Advanced (Cellular and Molecular) Approaches

### **Chapter 5**

Sustainable Management Plans in Fisheries and Genetic Tools: An Overview of the Challenge in Invertebrates' Fisheries at the Central Area of the Southern Bay of Biscay, Spain

*Marina Parrondo Lombardía, Lucía García-Florez, Eduardo Dopico Rodríguez and Yaisel Juan Borrell Pichs*

#### **Abstract**

The fishing and aquaculture sectors are an important source of development around the globe. In Asturias (Spain), the diversity and richness of the fishing grounds of the Cantabrian Sea favored the historical settlement of a large number of communities closely linked to the marine environment and fishing resources, forming an integral part of the region's cultural and natural heritage. However, aquatic ecosystems are facing, nowadays, important threats from anthropogenic activities. To address these problems and avoid their impact on fishing activities, it is essential to know the ecological and genetic status of the species. Despite this, the application of genetic tools is still incipient in many species of commercial interest; however, its use can help to generate data that allow better regulation and fisheries planning. Here, the use of genetic markers and educational strategies in the management of some shellfish species of great commercial and cultural value in Asturias are reviewed. Moving toward sustainable fisheries management is a priority that can only be achieved through R + D + i, educational strategies, and the development and implementation of a regional strategy oriented toward the sustainable management and exploitation.

**Keywords:** small-scale fisheries, shellfish, DNA, mitochondrial DNA, microsatellites, eDNA, traceability, mislabeling, fraud, management units, connectivity, stock management, mitigation aquaculture, invasive species, game-based learning

#### **1. Introduction**

In 1984, the United Nations (UN) established an independent group of 22 individuals from member states and charged them with identifying long-term environmental strategies for the international community [1]. In the resulting report of the World Commission on Environment and Development, entitled Our Common Future—also known as the Brundtland Report [2], the term "sustainable development" was used extensively and defined as "development that meets the needs of the present without compromising the ability of future generations to meet their own needs."

The fisheries, seafood, and aquaculture sectors are an important source of food and income for millions of people around the world [3]. Addressing the problems associated with fisheries is an essential purpose, not only in the development of marine conservation policies but also for the achievement of the sustainable development goals (SDGs) of the 2030 Agenda, a major agreement that was signed in 2015 among 193 countries [4]. The achievement of these SDGs should have a strong influence on the governance of sustainable fisheries and aquaculture, ensuring that fisheries and aquaculture adapt to the impacts of climate change and improve the resilience of food production systems [3].

In addition, aquatic ecosystems face today significant threats from anthropogenic activities. In global ocean systems, concerns include climate change, overfishing, dispersal of invasive species, fertilizer runoff, plastic pollution, ocean acidification, and general defaunation [5–10]. Only 65.8% of fish stocks are currently classified as being exploited within biologically sustainable levels, continuing a downward trend that has been occurring since 1974. Similarly, underexploited species account only for 6.2% –steadily declining from 1974 to the present—whereas stocks exploited at maximum sustainable levels account for 59.6% [11].

It has been demonstrated that when fisheries are properly managed, there are significant decreases in fishing pressure and important increases in stock biomass, with some stocks having reached biologically sustainable levels, underscoring the relevant role of fisheries managers and governments when willing to take strong action [12]. The UN Code of Conduct for Responsible Fisheries states that in adopting management measures, the "best available scientific data should be used to assess the state of fishery resources" [13]. However, most exploited stocks globally are classified as data-poor stocks [14] and their status, although highly uncertain, is generally considered to be worse than that of data-rich stocks [15]. Recently, it has been argued that stock estimates based primarily on historical catch series performed on average 25% better than a random estimate; but in turn, these methods assigned fisheries the wrong FAO status category 57% of the time [16]. Substantial improvements in estimates of the state of exploited stocks worldwide depend on the expansion of new information and efficient use of existing data [16].

The use of molecular genetic techniques in fisheries research has increased dramatically in recent decades, paralleling the awareness of the value of genetic data and mainly due to the increased number of techniques available and improvements in computer technology [17, 18]. However, the application of genetic techniques to invertebrate fisheries or related problems has been remarkably scarce. Thus, most of the invertebrate groups of fishery interest have been the subject of little or no genetic study in relation to these fisheries [19].

