Biological Control in Subtropical and Tropical Crops

C.E. KENNETT, ... J.W. BEARDSLEY, in Handbook of Biological Control, 1999

Helicidae

The brown garden snail. Helix aspersa Müller, of European origin, is the most important snail pest in California. Although most commonly a pest of landscape ornamentals and home gardens, this snail has also been an important pest of citrus for many years (Basinger, 1931).

Attempts to develop biological control of H. aspersa in California began with the introduction of predaceous snails and beetles during the 1950s and early 1960s. These efforts resulted in establishment of only one species, a staphylinid beetle (Fisher & Orth, 1985). In 1966, however, another (opportunistic) predaceous snail Rumina decollata Linnaeus (also of European origin) was found to have invaded California (Fisher, 1966). Subsequent observations suggested that R. decollata had been present there for 5 to 10 years before its discovery; later studies showed it to be established in at least 14 counties (Fisher et al., 1980; Fisher & Orth, 1985).

Experimental releases of R. decollata in southern California citrus orchards were begun in 1975 and, in most cases, resulted in complete control (displacement) of Helix within 4 to 6 years after the initial predator seedings (Fisher & Orth, 1985). These authors estimated that R. decollata is now used to control H. aspersa in some 20,000 ha of citrus in southern California. Methods for suppression of H. aspersa during the period preceding full control by R. decollata have been discussed by Fisher et al. (1983) and Fisher and Orth (1985).

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Voltage-Gated Proton Channels☆

Y. Okamura, in Reference Module in Life Sciences, 2017

1 Research History of Voltage-Gated Proton Channel

Voltage-gated proton channels (Hv channels) were first biophysically described by Thomas and Meech in giant neurons of snail, Helix aspersa1 (Fig. 1). Snail neurons were voltage clamped by two microelectrodes and HCl was microinjected through a third microelectrode. Both intracellular pH and membrane currents following microinjection of HCl were monitored under the condition where the effects of Na/H antiporters and Cl/HCO3 exchange were minimized by perfusion with a sodium-free and bicarbonate-free bath solution. Recovery of intracellular pH following microinjection of HCl was more rapid when membrane potential was depolarized (Fig. 1), suggesting the pathways of voltage-activated proton conductance (IHv). This conductance was inhibited by relatively high (1–10 mM) concentrations of heavy metal cations1,2 (Fig. 1).

Fig. 1. Activity of the voltage-gated proton current in snail neuron. Thomas and Meech1 for the first time recorded the voltage-dependent proton conductance by simultaneous measurement of membrane current, membrane voltage and intracellular pH using five microelectrodes in snail neurons. HCl was microinjected to induce a rapid decrease in intracellular pH. Snail neurons express an endogenous bicarbonate ion-dependent pathway to exclude protons. To inactivate this pathway, CO2-free solution was applied. Thereafter, recovery of intracellular pH following microinjection of HCl was slower at a holding potential of −50 mV. When the membrane potential was depolarized to 15 mV, however, recovery of intracellular pH is faster, and this recovery is suppressed with 2 mM Cd2+. Corresponding to the pH recovery was an outward current indicating the conductance of protons. Upon return of the membrane potential to −50 mV, recovery of intracellular pH became slower again, indicating the observed export of protons was activated by depolarization.

Properties of this proton conductance in snail neurons were further characterized by Byerly et al.3 using a perfusion-voltage clamp technique. Three important findings were obtained in this study. First, inward relaxation currents were elicited on repolarization, indicating that protons flow in both directions dependent on proton electrochemical gradients following the Nernst equation. This confirmed that IHv is not a transporter or pump current. Second, kinetics of activation became faster as the voltage increased (Fig. 2). Third, the conductance–voltage curve was found to be dependent on both intracellular and extracellular pH. Low external pH or high internal pH shifted the conductance–voltage (G–V) curve to a positive direction, whereas high external pH or low internal pH shifts the G–V curve to a negative direction. This shift was not explained by a simple screening effect of surface charges,4,5 since divalent cations such as calcium did not cause such an effect.

Fig. 2. Traces showing currents through voltage-gated proton channels in snail neurons (a), a human neutrophil cell line (HL60) (b), and mouse neutrophil (c). In (a), membrane currents were recorded using the internal perfusion method.158 Current were elicited by step pulses to 30, 10 and −10 mV from a holding potential of −50 mV. pHo/pHi=7.4/5.9. Tris-Cl solution was used. Taken from Ref. [3]. In (b), tail currents were measured by repolarizing the potential to a level ranging from −120 to −20 mV. Depolarizing prepulses were stepped to 60 mV for 2 s. pHo/pHi=7.5/5.5. NaCl solution was used. Taken from Ref. [8]. In (c), voltage was stepped to 180 mV from a holding potential of −60 mV. pHo/pHi=7.0/6.0. NMDG-methanesulfonate solution was used. Taken from Ref. [79]. Currents were recorded at room temperature in all the cases.

Another study6 using immature axolotl oocytes showed that IHv is not restricted to invertebrates. This study also reported that cadmium ions shifted the I–V curve rightward in a dose dependent manner, indicating that the decrease of IHv by cadmium is not via blocking the proton conduction pathway, but by suppressing channel gating.

