found in the vertebrate immune system (Franchi & Ballarin, 2017).

For oxidative enzymes, the peroxidase can produce reactive oxygen species (ROS) which demonstrate significant antibacterial activity, attributable to the ability of ROS to react with macromolecules of pathogens, such as lipids, proteins, and nucleic acids (Franchi & Ballarin, 2017; Yang et al., 2013). One enzyme that controls the activation of these cytotoxic pathways is PO, which reacts with phenol-containing peptides and produces quinones, which later synthesize into melanin and other ROS that bring cytotoxic effects to nearby cells and contribute to the cytotoxicity of hyaline amoebocytes (Franchi & Ballarin, 2017; Johansson & Söderhäll, 1996). To control PO activation, PO are stored in non-active form ProPO inside of cells and utilized by the serine proteases present in bacteria to complete activation (Cima et al., 2014; Johansson & Söderhäll, 1996). The reliance on bacterial enzyme activation provides PO with the cell identification abilities and made the immune response selective (Cima et al., 2018). As salps do not have the pathogen-specific adaptive immune system, the specificity provided by PO become their solution to incorporate distinguishing ability in their immunology.

As cells get recruited to the area of infection, adenosine monophosphates (AMPs) are released into extracellular space, and its expression increases (Bella et al., 2011). The full diversity and functions of AMPs in salps are not yet known, though two specific AMPs have been identified in a close ascidian relative Ciona intestinalis that are likely conserved in salps (Bella et al., 2011). These two AMPs, Ci-MAM-A24 and Ci-PAP-A22, display potent antibacterial activity by rapidly permeabilizing plasma membranes of Gram-positive as well as Gram-negative bacteria, indicative of salps’ non-specific innate immunity (Fig.9). Gram-positive bacteria have a thick peptidoglycan cell wall on the outside of a single plasma membrane, while Gram-negative bacteria have a thin peptidoglycan cell wall between two plasma membranes. The ability for these AMPs to target many different bacterial species allows for salps’ innate immune system to kill a wide range of microbial invaders. Permeabilizing the membrane of bacteria leads to the escape of ions and molecules from the bacteria’s cytoplasm as well as easier entrance for other cytotoxic molecules that disrupt intracellular processes. Ci-MAM-A24 is particularly salt-tolerant, suggesting a role in areas of the body exposed to saltwater, such as the siphons (Fedders et al., 2010).

Fig. 9. Activity of Ci-MAM-A24 inside an Acanthamoeba castellani cell infected with GFP-labeled pathogen Legionella pneumophila captured by confocal microscopy. A) Six hours after infection, cells were incubated in 12.5µM of Ci-MAM-A24. B) Remnants of dead L. pneumophila cells are found 48 hours after infection (Schlusselhuber et al., 2015).

2.2 Regulation

The regulatory molecules in granular cells are released through the mechanism of degranulation, as shown in Fig.10. (Cima et al., 2018). Granular cells contain two types of granules, each housing an important signaling molecule: type-1 granules—large ellipsoidal vesicles with electron-dense cores—contain heparin, while type-2 granules—small circular vesicles—contain histamine (Cima et al., 2018). Heparin is known for its anticoagulation abilities, but it mainly contributes to granular cells by regulating other molecules such as histamines and proteases as well as stabilizing proteases in heparin sulphate form, as shown in Fig. 11. The densely negatively charged heparin can balance the positive charge from histamine and serotonin, thus help maintain the neutral storage environment in the cell (Cima et al., 2014; Zehnder & Galli, 1999).

Cytokines are signalling proteins used in salps to induce diapedesis.

Fig.10. The degranulation of granular cells. A) Non-exposed Granular cell. B) Initial degranulation after exposure to bacteria. C) Type-1 granules move to the edge of the cell. D) Type-1 granule during release. E) Empty type-1 granule after release. Legend: g1: type-1 granule; g2: type-2 granule; RER: rough endoplasmic reticulum. Scale bars: A–E = 1 μm. Adapted from (Cima et al., 2018).

Fig. 11. The structure of Heparin Sulfate, generated by PubChem. The abondance of lone pairs from the oxygen and nitrogen greatly help stabilizing the positive chemicals in granular cells (National Center for Biotechnology Information, 2024).