We reviewed here the use of genetic markers as well as educative strategies in the fisheries management of some shellfish species with great commercial and cultural value in Asturias, the central area of the southern Bay of Biscay, Spain, to move forward with the relevant aim of generating data to support the design of sustainable fisheries management plans.

#### **2. Fisheries in the central Cantabrian Sea: evidence, needs, and actions aimed at reaching sustainable exploitation levels**

#### **2.1 Fisheries in the principality of Asturias**

The Principality of Asturias is an autonomous community located in northern Spain (SW Europe) bordered by the Cantabrian Sea to the north and the autonomous regions of Castilla y León, Cantabria, and Galicia to the south, east, and west, respectively. The Cantabrian Sea is the transition from the Atlantic Ocean to the Bay of Biscay, between Spain and France. The coast of Asturias covers about 30% of the Cantabrian coast and presents a general E-W trend along approximately 335 km. In general, this coastline is eminently rocky and abrupt—with a predominance of northfacing cliffs, dotted with small coves, beaches, and dune systems associated with the wider beaches [20].

Currently, the coast of Asturias is one of the most populated areas in the region, which is linked to the presence of industrial activity, such as fishing industry, factories, and ports, and to the tourist exploitation of natural resources. The historical settlement of a large number of coastal communities strongly linked to the marine environment and fishing resources was favored by the diversity and richness of the Cantabrian fishing grounds [20, 21]. These regional fishing grounds have been recently redefined and mapped, totaling 226 and occupying 984,038 hectares.

Fishing is a traditional activity linked to the Asturian coast since prehistoric times, as evidenced by archeological excavations in which numerous mollusks, crustaceans, salmonids, and other marine remains have been found [22–27]. Nineteen fishing seaports currently operate along the entire coast, and all of them are strategically located taking advantage of the sheltered location of the cliffs. These ports differ in terms of total landings, species marketed, and number of vessels. Of the total number of fishing ports, 17 of them record sales for at least 6 months of the year [21]. The National Strategic Plan of the European Fisheries Fund established that of the total number of councils that form the Asturian coastal belt, only Muros, Castrillón, and Caravia do not depend on fishing activity [28].

In Asturias, specialists agree in considering artisanal vessels registered in the category of the national census of the operational fishing fleet of "minor gears" [29], and, in 2010 there were 233 vessels registered in that category. However, according to the data collected in the Census of the Fishing Fleet registered in Asturias, in 2016 the number had decreased to 202 and, although at present (latest data from 2021) the total number of artisanal vessels totals 192—out of a total of 248 active vessels—distributed in 19 ports—representing 77.4% of the total fishing fleet with home port in Asturias –, the downward trend in the fleet of minor gears is not so pronounced, even stabilizing.

In terms of fishing strategies, most of this regional artisanal fleet switches between different gears throughout the year and exploits areas that can be reached in a few hours from fishing ports. Although the landings of the artisanal fleet—in kilograms—only represent around 10% of the total regional catch, they account for approximately 30% of the total economic value generated by the landings, due to the higher first sale price of these catches (high-value seafood products) compared to those of other fleet segments—average 4.25 €/kg compared to 1.68 €/kg –. The regional artisanal fleet targets many species, including high-value species, and landings are characterized by quality, freshness, and higher first-sale profit. This partly compensates for the lower weight of landings and lower fishing power [21, 30].

The main species landed by the Asturian artisanal fleet are hake (*Merluccius merluccius*), mackerel (*Scomber scombrus*), conger eel (*Conger conger*), sea bass (*Dicentrarchus labrax*), and octopus (*Octopus vulgaris*), in addition to the shellfishing of stalked barnacle (*Pollicipes pollicipes*). Eel (*Anguilla anguilla*) fishing, and other fishing activities, such as stalked barnacle (*P. pollicipes*) harvesting, play a fundamental role as alternative fishing activities during certain periods when it is difficult and unprofitable to carry out other activities, that is, the first months of the year when many fishing activities cannot be carried out due to bad weather conditions [30].

Artisanal fisheries have traditionally received less research effort than industrial fisheries and have generally received little attention in Europe [31, 32]. This lack of information has reduced the potential for developing effective and integrated management measures aimed at improving the long-term sustainability of artisanal fisheries, considering the complex interactions and linkages between the human and natural dimensions within these fisheries [33]. Despite the comparatively low volume of catches and its economic relevance, artisanal fisheries are important in terms of employment and must be considered in economic terms at the local level. It contributes to strengthening people's attachment to their territory, increasing social stability in rural and peripheral areas [32].