In these early pioneering studies, however, the physiological roles of IHv remained a mystery. The first indication of a physiological function of IHv was obtained in milestone studies with denucleated ghost membranes, called cytoplasts, prepared from neutrophils.7 In phagocytes, such as neutrophils and macrophages, NADPH oxidases transfer electrons to produce superoxide anions but leaves protons in the cytoplasm. This causes a charge imbalance, thereby depolarizing the membrane. Henderson et al.7 inferred that there must be a specific mechanism for charge compensation to avoid extreme membrane depolarization. The membrane potential of neutrophil-derived cytoplasts, measured with an organic voltage dye, oxonol, was altered dependent on pH and was affected by heavy metal ions, indicating that neutrophils have a proton conductance similar to IHv in snail neurons.

IHv in mammalian blood cells was directly shown by patch-clamp recording from neutrophils7–9 (Fig. 2), macrophages,10,21 lymphocytes,11 and eosinophils.12 IHv has also been recorded from lung alveolar type II cells,13 amphibian oocytes,6 amphibian renal epithelium cells, cultured mammalian microglia,14–16 osteoclasts,17,18 fibroblasts19 and sperm.20 Through intensive patch clamp studies from these mammalian cells, unique characteristics of Hv channels have been established; strict proton selectivity, extremely low single channel conductance,21 pH-dependent modification of voltage-activated gating,13,22 and large effects of deuterium and temperature on proton permeation.17,23,16,24

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Epigenetic Control of Reproduction

Nelson R. Cabej, in Epigenetic Principles of Evolution, 2012

Role of the Ovarian Innervation in the Ovarian Function

From the view of the role of the CNS in the physiology of gonads, a recent study on the sensory innervation of the OT in the snail Helix aspersa (Figure 3.5) is very illuminating. The brain of the snail consists of ~40,000 neurons, but, as already mentioned, the OT branch of the intestinal nerve alone, which innervates the OT, contains 3,025 axon profiles representing almost 8% of the total number of the brain neurons. This by far exceeds that expected from the size of the nerve (Antkowiak and Chase, 2003).

To sense the stretch resulting from the growth of oocytes is a task that would only need a few neurons, rather than thousands of them. There is a clear threshold of 87 mature oocytes accumulated in the gonad, which marks a ~10-fold increase in afferent electrical spikes, and a correspondingly higher efferent spike rate, and the beginning of oviposition. The clear correlation between the afferent activity of the OT innervation and the efferent response from the CNS indicates that this innervation is of the sensory type (stretch sensors for monitoring the oocyte growth), but other evidence suggests that it might also have motor functions (Antkowiak and Chase, 2003) for oviposition in this snail species.

The endocrine function of the ovary has generally been considered to be under control of the CNS via the hypothalamic–pituitary axis, where the secretion of androgens and estrogens is regulated by the pituitary FSH, LH, prolactin, and corticotropin. However, it appears that, important as it is for the reproductive function, the neurohormonal control would be insufficient for the regulation of the complex process of production and deposition of maternal factors in the oocyte. A closer, immediate monitoring of the process of deposition of parental factors seems to be a sine qua non. Indeed, in vertebrates, innervation by the SON, ovarian plexus, and vagus nerve form a dense network of nerve fibers coming into close contact with follicle cells of the theca externa and theca interna surrounding the Graaf follicle. It is acknowledged that the ovarian innervation plays important role in the physiology of this organ (Aguado, 2002). Besides, a local network of intrinsic neurons and innervation has been discovered in the ovary (D’Albora et al., 2002).

Viral transneuronal tracing studies have shown that extrinsic ovarian innervation and the network of intrinsic ovarian neurons, besides the spinal cord (e.g., IML cell column, dorsal horn) are connected, via multisynaptic neuronal pathways, with most different regions of the brain (various structures of the brain stem, pons, mesencephalon, telencephalon, diencephalon, especially hypothalamic structures; Gerendai et al., 1998, 2002). These multisynaptic connections are necessary for continually monitoring the physiological status of the ovary, Graaf follicle, and oocyte and probably for the efferent CNS output for controlling and coordinating the activity of various cell types for the development of the oocyte.

Secretion of ovarian hormones is under parallel local control of a second mechanism from the CNS, via vagus and SON. Involved in this mechanism are nervous fibers descending from the hypothalamus to the spinal cord, sympathetic and vagal preganglionic neurons, as well as sympathetic neurons in para- and prevertebral ganglia (De Bortoli et al., 1998), which influence the peripheral neurons of the autonomic system. Electrical stimulation of a number of brain centers (ventromedial hypothalamus and medial basal prechiasmatic area), even in absence of FSH, LH, and ACTH (hypophysectomized and adrenalectomized animals) stimulates significant increase of plasma E2 and progesterone in the contralateral ovarian venous blood. Ovarian denervation blocks estrogen secretion induced by stimulating the medial basal prechiasmatic area. The surprising differences observed in the secretion of ovarian hormones and functions in left and right ovaries are also explained with differences observed in the neurotransmitters released by innervation of the left and right ovaries, which modulate effects of pituitary gonadotropins (Cruz et al., 2006).