One hypothesis suggests heparin affects the chemotaxis (use of chemical concentration gradient to direct locomotion) of hyaline amoebocytes, thereby manipulating the diapedesis (a mechanism of hemocyte migration from hemocoel to nearby tissue) of these phagocytes, potentially via the cytokine Interleukin 8 (IL-8) (Cima et al., 2018; Jin et al., 2008; Schlorke et al., 2012). Histamines in type-2 granules are proposed to be involved in the recruitment of immune cells as well as the negative regulation of phagocyte activity (Cima et al., 2018; García-García et al., 2014). For the recruitment of immune cells, histamine is believed to bind to H4 receptor and trigger the release of Ca²⁺, the cytokine tumor necrosis factor-α (TNF-α), ands Interleukin-8 (IL-8). (Cima et al., 2018; Czerner et al., 2014; Thangam et al., 2018). The negative regulation can be helpful as it can reduce collateral damage to healthy cells caused by an overactive immune system. Histamines also show the ability to constrict tunic vessels as well as mucus production in the digestive tract in salps, potentially through the H2 receptor, shown in Fig.12. (Cima et al., 2018; García-García et al., 2014).

Fig. 12. Interaction of Histamine and H2 receptor. Areas marked with Roman numerals are receptor domains. The Aspartic acid (Asp) is connected to a counter anion that matches the positive NH3 group of the histamine and uses such intermolecular force for interaction. (Gantz et al., 1992).

Phagocytes synthesize and release lectins, proteins that bind to specific carbohydrates that serve to recognize foreign objects and modulate immune response (Franchi et al., 2024). Many lectins bind to foreign objects and act as opsonins, aiding phagocytes in nonself recognition and digestion (Franchi et al., 2024). In tunicates, rhamnose-binding lectin binds to the rhamnose sugar on microbial cell walls, triggering respiratory burst in phagocytes to switch to spreading, active-searching morphology and degranulation of cytotoxins into the tunic (Cima et al., 2016).

Another important molecule released by granular cells is the TNF-α inflammatory cytokines, secreted when common bacterial components such as lipopolysaccharide and bacterial DNA are detected (Cima et al., 2018; Locksley et al., 2001). TNF binds to several receptors and triggers inflammatory response as shown in Fig.13. (Locksley et al., 2001). The release of TNF-α stimulates phagocytosis by hyaline amoebocytes, the same way as mast cells stimulate macrophages in mammals (Cima et al., 2018). TNF-α also helps with the migration of hyaline amoebocytes as well as in their transcellular diapedesis (Cima et al., 2018). Both granular cells of salps and mast cells in mammalians regulate other immune cells by releasing the immune factors, showing the close evolution relationship between the two species.

Fig. 13. Models of TNF and its receptor, with data from x-ray crystallography (Eck, M. J., & Sprang, S. R., 1989). A) Trimeric structure of TNF. B) Trimeric structure of the TNFR1 (TNF receptor). C) The TNFR1 is viewed from the “top” side of the membrane. Adapted from (Locksley et al., 2001).

3. Chemical Communication

Salps coordinate within chains to survive, such as swimming backward when touched at the front, accelerating forward when touched at the back, and splitting when touched in the middle (Mackie & Bone, 1977; Fedele, 1923). This coordination relies on electrical communication between epidermal cells, as well as chemical signaling and synaptic activity.

3.1 Arrangement of Zooids in a Chain

Chemical transmission specifically occurs at the junctions between individual salps, but a broader picture of how different zooids are attached together is required before describing the transmission mechanism. On this note, due to the limited research available on salps, detailed findings on the specifics of their chemical transmission remain constrained.

Each zooid is connected to its neighbors through two well-defined attachment plaques joining adjacent zooids together (Mackie & Bone, 1977). A zooid is linked to four neighbors, resulting in a total of eight plaques per zooid as seen on Fig. 14. (Anderson et al., 1980). The edges of these plaques are thickened, and the epithelial cells in these regions extend branching processes towards the midline of the plaque as seen on Fig. 15. (Anderson et al., 1980). These processes contain fine fibrils, approximately 6-10 nm in diameter, which are absent in the epithelial cells of the rest of the plaque (Mackie & Bone, 1977). Although these processes approach the midline, they do not cross it, and there is no direct contact between the epithelial layers of neighboring zooids at the plaque edges.

Fig. 14. Small portion of blastozooid chain (anterior to left), showing attachment plaques pointed by the arrows between zooids. Scale bar: 1 mm. (Anderson et al., 1980). Technique: Normarski interference microscopy.