#### **2.2 Traceability as a cornerstone of sustainable fisheries management in Asturias: a case study on scallops (family Pectinidae)**

The UN Code of Conduct for Responsible Fisheries stated in 1995 that the "best available scientific data for assessing the state of fishery resources" should be used for effective fisheries management measures [13]. Traditionally, fisheries conservation and management have been conducted on the basis of abundance data, productivity estimates, and information on stock dynamics—that is, an intraspecific group of randomly mating individuals with temporal and spatial integrity [34–37]. However, managers need to be aware that the implementation of legislation, stocking strategies, and other management activities affect the genetic composition of populations [38–40]. Genetic factors play a role in the conservation of fishery resources because fishery resources are the product of their genes, the environment, and the interactions between them [41]. Although understanding of the state of global fisheries has now improved over the last decade, there is a consensus that data remain incomplete, with most of the world's fish stocks lacking formal statistical assessments [16]. In addition, this lack of biological data is compounded by the fact that not all fishery catches are properly reported or recorded by governmental or non-governmental agencies. These unreported catches may be illegal, of unregulated species, or simply not monitored due to logistical barriers [42]. Mislabeling, inaccurate species identification in landings [43, 44], or the modification of the catch area are other factors that also contribute to the unreported exploitation of stocks and the consequent reduction of fishery resources.

The Common Market Organization (CMO) for fishery and aquaculture products is laid down in Regulation (EU) No 1379/2013 of the European Parliament. In Article 35, the commercial designation of the species and its scientific name (among other relevant data) are included as mandatory information on the relevant labeling. Furthermore, Article 37 stipulates that the Member States shall draw up and publish a list of the commercial designations accepted in their respective territories, together with their scientific names [45]. Informative labeling is particularly important for processed products, as any recognizable external morphological characteristics are

*Sustainable Management Plans in Fisheries and Genetic Tools: An Overview of the Challenge… DOI: http://dx.doi.org/10.5772/intechopen.105353*

often removed, so consumers rely on product labeling for information about the contents of the product.

In recent years, molecular biology techniques—based on DNA and sequencing have gained notoriety in the study of mislabeling and food fraud, allowing species identification even if the product under suspicion is highly processed [46, 47]. A combined assessment of the levels of fraud in the commercialization of fresh and processed specimens of the family Pectinidae, both in retail establishments and restaurants, has been carried out using genetic methods based on mtDNA—16S rRNA gene–and taxonomic methods in Asturias [48]. That research showed that out of 148 samples of 15 commercial products analyzed, 73 samples (49%) and 9 (60%) of the 15 commercial products studied were mislabeled. In the case of the 20 samples purchased in 20 restaurants, all specimens labeled with the common name "zamburiña" were assigned to the Pacific scallop (*A. purpuratus*), resulting in 100% substitution fraud in the samples analyzed. These results are in agreement with the numerous works previously carried out that used molecular markers as an effective tool for the identification of species—both in fresh and frozen products as well as in highly processed ones—that, in many cases, were impossible to identify without the use of this type of tools [49–52]. At the same time as Parrondo et al. study [48] was published, Klapper and Schröder report the development of a multiplex qPCR assay using a TaqMan probe that allowed the rapid and reliable identification of three commercially important scallop species in German supermarkets and fishmongers—the scallop (*P. maximus*), the Atlantic scallop (*Placopecten magellanicus*), and the Japanese scallop (*Patinopecten yessoensis*) [53]. Using this new tool, Klapper and Schroeder revealed a mislabeling rate of 48%—especially high for products purchased in fish shops. Furthermore, they showed that only 18 out of 33 (52%) samples were correctly labeled and in 12 (36%) samples the scientific name was not provided. Where appropriate, the rate of mislabeling in supermarkets was relatively low (5 out of 21, 24%) compared to fishmongers (8 out of 10, 80%) and restaurants (2 out of 2, 100%) [53]. Despite differences in methodology and target species, the results obtained for Germany by Klapper and Schröder are consistent with the data produced by Parrondo et al. [48] for Asturias.