Granulosa cells in the ovary (Mayerhofer et al., 2003, 2006) and epithelial oviducal cells in pregnant pigs (Steffl et al., 2006) are endocrine nonneuronal cells secreting the neurotransmitter Ach, but it is again a central neural mechanism that, via the hypothalamic–pituitary axis, with the FSH as proximate inducer, stimulates secretion of this “cyto-transmitter” and ensuing growth of the ovarian follicle (Mayerhofer et al., 2006).

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Molluscan Bioactive Peptides

Anna Di Cosmo, Gianluca Polese, in Handbook of Biologically Active Peptides (Second Edition), 2013

Discovery

APGWamide is a tetrapeptide with the sequence Ala-Pro-Gly-Trp-NH2 and is cleaved from a longer precursor peptide. It was isolated from ganglia of prosobranch mollusc Fusinus ferrugineus. In Lymnaea stagnalis and in Helix aspersa, APGWamide seems to be localized mainly in the neurons of asymmetrical right lobe of the cerebral ganglion. APGWamide-related peptides are also found in bivalves. In the cephalopod Sepia officinalis, the dipeptide GWamide has been purified from the optic lobe. Immunoreactivity occurs in the nervous system and in the oviducal gland of Octopus vulgaris APGWamide and in neurons and fibers of Idiosepius pygmaeus with a greater number of immunoreactive cells in the male.28 It is a common neurotransmitter/neuromodulator peptide found in many species of molluscs and is often related to sex organ growth or reproductive behavior.

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Chemical Ecology

Falko P. Drijfhout, E. David Morgan, in Comprehensive Natural Products II, 2010

4.11.4.2 Glycoalkaloids

α-Solanine (306) from the potato, Solanum tuberosum, and the tomato Lycopersicon esculentum (both Solanaceae) and α-chaconine (307), also from the potato and other Solanum species, are known antifeedants toward snails, but recent tests showed that these two glycoalkaloids act synergistically. Tested alone, both compounds deterred feeding of the test snail, Helix aspersa, with chaconine (307) being more effective than solanine (306). But when they were tested as a mixture, the inhibition increased significantly more than that of each compound on its own.134 At 0.2 mmol l–1, chaconine inhibited feeding by 30% whereas 0.2 mmol l−1 of solanine did not affect feeding at all. Yet a mixture of solanine and chaconine, both at 0.2 m mol l−1 inhibited feeding by 60%. It is worth noticing that when the mixture was diluted and tested against the extract of the peel of the potato variety Home Guard, the 10 times diluted peel extract was still active as antifeedant, while the authentic glycoalkaloid mixture was not active at this dilution. This gives rise to the question whether other glycoalkaloids maybe present and also working synergistically.

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Epigenetic Control of Reproduction

Nelson R. Cabej, in Epigenetic Principles of Evolution (Second Edition), 2019

Local Neural Control of the Reproductive Function in Insects

In insects, the CNS is involved in the reproductive behavior and in the reproductive activity not only via neuroendocrine pathways but also directly via the local gonadal innervation.

Octopaminergic neurons innervating the female reproductive system in Drosophila, via Ca2 + as a second messenger, activate the enzyme Mmp2 (matrix metalloproteinase2) thus regulating follicle wall degradation, follicle rupture, and ovulation (Deady and Sun, 2015) (Fig. 3.3).

Fig. 3.3

Fig. 3.3. The model of follicular adrenergic signaling in Mmp activity and follicle rupture. Octopaminergic (OA) neurons are shown in the right of the figure.

From Deady, L.D., Sun, J., 2015. A follicle rupture assay reveals an essential role for follicular adrenergic signaling in Drosophila ovulation. PLoS Genet. 11 (10), e1005604.

For almost one century, since it was first described, no specific function was attributed to the nerve innervating the ovotestis (OT) in pulmonate snails, but recent studies on the slug Helix aspersa have shown that the OT branch of the intestinal nerve consists of as many 3025 axons, suggesting that approximately 8% of the total number of neurons of the CNS are involved in the innervation of the slug ovotestis.

The innervation is necessary for maturation of oocytes and ovulation. It has both sensory and motor functions. The fine terminals of the OT nerve branch have stretch sensors in the acinar walls, which monitor the degree of stretching as a result of the concurrent growth of over 100 oogonia. That information on the stretch, via afferents, is sent to the brain, where it is assessed and, when it exceeds a threshold, efferent brain signals are sent back for increasing peristaltic contractions in the hermaphroditic duct and the beat of the cilia in the internal lining of the duct, thus facilitating the movement down the duct and oviposition of 58 to 108 egg cells (Antkowiak and Chase, 2003; Chase et al., 2004; Geoffroy et al., 2005).