Fig. 15. Median section through attachment plaque, showing processes of cells bordering the plaque and the position of the sensory cell (s.) and button cell regions (b.) (Anderson et al., 1980).

Near the plaque edge, a small and rounded area appears thicker than the rest of the epithelium and varies in prominence among the plaques of a zooid due to the asymmetrical nature of the plaques (Anderson et al., 1980). A closer examination reveals that this thickened area consists of a small button of modified epithelial cells on one side and a small group of sensory cells on the other (Anderson et al., 1980). The button cells protrude from the plaque’s epithelium, resembling a small button or mushroom, and extend into the test material that separates the two epithelial layers as seen in Fig. 16.

Fig. 16. Semi-thin section along long axis of plaque, showing button cells (below) and their processes. Scale bar: 10 μm. (Anderson et al., 1980). Technique: Fixation with glutaraldehyde, post fixation with osmic acid, section stained in uranyl acetate and lead citrate to be examined with electron microscope 200 or 300.

Opposite to the button cells are the sensory cells, whose axons travel towards the brain, nestled within the bases of the epithelial cells as seen on Fig. 17. (Anderson et al., 1980). This arrangement is consistent across zooids: in each pair of plaques connecting a zooid to its neighbor, one plaque contains sensory cells, while the other contains button cells. The plaques located at the outer ends of the horns and near the horn bases are innervated, while those on the ventral surface and at the inner ends of the horns are non-innervated and feature button cells instead (Anderson et al., 1979).

Fig. 17. Portion of edge of innervated plaque, showing group of sensory cells and their axons passing towards the brain. Scale bar:
 10 um. Technique: Fixation with glutaraldehyde, post fixation with osmic acid, section stained in uranyl acetate and lead citrate to be examined with electron microscope 200 or 300. (Anderson et al., 1980).

3.2 Non-innervated side of the plaque: Button cells

The button cells form small protrusions on the side of the plaque opposite the sensory cells. These projections are rich in mitochondria and contain numerous small vesicles, along with short villi as seen in Fig.16. (Anderson et al., 1980). Each button cell is connected to its neighboring cells through gap junctions, and the cells at the button's edge are also connected to the regular epithelial cells of the plaque (Anderson et al., 1980).

Fig. 18. Button cells and their projections. The midline of the plaque is just above the top of the photograph. Note numerous processes in the upper part of the photograph and pointed by the arrows are connections with the button cells (Anderson et al., 1980). Scale bar: 1 μm. Technique: Fixation with glutaraldehyde, post fixation with osmic acid, section stained in uranyl acetate and lead citrate to be examined with electron microscope 200 or 300.

The most notable feature of button cells is their ability to send signals that initiates transmission to sensory cells on the opposite side. They establish contacts on their outer surfaces with another cell type called supporting cells, which are directly connected to the sensory cells, as indicated by the arrows on Fig.18. These contacts resemble chemical synaptic junctions, with a junctional gap of approximately 5-10 nm separating the membrane of the button cells from the profiles of the supporting cells (Anderson et al., 1980). The signal originates from the button cells and travels across this gap towards the sensory cells.

Before receiving the signal from the button cell, the supporting cell are usually "empty" and contain electron-lucent vesicles that are 30-40 nm in diameter (Anderson et al., 1980). When there is an action potential (a movement of charge), it sends a signal from the button cells to the electron-lucent vesicles of these supporting cells (Anderson et al., 1979). These vesicles respond, and the previously "empty" profiles fill with tubules that comes from numerous thinner cell processes (Anderson et al., 1979). The tubules then reach the sensory cells on the innervated side to complete the transmission.

Research by Anderson et al. (Anderson et al., 1980) demonstrated that these tubule-containing processes originate from the cilia of the sensory cells on the opposite side. However, the initiation of the transmission occurs at the button cells which can be a design solution, for example, if the signalling side is dysfunctional, the sensory side can still be activated by artificially initiation if possible (Anderson et al., 1979).