These investigations join those previously presented by other authors, both in the development or use of forensic biology techniques and in the creation of a threshold of knowledge in the study of mislabeling and fraud of fishery products from marine invertebrates, specifically scallops [54–59]. Despite the difficulty in making accurate estimates of the extent of mislabeling, especially in invertebrates due to lack of data and bias toward certain taxa and geographical areas [60], the high percentages of errors in product labeling found in Parrondo et al. [48] are not unusual in other European countries, such as Iceland, Finland, or Germany, where similar results of between 40 and 50% were obtained [51]. These percentages are alarming if we take into account that most of the samplings carried out for these studies (including the present one) lack temporality, being performed only once in a specific locality, so the approximations tend to be always conservative [49].

Research regarding mislabeling and food fraud in seafood products are increasingly extensive at all levels of the production chain [49, 50, 61], the urgent need for control measures throughout marketing to avoid consumer confusion, mislabeling, or potential health problems—such as allergies due to substitutions [62]—is evident. It is worth mentioning that, in general, there is an increased awareness of the industry to improve the transparency of the food chain, as well as the growth in the number of inspections put in place by European official control bodies, which have achieved

a significant reduction in the number of incidences of misdescriptions [63]. This lack of monitoring plays an important role in threatening the sustainability of fisheries, despite international efforts, and may even imply the eventual extinction of more vulnerable overfished species [64].

In Asturias, the existing regulation gives legal and regulatory support to food quality—especially to differentiated quality and organic production—and establishes regulation of infractions and sanctions, with the aim of tackling intrusion and fraud [65]. However, the Asturian law is framed within Community regulations and applies European legislation on labeling. That is why, from a legislative point of view, Article 35 of Regulation (EU) No 1379/2013 of the European Parliament should be amended to include the fishery and aquaculture products listed in points h) and i) of Annex I to that Regulation, which refers to "prepared or preserved fish; caviar and caviar substitutes prepared from fish eggs" and "crustaceans, molluscs, and other aquatic invertebrates, prepared or preserved." It is indisputable that to implement correct management and planning of the exploitation of marine resources and to watch over the rights of consumers, it is necessary to increase routine controls and sanctions both on fishery products and those prepared and processed throughout the whole production chain—with emphasis on those stages where there is greater evidence of fraud and on those species to which, perhaps, less attention has been paid, such as marine invertebrates.

#### **2.3 Sustainable fisheries management, certifications, and the scarcity of biological data in Asturias**

#### *2.3.1 The MSC octopus fishery in western Asturias: dilemma and challenges*

Labeling to provide additional ecological information about a product is usually voluntary. FAO recognized that it could contribute to improved fisheries management and convened a technical consultation in 1998, which led to the development of the "Guidelines for the Ecolabeling of Fish and Fishery Products from Marine Capture Fisheries" [66]. Since then, numerous programs have been proposed for ecolabelling seafood products in an effort to encourage fisheries managers to create sustainable fisheries. One of the most recognized today is the Marine Stewardship Council (MSC)—created in 1997, thanks to a collaboration between the World Wildlife Fund (WWF) and Unilever, a multinational company that markets several international brands [67]. The aim of these initiatives is to provide a market-based incentive for sustainable fisheries management. Processors, wholesalers, and retailers who purchase products from these accredited fisheries may acquire the right to affix an eco-label, informing consumers that the product has been caught in a sustainable fishery. Hypothetically, if there were a demand for environmental quality, consumers would respond by purchasing those products with an eco-label, thereby reducing demand for those without and causing price devaluation on unlabeled products. This may result in fishermen putting pressure on fishery managers to achieve sustainability accreditation and thus receive a higher percentage of the price [67].

In February 2016, the MSC awarded the Tapia, El Porto, Ortigueira, and Veiga fishermen's associations the first certification for the octopus fishery worldwide. *O. vulgaris* (Cuvier, 1797) is the cephalopod species with the largest landings in Asturias, being this resource of great importance for the artisanal fleet of the region [68]. This fishery operates with a "management plan for the common octopus

#### *Sustainable Management Plans in Fisheries and Genetic Tools: An Overview of the Challenge… DOI: http://dx.doi.org/10.5772/intechopen.105353*

(*O. vulgaris*) in the Principality of Asturias," developed by the Directorate General of Maritime Fisheries of the Government of Asturias with the collaboration of the fishermen's guilds, which is revised every year [69]. From that moment on, all octopus caught by certified vessels is eligible to display the blue MSC ecolabel, allowing consumers to enjoy this species with the security that it comes from a sustainable and environmentally friendly fishery [70]. The MSC report accrediting such sustainability highlighted some weaknesses in certifying this fishery, warning that "biological information on the resource was still scarce" and explicitly recommending that "information on the knowledge of octopus stocks should be improved." This means that relevant parameters, such as maximum sustainable yield and stock definitions, were still lacking or incomplete for octopus fisheries.