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Sperm Competition in Molluscs

Bruno Baur, in Sperm Competition and Sexual Selection, 1998

3 Dart shooting

In marine opisthobranchs and at least 10 families of terrestrial pulmonates, the animals form a sharp, hard, calcified or chitinous structure (the so-called love dart) in the female part of the reproductive organ (Pruvot-Fol 1960; Tompa 1980; Fig. 8.6). The dart is used to pierce the body of the mating partner during courtship. This peculiar behaviour has been hotly discussed for more than 250 years, but its adaptive significance is still unknown (Kothbauer 1988; Leonard 1992). Even though darts may wound or even kill a partner, the elaborate structure of the dart apparatus suggests that it serves some adaptive function.

Fig. 8.6. Love darts (with sections) of three simultaneously hermaphroditic land snails. (A) Eobania vermiculata (adult shell breadth 2.6 cm); (B) Helix aspersa (3.5 cm); (C) Chilostoma tacheoides (2.5 cm). Scale bar: 24 mm.

(after Giusti and Lepri 1980, with permission)Copyright © 1980

Dart shooting is best studied in Helix pomatia and Helix aspersa (Jeppesen 1976; Lind 1976; Chung 1987; Adamo and Chase 1988). In both species, dart shooting is a facultative element of courtship behaviour; it occurs when a snail quickly everts the basal tubercle of the dart sac out of its everted genitals (Chung 1987). The dart is never propelled through the air, because it is firmly attached by its base to the tubercle until it is lodged in the partner's tissue. Occasionally, the dart does not hit the partner. A new dart is produced within 5–6 days after dart shooting (Dillaman 1981; Tompa 1982). During the expulsion of the dart, a globule of whitish mucus from the digitiform glands adheres to the dart. It has been shown that the mucus transferred with the dart decreases the duration of the courtship (Chung 1986; Adamo and Chase 1990; see also below). It is assumed that a pheromone in the mucus may cause a behavioural synchrony between the mating partners to assure a faster courtship and subsequent copulation, which might decrease both the chances of failure to complete mating and of predation (Adamo and Chase 1990). However, the fact that dart shooting results in 15% of the recipients breaking off courtship contradicts the stimulatory function of this behaviour (Lind 1976).

Virgin individuals of H. aspersa possess no dart (Chung 1987). Thus, mating sequences without dart shooting occur naturally in H. aspersa. Furthermore, in 10 of 23 observed copulations in Helix aperta the dart fell on the ground (Giusti and Lepri 1980).

Other hypotheses have been proposed to explain the adaptive significance of dart shooting (Leonard 1992; Adamo and Chase 1996). The hypothesis that the dart is a gift of calcium to the partner for the production of eggs (calcium is a limiting factor in the reproduction of most land snails) has recently been rejected (J. M. Koene, personal communication). Simultaneous hermaphrodites that mate in pairs are thought to experience a conflict over which individual will assume which sexual role (Fischer 1980,1984; Leonard and Lukowiak 1984b, 1985). This conflict can be resolved by some sort of parcelling behaviour, where individuals repeatedly alternate sexual roles (Leonard 1991). According to Leonard (1992) the dart may serve to induce the partner to act as a male, by acting as an honest signal of an individual's Willingness to reciprocate in a sperm-trading mating system (for a discussion of gamete-trading models see chapter 7). An alternative hypothesis suggests that dart-shooting behaviour might have evolved in order to manipulate sperm utilization and/or oviposition in the mating partners (Charnov 1979; Tompa 1980). In other animals from different phyla, males influence female reproduction indirectly by stimulating the female's sensory systems (Adamo and Chase 1996). In a variety of terrestrial gastropods, copulation per se and/ or the transfer of the spermatophore stimulates egg production via hormones in both partners (Griffond and Gomot 1989; Saleuddin et al. 1991; Baur and Baur 1992b). Thus, a copulation might be beneficial even if the sperm delivered are not used by the partner for the fertilization of eggs. Adamo and Chase (1996) suggest that dart shooting is an additional ability to influence fertilization directly by manipulating the endocrine system of the partner. The dart serves to increase the likelihood that the recipient will use the shooter's sperm to fertilize its eggs. Adamo and Chase (1996) reviewed features of snails reproduction which are consistent with this hypothesis. However, detailed experimental studies that consider anatomical, physiological and behavioural specializations are needed to understand the significance of dart shooting.

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Mucus from Marine Molluscs

Mark S. Davies, S.J. Hawkins, in Advances in Marine Biology, 1998

5.4 Protection

The use of mucus to isolate an animal from its environment, or to actively counter some facet of environment is common in marine Mollusca and it is perhaps here that mucus is most diverse in function. Mucus is produced from most molluscan epithelia (Simkiss and Wilbur, 1977), acts as a barrier to diffusion (Grimm-Jørgensen et al., 1986) and may function in selective ion transport (Ahn et al., 1988).