3.3 Innervated Side of the Plaque: Sensory Cells

On the opposite side of the plaque, five to ten sensory cells are located, receiving signals transmitted from the button cells (Anderson et al., 1980). These large sensory cells are paler than the adjacent epithelial cells and contain numerous mitochondria, an extensive smooth endoplasmic reticulum, a large lobed nucleus, and a well-developed Golgi apparatus as vaguely seen on Fig.19. (Anderson et al., 1980). A crown of microvilli surrounds a cilium on the outer surface, originating from a crypt in the cell's apical region, which is densely packed with small vesicles (Anderson et al., 1980). At the base of the cilium, scattered electron-dense bodies represent remnants of the ciliary rootlet, along with a reduced basal apparatus (Anderson et al., 1980).

Fig. 19. Single sensory cell from the plaque with its axon towards the brain of the salp (Anderson et al., 1980). Technique: Fixation with glutaraldehyde, post fixation with osmic acid, section stained in uranyl acetate and lead citrate to be examined with electron microscope 200 or 300.

At the tip of the sensory cells are supporting cells that react with them signals coming from the button cells. These supporting cells do not have a gap junction with the sensory cells, in fact, they seem stemming out of the sensory cells. However, they connect via gap junctions to the button cells on the non-innervated side.

During the transmission from button cells to sensory cells, the ciliary structure undergoes changes. The cilium expands, forming a large cytoplasmic mass filled with multivesicular bodies and vesicles which becomes the supporting cells as seen on Fig.20. (Anderson et al., 1979). This structure receives input from the button cells, then fills its electron-lucent vesicles with microtubules and sends those vesicles toward the sensory cells (Atema, 1975). The supporting cells are flattened in the plane of the plaque when transmission happens to facilitate microtubule collections and to help guiding the transmission from the button cells to the sensory cells (Atema, 1975).

Fig. 20. Schematic diagram showing details of link between the cilia of the sensory cells on one side of the plaque (s.) and the button cells (b.) on the other. The arrows indicate inferred direction of transmission (Anderson et al., 1980).

Fun Fact! In addition to synaptic activity at the plaques, synapses located near the brain also release neurotransmitters like acetylcholine, which help process incoming signals and coordinate the zooid's behaviour and movement (Anderson et al., 1979).

4. Ecological Impact of Salps on the Carbon Cycle

Salps engage in filter feeding and eat floating organic particles and phytoplankton such as diatoms. It is known that at least 149 species of fish have salps as their preferred food source with many more marine predators being known to consume them (Ariffian et al., 2024). Sinking marine particles like salp carcasses and fecal pellets are one of the major contributors of biogeochemical cycling in oceanic environments. Some species of salps can contribute by mixing carbon and nutrients from deeper water up to the surface during their diurnal vertical migration (Ariffian et al., 2024).

Diel vertical migration, or diurnal vertical migration, is used to describe the daily migration pattern of marine organisms. It usually describes the migration of marine organisms towards the surface at night and towards the depths during the day.

Salps have long been proposed as a sort of “vacuum cleaners” for oceans, responsible for clearing up small floating particles in water columns and compacting them into large fecal pellets that will eventually serve to enrich the seafloor for other marine organisms (Ariffian et al., 2024). These fecal pellets rich in organic carbon and nitrogen may also serve as a useful food source for heterotrophic organisms, organisms that cannot produce their own food and must consume other sources of organic carbon (Iversen et al., 2017). As some zooplankton are unable to feed on small picoplankton, the undigested remains of salp filter feeding may serve as an alternate method to get that extra organic matter necessary for growth and survival (Iversen et al., 2017). Individual salps were noted to be able to pack 0.33% to 0.65% of the currently available particulate organic matter into their fecal pellets each day, which is the equivalent to 16.3μg of ocean carbon captured per hour for each individual (Ariffian et al., 2024). This represents four times the carbon capturing of krills, another important zooplankton involved in biogeochemical cycling (Ariffian et al., 2024).

Depending on their environment, salp fecal pellets are also known to contain various trace elements useful for marine life, as seen in Table 1. For instance, iron, an element vital for biogeochemical cycling and ocean ecology, has been recorded in significant concentration within salp fecal pellets. As such, the breakdown of the fragile salp fecal pellets by other marine grazers may be an important factor contributing to the bioavailability of iron within the environment. This would in turn increase the primary productivity of oceans and make salp abundance a factor contributing to ocean health (Ariffian et al., 2024).

Table 1. Trace elements concentrations (mg/g dry weight) detected in the whole body, guts and faecal pellet of salps determined by atomic absorption spectrophotometry (from Ariffian et al., 2024).