Stock assessment is crucial, being an integral part of fisheries management. However, it can be challenging due to the methodological difficulties arising from marine monitoring using traditional methods—such as individual capture (with trawls, nets, or traps) or visual identification of species based on their distinctive morphological characteristics—and the amount of time consumed. Moreover, its financial cost is high, and, in some cases, it is simply unfeasible. The need to overcome these impediments has stimulated the search for new tools and approaches to integrate different environmental dimensions into decision-making in a data-driven policy approach [71].

Since 2016, improvements have been made in those areas where information was lacking for the management of the octopus fishery in Asturias. Particularly important has been the development of a model for octopus stock assessment, and the determination of annual reference points for the fishery, allowing the estimation of an annual TAC per campaign that responds to the situation of the octopus stock in the area [72], as well as making it possible for octopus fished with traps in western Asturias to have achieved—in this year 2021 and for five more years—the recertification of this MSC ecolabel. This work is part of a research project (ECOSIFOOD; MCI-20-PID2019- 108481RB-100) funded in 2020 by the Spanish National State Program of Research and Development oriented to the challenges of society.

This last research project is also included as a target to work on obtaining new molecular traceability matrices based on new genomic and environmental DNA (eDNA) data from octopus and their application in temporal and spatial samples to help in detection, quantification, defining historic and contemporary patterns of genetic variation data, stocks and management units. The concept of the biological stock as the basic population unit of exploited species is fundamental to the management of wild fisheries. The delimitation of appropriate conservation units—which is the core of short-term management programs—is also a difficult task in marine systems that have traditionally been characterized as genetically undifferentiated populations [73] due to the large population size, high dispersal potential, and high fecundity of these species [74, 75].

The collection and analysis of water samples for eDNA has, in many cases, proven to be a cost-effective, sensitive, and noninvasive method for species presence/absence surveys, in contrast to established monitoring techniques that rely on the capture of whole organisms [76–78]. Studies now abound listing the many qualities of eDNA analyses for aquatic species detection and distribution assessment using DNA released into the environment by marine organisms, both vertebrates [79–81] and invertebrates [82–84]. The development of more reliable and cost-effective procedures for monitoring commercial species populations may, therefore, improve stock assessment [85]. An eDNA-based method was developed

for stock assessment of *O. vulgaris*, pioneering work in the use of this methodology to estimate the sustainability of common octopus fisheries [86]. Furthermore, in that work it was found a positive and significant correlation—Pearson correlation coefficient 0.38627, p-value = 0.01261—between octopus biomass and eDNA abundance detected in tank experiments [86]. These results are in agreement with previous studies that showed a strong linear relationship in aquaria: Takahara et al. found that eDNA concentration was positively correlated with carp biomass in both aquaria and experimental ponds, furthermore, they used this method to estimate carp biomass and distribution in a natural freshwater pond [87]; Maruyama et al. examined the effect of the developmental stage of sunfish (*Lepomis macrochirus*) on environmental DNA release rate, finding a positive relationship between fish size and eDNA release rate [88]; and Klymus et al. used controlled laboratory experiments to measure the amount of eDNA that two invasive carp species (*Hypophthalmichthys nobilis* and *Hypophthalmichthys molitrix*) shed into the water, finding a positive relationship with fish biomass by finding that fed fish, compared to non-fed fish, excrete more eDNA [89].

Other authors have found this relationship to be less pronounced [90–92] or even nonexistent [93] in natural ecosystems. However, in more recent studies, Spear et al. have shown that pikeperch density explains most of the variance in eDNA recovered in lake surface waters in natural systems [94]. Quantification of eDNA abundance is based on the assumption that local population size can be inferred by measuring eDNA concentration at a given locality and that this estimate represents the quantitative relationship between eDNA concentration and underlying population size [95]. However, such a relationship may not always be true, or even present in most cases. The results of Mauvessau et al. [86] show significant variations in the amounts of eDNA detected in the different sampling points located in the Cantabrian Sea. In any case, the observed variation in the amount of eDNA may be due to different and even unknown factors. Compared to eDNA sampling in river systems—which also poses its own set of problems, often difficult to address—factors such as tides, currents, large depths, and rapid movements of individuals in three dimensions may affect the collection of unbiased samples [71, 96].