Gastropod epithelial mucus is often a first line of defence and has been shown to reduce exposure to physical stress and predation. Wilson (1929), Bingham (1972), Morris et al. (1980), Denny (1984), McMahon and Britton (1985), Britton (1995) and Davies and Hawkins (pers obs.) have noted the habit of littorinids and amphissids of attaching themselves to vertical rock using a strand of pedal mucus as a glue between substratum and shell. The animal’s head is then retracted behind the operculum. This behaviour is thought to render the animal less susceptible to desiccation and overheating. By secreting a veil of mucus (which then dries to form a wall) between shell and substratum, the limpets Acmaea (Tectura) digitalis, A. (Macclintockia) scabra and A. persona can reduce desiccation stress (Wolcott, 1973). A similar phenomenon occurs in terrestrial snails which secrete a CaCO3/ mucus matrix across the shell aperture which dries to form a water-tight seal (e.g. Helix aspersa, Otala lactea, Sphincterochila boisseri,Machin, 1967; Schmidt-Nielsen et al., 1971). Mucus has also been implicated in protecting antarctic limpets from extreme cold (Hargens and Shabica, 1973). The role of mucus in fish as a barrier to pollution has been extensively studied (Shephard, 1994), but in molluscs this has not been directly assessed. However, excess mucus production by bivalves after exposure to heavy metals (Lakshmanan and Nambisan, 1985; Moraes and Silva, 1995; Sunila, 1987; Hietanen et al., 1988; Sze and Lee, 1995) and hydrocarbons (Axiak and George, 1987) has been reported; and excess pedal (Mills et al., 1990) and intestinal (Triebskorn, 1989; Triebskorn and Ebert, 1989) mucus production by slugs in response to metaldehyde (a molluscicide) has been observed. Davies (1992) described a reduction in pedal mucus production in limpets, Patella vulgata, exposed to single heavy metals, although this reduction was probably owing to an accompanying lack of activity. Mucus can function in predator avoidance by rendering the gastropod distasteful and/or toxic (e.g. the dorsal secretions of Doriopsilla albopunctata,Reel and Fuhrman, 1981 and Phyllidia varricosa,Johannes, 1963; the hypobranchial mucus of Calliostoma canaliculatum (pers. comm., N. Smaby); the secretions of the mantle edge in Cellana spp., Branch and Branch, 1980); by anaesthetizing the predator (Trimusculus reticulatus,Rice, 1985); by fouling the predator’s feeding apparatus (e.g. Ariolimax columbianus,Richter, 1980); or by making the animal too slippery to handle (e.g. Calliostoma species, Sellers, 1977; Harrold, 1982). Handling of molluscs can also induce copious mucus secretion (e.g. Buccinum undatum, Strombus gigas pers. obs.), as can tissue disruption upon dissection (e.g. Milax sowerbii,Barr, 1926; Archidoris pseudoargus,McCance and Masters, 1937). Interestingly, some nudibranchs are able to sequester poisons from their food which subsequently emerge in their mucus, providing a defence for these molluscs which cannot retreat into a shell. Indeed the evolution of loss of shell in this group may well have coincided with the ability to use mucus in a defensive capacity. Poisons or deterrents will probably all emerge with mucus, but few studies have specifically recognized this. Examples include: Avila et al. (1991) who observed that Hypselodoris webbi secretes the allomone longifolin (an ichthyodeter-rent) in its dorsal mucus, the allomone originating in the sponge Dysidea fragilis;Paul et al. (1990) who observed that Nembrotha spp. secrete tamb-jamines (ichthyodeterrents from the ascidian Atapozoa sp.) in their mucus; Gustafson and Andersen (1985) who discovered terpenoids from sponges, bryozoans and coelenterates in the mucus of Archidoris montereyensis and Anisodoris nobilis.

The African land snail Achatina fulica produces an agglutinin (lectin) in its mucus (Iguchi et al., 1985) which is a 70 000 MW glycoprotein (Mitra et al., 1988). Lectin activity has also been reported for the mucus of the terrestrial gastropods Arion empiricorium (Habets et al., 1979), Helix aspersa (Fountain and Campbell, 1984; Fountain, 1985) and Archachatina marginata (Okotore and Nwakanma, 1986). Astley and Ratcliffe (1989) examined the mucus of some species of marine mollusc but could find no lectins, although they were present in the epithelial mucus of Loligo vulgaris (Marthy, 1974). Whilst these discoveries present numerous potential roles for mucus (slug mucus is apparently used in some human therapy, Habets et al., 1979), it may be that the lectin has no function other than structural within the mucus matrix (Fountain, 1982). McDade and Tripp (1967) recorded the presence of lysozyme in oyster mucus and hypothesized that this formed an antimicrobial defence, although lysozyme may merely prolong the functional life of the mucus by slowing down bacterial breakdown. Kubota et al. (1985) purified a glycoprotein (“achatin”, 140 000 MW) from the pedal mucus of Achatina fulica which showed no lysozyme activity but did kill both gram-positive and gram-negative bacteria by acting on cytoplasmic membranes (Otsuka-Fuchino et al., 1992). The use of mucus as a carrier for these compounds provides an unstirred layer on the surface of the animal in which the compounds can be held and prevents them from dispersing in an aquatic environment (Denny, 1989). Bakus et al. (1986) reviewed the chemical ecology of marine organisms and whilst they rarely mentioned mucus it is likely that mucus is employed as a carrier of secretable chemicals in most of the taxa they describe.