A study found that during Southern Ocean phytoplankton blooms, when salp abundance ranged from 63 to 217 individuals per 1000 m³ within the upper 250 m of the water column, individual salps produced 0.1–0.5 fecal pellets per hour in environments containing approximately 1.5 µg/L of chlorophyll a (Iversen et al., 2017). This result was in accordance with previous studies which suggested that individual salps produced 0.1-0.6 fecal pellets from chlorophyll a concentration of 1-3.7 µg/L (Iversen et al., 2017). Meanwhile, lower chlorophyll a concentration has been shown to decrease salp excretion rates (at 0.08-0.6 µg/L concentrations of chlorophyll a, 0.05-0.1 fecal pellets/hour were produced for one individual salp).

Though salp carcasses rarely reach the seafloor due to decomposition or interception by scavengers, they still somewhat contribute to the carbon flux by exporting dispersed and floating carbon onto the seafloor (Ariffian et al., 2024). In the coastal Mediterranean region, salp fecal pellets and carcasses were measured to make up 72% of the carbon flux while data from the Bermuda Atlantic Time Series, a monthly long-term study of a sample area in the western Atlantic Ocean, showed that fecal pellets represent 78% of the generated carbon flux with carcasses took up a modest 3% (Ariffian et al., 2024). This was to be expected as the sinking rate and degradation rate of salp fecal pellets and carcasses vastly differ with pellets being clearly more adapted to sinking than carcasses. Fig.21 illustrates the role of salps in biogeochemical cycling. As for the sinking rates of both salp fecal pellets and carcasses as well as their degradation rates, please refer to Salp Physics.

Fig. 21. A schematic showing the role of salps in biogeochemical cycling, highlighting the fate of fecal pellets and carcasses of salps (from Ariffian et al., 2024).

With changes in the environment happening due to increased human activity and the advent of new technologies, marine organisms are bound to be affected too. With more and more microplastics in the ocean, salp fecal pellets have a more difficult time reaching the seabed as salp fecal pellets that were discovered to contain microplastics had their velocities reduced by 30-50% as shown in Fig.22 (Ariffian et al., 2024). It is unclear as of now what impacts this will cause in the wider biogeochemical cycle as this area of study is mostly undocumented, but this is something to look out for (Ariffian et al., 2024).

Fig 22. The effect of microplastics on the sinking rate of salp fecal pellets in transporting POM to deeper waters (from Ariffian et al., 2024).

Conclusions

In conclusion, salps possess unique chemical adaptations crucial for their survival and the marine ecosystem. Their light and transparent tunic allows for easier movement while minimizing absorption and reflectance to stay invisible from sighted predators. At the molecular scale, their tunic is composed of Iβ cellulose, which is insoluble and has high tensile strength due to its crystalline structure and hydrogen bonding system. The high tensile strength and durability of nano-crystalline cellulose provides an outer structure similar to the cell wall of plants, giving salps both protection and mobility.

Furthermore, salps require strong innate immune systems to prevent the spread of diseases through chains and have developed a robust innate immune system to minimize such damage. At the cellular level, amoebocytes contain digestive enzymes and recognition factors such as acid phosphatase that break down ingested material and lectins that identify sugars from invading pathogens. Granular cells release bioactive molecules like TNF-α and heparin to regulate the immune response and cytotoxic material like PO and antimicrobial peptides to destroy microbes extracellularly. The PO enzyme also provides additional recognition between cells and bacteria to compensate for the absence of adaptive immune system. The similarities between salps and vertebrates’ immune systems show the close evolutionary relationship between the two species and calls for further investigation.

In addition to their efficiently evolved bodies, salps also display remarkable coordination between other salp bodies. Chemical transmission allows them to synchronize actions, starting with button cells that send signals to supporting cells. These supporting cells, filled with microtubules, transmit signals to sensory cells. This enables communication directly to the brain through axons, which allows the salp to function as a cohesive unit.

Beyond their physical structure, salps contribute significantly to marine ecosystems. Their fecal pellets and carcasses nourish the environment in multiple ways. They serve as food for grazers and predators and aid in the transport of carbon to the seafloor, while also dispersing important elements like iron. However, the increasing presence of microplastics is causing salp fecal pellets to sink more slowly, raising concerns about unknown impacts on the marine environment and biogeochemical cycling. Altogether, these features highlight the critical role of salps in marine ecosystems and offer insights into biological and evolutionary processes.