The results presented by Mauvisseau et al. [86] were obtained using speciesspecific primers and the qPCR technique using the SYBR Green compound double-stranded DNA-binding dye that allows the detection of the PCR product as it accumulates during PCR, as it is a simple, easy, and economical option. However, other types of technical approaches are now common, such as qPCR using TaqMan probe or recently digital droplet PCR (ddPCR) [97]. More recently, intraspecific diversity assessments have been performed in several species [98, 99], finding multiple haplotypes that had previously been identified from tissue-derived DNA by Sanger sequencing. This is a revolutionary tool for fisheries and population management, as the use of eDNA could allow detection, quantification, and estimation of diversity with minimal sampling efforts.

The use of environmental DNA-based tools to quantify commercial species populations is of great interest to fisheries managers and policy makers, as stock assessment is a central component of any management and/or conservation program [71]. There is a strong need to inform researchers, advisors, managers, and other stakeholders about the many challenges (and opportunities) associated with the application of environmental DNA analyses in routine marine fisheries management [85].

*Sustainable Management Plans in Fisheries and Genetic Tools: An Overview of the Challenge… DOI: http://dx.doi.org/10.5772/intechopen.105353*

#### *2.3.2 Getting data from a potentially eco-certifiable fishery in Asturias: the highly valued goose barnacle P. pollicipes*

The stalked barnacle fishery in Western Asturias has been co-managed by the fishermen's guilds and the Center for Fishing Experimentation (General Directorate of Maritime Fisheries of the Principality of Asturias) since the eighties. On the west coast of Asturias, harvesting is not open, but rather each fishery guild or group of guilds is granted the right to exclusively exploit barnacles in a strip of the coast, a system known as "exploitation plans." In exchange, the guilds commit to exploiting the barnacle in a sustainable manner (complying with the rules established in the exploitation plan) and to report in minute detail the date, place, and amount of barnacle extracted, which is an extraordinary source of information for research and management [100]. In these systems, fishermen become co-responsible for management, intervening in the design of the exploitation plans. The DGP collects and analyzes the data on daily catches per shellfish gatherer and extraction area collected by the authorized fish rangers in each exploitation plan. At the same time, in collaboration with shellfish harvesters and fish wardens, the total and partial closed seasons and areas for each season are evaluated and proposed.

The potential of genetic approaches for the identification of fish stocks has long been recognized. However, in practice, stock assessments for management purposes often do not incorporate information on the biological stock structure because genetic and biological data are unavailable or ambiguous [101]. Even today, the isolation and characterization of new molecular markers remain difficult and costly for many non-model species [102]. Although microsatellite markers—and now also SNPs—are increasingly available for more species, studies on most marine organisms are still limited by marker availability and biased toward those of greatest commercial interest. A good example of this could be the barnacle fishery, which has an annual economic value of 10 million euros, with about 500 t of landings and 2100 professional fishermen involved [103] and yet has hardly been studied from a genetic point of view [100, 104, 105]. Although this may be alarming due to their importance, crustaceans—and other marine invertebrates as well—still lack genetic and genomic resources compared to other widely studied groups [106, 107]. The research projects PERCEBES (PCIN-2016-120) (funded by the EU Biodiversa call in 2016) and ECOSIFOOD (MCI-20-PID2019-108481RB-100) have targeted this objective of developing new genetic tools (microsatellites and SNPs) to assess genetically the fishery stocks of the stalked barnacle *P. pollicipes*.

Microsatellites are established as the most popular and versatile marker type [17]. Their hypervariable nature confers sufficient power to compare gene pools between populations, as unique alleles appear at low frequencies that are useful for discriminating populations. Under a standard set of parameters that includes 20 highly mutational microsatellite loci and approximately 50 individuals from each of the subpopulations to be sampled, the power to detect deviations from panmixia is very high—even with high gene flow [73]. This is the case of barnacle populations, where patterns of spatial and temporal structuring have been observed at a scale where variability should be homogenized by gene flow through larval dispersal and coined as chaotic genetic patchiness (CGP) [108–110].