The functioning of limpet pedal mucus in limpet tenacity (to prevent dislodgement by, for example, predation) is a subject of debate. Smith (1991, 1992) concludes that a glue-like adhesion is responsible for the great tenacities observed in limpets by Grenon and Walker (1981) and Denny (1984), although whether this glue is related to, or is part of, mucus is not clear. It may be that these tenacities are is not a product of a glue or mucus at all, but are owing to a very flexible muscular foot which can effectively mirror, and hence grip, the microscale contours of substrata. This would explain the way in which limpets can increase their tenacity when disturbed. Davies and Case (1997) who studied the tenacity of two littorinid species, concluded that “muscular grip” does not play a role in adhesion. They suggested that the mechanism of adhesion in these animals involves mucus. Grenon and Walker (1982) measured the thickness (3 μm) of the mucus layer under the foot of Patella vulgata after it had been attaching to an alga for 2 d. Grenon and Walker suggested that adhesion was afforded by the thinness of the layer (cf. Branch and Marsh, 1978) caused by the slow uptake of water and mucus from the sole of the foot by epithelial cells, a mechanism proposed by Zylstra (1972) and Machin (1975).

Cephalopods also use mucus in escape from predators. Their “ink”, squirted at predators to confuse them is bound with mucus to prevent its rapid dispersion in water (Denny, 1989).

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Mucosal immunity in mollusks

Bassem Allam, Emmanuelle Pales Espinosa, in Mucosal Health in Aquaculture, 2015

12.7.2 Lectins

The most significant progress in the understanding of immune recognition in molluscan mucosal tissues was made on lectins. The term “lectin” commonly refers to a wide range of carbohydrate-binding proteins and glycoproteins. Most of these molecules are organized as homo-oligomers or hetero-oligomers of noncovalently bound polypeptide subunits displaying one or more carbohydrate-recognition domains (CRD) that bind to their corresponding sugar ligands, usually a nonreducing terminal monosaccharide or oligosaccharide (reviewed in Vasta, 2009). They recognize and bind carbohydrates present on cell surfaces of several unicellular organisms, agglutinating and enhancing the destruction of microbes by specialized immune cells (reviewed in Olafsen, 1986). Lectins, including C-type lectins and galectins, are known to serve multiple purposes in the immune system (Weiss et al., 1998; Zelensky and Gready, 2005; Vasta, 2009). In molluscan mucosa, lectins or lectin-like activities have been described in gastropods (Fountain and Campbell, 1984; Iguchi et al., 1985), in cephalopods (Marthy, 1974) and in bivalves (McDade and Tripp, 1967b; Fisher, 1992; Tripp, 1992; Pales Espinosa et al., 2009, 2010b, 2011; Jing et al., 2011). These molecules were shown to be involved in microbial binding, for the most part, and no bactericidal activity has been demonstrated for mucosal lectins so far.

Fountain and collaborators (Fountain, 1985; Fountain and Campbell, 1984) purified and characterized a lectin-like molecule in the mucus of the snail Helix aspersa. This molecule was able to agglutinate rabbit red blood cells, and this agglutination activity was inhibited by N-acetyl-galactosamine (Ga1NAc) and, to a lesser extent, by N-acetyl-glucosamine (GlcNAc) and galactose. The authors concluded that this agglutinin represents an important component of the mucosal immunity of the snail. Mitra (Mitra et al., 1988) identified an agglutinin from the mucus of another snail species, Achatina fulica. In this case, inhibition tests showed specificity for galactose residues. A C-type lectin (achatinin) was also isolated from the mucus of the giant African snail (Achatina fulica). This lectin did not show any bactericidal activity but was able to agglutinate vertebrate red blood cells, and its activity was inhibited by N-acetyl-neuraminic acid (Iguchi et al., 1985). More recently, another lectin, AfHML, was also purified from the mucus of A. fulica (Itoh et al., 2011). In the presence of calcium, this lectin was able to agglutinate rabbit red blood cells as well as Gram-positive and Gram-negative bacteria, and its activity was inhibited by galactose. AfHML, however, did not inhibit bacterial growth. Tissue immunolocalization of AfHML revealed that the lectin was expressed in the tissues of the mantle collar. Based on these results, these two lectins were suggested to play a role in the agglutination and immobilization of pathogens, but not in their direct destruction.

References are very rare on the presence of mucosal lectins in cephalopods, and only one study reports the presence of a hemagglutinin from the epithelial mucus of the European squid Loligo vulgaris (Marthy, 1974). In contrast, several lectins or agglutinins have been found in bivalve mucus. The presence of “agglutinins” in the pallial mucus of the eastern (American) oyster (Crassostrea virginica) was initially reported in 1967(b) by McDade and Tripp. In 1992, Fisher reported that C. virginica pallial mucus agglutinates a variety of bacteria, including Aeromonas hydrophila, Vibrio cholerae, and V. fluvialis. More recently, studies have shown that mucus covering the gills and labial palps of C. virginica (Pales Espinosa et al., 2009) and Mytilus edulis (Pales Espinosa et al., 2010a) agglutinates red blood cells and several microalgae species. Agglutination was inhibited to various degrees by different carbohydrates, suggesting the presence of several lectins in mucus. Further, several putative C-type lectins have been identified in the transcriptomes of the oyster C. virginica (CvML) (Jing et al., 2011), C. gigas (CgCLec-1) (Yamaura et al., 2008), and the mussel M. edulis (MeML) (Pales Espinosa et al., 2010b). Gene transcription analysis coupled with in situ hybridization revealed that CvML and MeML were specifically localized in mucocytes lining the epithelium of the pallial organs (Figure 12.5), while CgCLec-1 was expressed only in the digestive gland. In addition, CvML and MeML gene transcription levels were significantly up-regulated after bivalve starvation, suggesting that these molecules are involved in food particle capture (Pales Espinosa et al., 2010b, 2013; Jing et al., 2011). More interestingly, CvML mRNA levels increased significantly in oysters exposed to a bacterial challenge, Vibrio alginolyticus, but only following bath exposure and not bacterial injection into the circulatory system, which shows that this lectin responds to external cues associated with the presence of pathogens in the pallial cavity, but not in tissues, and highlights its role in mucosal immunity (Jing et al., 2011). In the symbiotic clam Codakia orbicularis, which harbors sulfo-oxidant bacteria in its gills, a calcium-dependent C-type lectin (codakine) was identified and purified from gill tissues (Gourdine et al., 2007). These authors hypothesized this lectin to be involved in the recognition of symbiotic and possibly pathogenic bacteria alike. Finally, several lectins (including a galectin and a c-type lectin) were up-regulated in mantle tissues, and hemocytes transmigrated to the extrapallial cavity in clams affected by brown ring disease, a shell disease caused by Vibrio tapetis (Allam et al., 2014). Similar trends were also identified for several immunoglobulin-like sialic-acid-binding lectins (c1q domain-containing proteins), which seem to display an elaborate, tailored response to various microbial challenges affecting mucosal tissues (Allam et al., 2014). Overall, molluscan mucosal tissues and secretions appear to contain a large repertoire of lectins that seem to vary with the reproductive cycle and overall physiological conditions of the animals (Pales Espinosa and Allam, 2013).

Figure 12.5. A. Detection of Crassostrea virginica mucocyte lectin (CvML) in mucocytes (arrows) lining oyster mantle epithelium by in situ hybridization using specific cRNA probes. B. Relative expression of CvML (Log scale, normalized to 18S) in different organs determined by qRT-PCR after exposure to V. alginolyticus. For each organ, *indicates significantly higher expression (t-test, p < 0.05) in challenged oysters compared to controls represented by the x-axis (from Jing et al. 2011).

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Prospects of nanotechnological development for wound management

Pooja Singh, ... Ravindra Pratap Singh, in Nanotechnological Aspects for Next-Generation Wound Management, 2024

15.2.2 Metal and metal-oxide based nanomaterials

Metal and metal-oxide-based nanoparticles have received considerable attention in the biomedical domain due to their antibacterial property, low toxicity, and antiinflammatory properties. The most studied, metals such as gold, silver, copper, and zinc compounds are widely used in treating skin-related disorders. Due to hydrolytic stability, porosity, heterogeneity, and chemical structure, gold nanoparticles widely used as effective nanomaterials in wound healing and treating damaged skin cells. Leu et al. (2012) reported gold nanoparticles, epigallocatechin gallate (EGCG), and α-lipoic acid (ALA) effects on human dermal fibroblast (Hs68) and HaCat migration and proliferation which significantly accelerates its antiinflammatory effects in cutaneous wound healing. Silveira et al. (2014) demonstrated an amalgamation of microcurrent (MIC) and gold nanoparticles (GNP gel) (MIC+GNP gel) had improved the mitochondrial functioning and oxidative stress marker activity, which contributes to tissue repairing and wound healing. Gubitosa et al. (2020) studied the potential effect of snail secretion (SS) from Helix aspersa, in gold nanoparticles synthesis (AuNPs-SS) by adopting several techniques such as XPS, UV-Vis, ATR-FTIR spectroscopy, DLS, and high-resolution MALDI-MS analysis. The SS protein plays a very crucial role in cell growth and regeneration, which prevents the effect of inflammatory diseases and many skin-related disorders. As a result, the formation of AuNPs-SS is used as antiinflammatory agent in the biomedical domain which accelerates its efficiency in wound healing and in treating many painful inflammatory disorders such as rheumatoid arthritis. Al-Musawi et al. (2020) reported, in vitro antibacterial activity of honey/tripolyphosphate (TPP)/chitosan (HTCs) loaded with capsaicin derived from the natural extract of hot pepper (Capsicum annuum L.) and gold nanoparticles (AuNPs) against Klebsiella rhinoscleromatis, Pasteurella multocida, Vibrio vulnificus, and Pasteurella multocida shows promising antimicrobial wound healing property. Xie et al. (2020) reported excellent antibiotics activity of AuNPs against multidrug-resistant bacteria (MDR) bacteria. According to strategy, antibacterial spectrum of small molecules (4,6-Diamino-2-pyrimidinethiol-improved AuNPs (DGNPs, DAPT-AuNPs)), with ultrasmall (Us) size shows antibacterial efficiency against gram-positive bacteria (G+) bacteria. Moreover, Us DAuNPs-functionalized scaffolds (agarose gel) shows wound healing properties against burn infection. Hu et al. (2020) reported UsAuNPs modified 2D metal-organic frameworks (MOFs)-based nanoenzyme with good biocompatibility that shows excellent antibacterial property against gram-positive bacteria can effectively facilitate wound healing. Moreover, silver nanoparticles (AgNPs) have also received great attention in wound healing due to properties such as high surface area to volume ratio and very efficient drug administration. AgNPs have explored its antiinflammatory potentialities in antimicrobial wound dressing and serves as a real topical bullet for wound repairing (Gunasekaran et al., 2011). Khan et al. (2022) reported antibacterial, antioxidant, and catalytic activity of biosynthesized AgNPs of Rumex hastatus (Polygonaceae) plays a promising role in treating skin diseases, rheumatism as well as promotes wound healing. Liu et al. (2022) reported, antiscarring, and antibacterial property of electrospun chitosan nanofibers containing AgNPs associated with curcumin nanoparticles (Cur@β-CD/AgNPs) possessed synergistic potential effects on wound healing. Tang et al. (2020) reported hemostatic and antibacterial activity of adhesive, prepared by using methacrylated hyaluronan-polyacrylamide (MHA-PAAm) hydrogels, gelatine bonded, integrated with AgNPs plays a potential role in wound healing. Mehwish et al. (2021) reported, therapeutic potential application of Moringa oleifera seed polysaccharide-enclosed AgNPs (MOS-PS-AgNPs) displayed strong antimicrobial activity against pathogenic bacteria and used as a wound dressing material. Zhang, Wang, et al. (2021) reported due to excellent poor biocompatibility and antibacterial activity upper layers of Ag-MOF-loaded chitosan NPs (Ag@MOF/CSNPs) and lower layers of polyvinyl alcohol/chitosan/sodium alginate (PACS) with good water retention, biocompatibility, uniform poor size distribution, water vapor permeability, and antibacterial activity were employed as upper and lower layers to successfully develop a bilayer composite dressing for wound treatment. El-Aassar et al. (2020) reported natural absorbable material such as hyaluronic/polygalacturonic acid-embedded antimicrobial AgNPs, which is further designed to shape nanofibrous mat, using electrospinning which shows its advanced wound healing property. Maghimaa and Alharbi (2020) reported antimicrobial potential of biologically synthesized AgNPs from Curcuma longa L. coated on cotton fabrics utilized for wound healing applications. Moreover, copper (Cu) plays a crucial role in all stages of wound healing process and it modulates several growth factors as well as cytokines mechanisms of action. Salvo and Sandoval (2022) investigated that Cu shows good efficiency in angiogenesis and regenerating damaged skin cells, and expedites the process of recovery through induction of angiogenesis and endothelial growth factor (VEGF) by hypoxia-induced factor- 1-alpha (HIF-1α) action, where HIF-1α expression binds to the crucial motifs in the promoter and enhancer region of HIF-1α-regulated genes. Wang et al. (2021) reported antibacterial activity of Cu MOF-embedded carboxymethyl chitosan-g-polyacrylamide/glutathione hydrogels accelerates in vivo wound healing by inhibiting oxidative stress and killing bacteria. Lin et al. (2021) reported excellent antibacterial and hemostasis properties of Cu-loaded melanin and polydopamine (PDA) NPs with low toxicity and good blood compatibility promotes healing as well as prevents wound infections. Lemraski et al. (2021) reported double layer nanofibers composed of chitosan, poly(vinyl alcohol) as well as polyvinylpyrrolidone (PVP) show its antimicrobial efficacy in wound dressing against gram+ve and gram−ve bacteria. Bayrami et al. (2020) reported green synthesis of CuO nanocomposite using propolis extract with gauze coat utilizes its potential antibacterial application in wound healing. Moreover, zinc oxide nanoparticles (ZnO NPs) have been investigated for its antioxidant, antiinflammatory, and antibacterial activity for wound healing applications. It stimulates re-epithelialization, cell migration, skin repair as well as angiogenesis, which is the most relevant to wound dressing and healing (Rayyif et al., 2021). Irfan et al. (2022) investigated biological synthesis of ZnO NPs by translucent and yellow colored gum Acacia modesta, coated with suture (sterile surgical threads) holds good antibacterial potential applications against Methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli and shows excellent wound healing property. Zhang et al. (2022) reported zinc silicate NPs (Zn2 SiO4 NPs) with spindle-like morphology integrated with bioactive nanofibrous scaffolds possess promising vascularization, re-epithelization, and innervation ability via local release of bioactive Zn and Si ions from ZS NPs leads to rapid tissue regeneration and rapid wound healing. Hasanin et al. (2022) investigated novel green synthesis of cotton pads doped with ZnO NPs exhibits antimicrobial property against toxic microorganisms and shows the most relevant applications to design antimicrobial wound bandages. Zhang, Qiao, et al. (2021) reported a novel sodium alginate-chitosan oligosaccharide-based ZnO (SA-COS-ZnO) is fabricated using aldehydated sodium alginate (SA), chitosan oligosaccharide (COS), and zinc oxide (ZnO) NPs showed a promising antimicrobial property in wound care management.

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