The detection of genetic differences between samples far apart in space or time implies the existence of some level of demographic independence and the presence of separate populations [34, 73, 111]. The ongoing analyses carried out with 20 new microsatellite loci aimed to define, with greater precision, the spatiotemporal evolution of the genetic structure of the barnacle *P. pollicipes* [112]. Preliminary results using microsatellites pointed out a population dynamics where *P. pollicipes* displays high genetic diversity along the Iberian Peninsula, which is attributable to large effective population sizes representing a well-connected network of local populations. However, temporal and spatial genetic differentiation of populations over regional scales, on one hand, and a significant reduction in genetic diversity in juveniles, on the other hand, clearly indicate that patterns of exchanges together with seasonal wind-induced upwelling may induce genetic differences between settlers throughout generations. Such patterns of chaotic genetic patchiness are likely due to sweepstake reproductive success with possible collective dispersal or episodic self-recruitment events [112]. In the specific case of the barnacle fishery, the future use of SNP markers would allow a more precise review of concepts such as population structure—which has been described as patchy [112]—and larval connectivity—which on the Asturias coast seems to take place on a small scale [100]; also if there are differences or not between the phenotypes considered of better and worse quality—in previous reports, these differences were not significant [113]—or how landscape components affect the resource on its quality [114]; allowing to know the evolutionary forces that drive the spatio-temporal heterogeneity of barnacles, and thus being able to assess and preserve the evolutionary potential of this fishery resource in a context of global change. Although these boundaries are usually spatially defined, they may also have a temporal component [115].

#### *2.3.3 The management of declining or depleted stocks in Asturias: restock or restore? The sea urchin case study*

*Paracentrotus lividus* is a sea urchin with an important ecological role in the Cantabrian Sea ecosystem. In the same context of global change and triggered in recent decades by overfishing, there has been a definitive decline in sea urchin (*P. lividus*) populations on the Asturian coast. Among the measures to mitigate this population collapse, the Government of Asturias decided to establish a year-round ban on the capture of this species and to undertake a population restoration experience with the aim of preserving this marine resource. However, these practices may entail a series of genetic risks that are widely recognized and documented in the literature and that can be summarized as follows: (i) loss of genetic diversity, (ii) loss of fitness, (iii) change in population composition, and (iv) change in population structure. Moreover, although adverse genetic impacts on wild populations are recognized and documented, little effort is devoted to their actual monitoring [116]. Because of this, these practices are highly controversial [117] and their utility is not always clear to fisheries and conservation goals [118]. Genetic monitoring of *P. lividus* populations in the central Cantabrian Sea (Asturias) was carried out using the mitochondrial DNA cytochrome B gene and microsatellite markers previously described by Calderón et al. [112, 119].

The results of a genetic diversity study based on mtDNA on this marine invertebrate show that the Asturian populations could constitute, at present, a peculiar and so far, undiscovered management unit (MU) of the Bay of Biscay, separate from that of the Atlantic populations. Microsatellite marker data—which reflect recent processes of population dynamics—did not reveal controversial results. However, both markers suggested genetic heterogeneity in the Mediterranean and Candás samples [112]. These results are fundamental, as an essential requirement for sustainable

#### *Sustainable Management Plans in Fisheries and Genetic Tools: An Overview of the Challenge… DOI: http://dx.doi.org/10.5772/intechopen.105353*

exploitation is the adequacy of biologically relevant processes and the scale of management: mismatches between the two often occur [120]. The *P. lividus* fishery in Galicia has been an example of how the mismatch between biological, fishery, and management scales causes governance failures, leading to overexploitation. *P. lividus* is spatially distributed in nested biological units: patches, micropopulations, local populations, and metapopulations. Fishing operations are local and exploit microstocks; however, management units in Galicia often comprise more than one local population. This pattern allowed the depletion of several micro-stocks with no shortterm signs in exploitation rates across the managed territory [121]. Identification of MUs is fundamental for the management of natural populations and is crucial for controlling the effects of human activity on species abundance [122]. Local sea urchin populations may be more isolated than suggested by larval dynamics [123–127].

The number of broodstock used in the sea urchin restocking experience carried out so far in Asturias—average number of spawners per event = 14.42—has been clearly far from what is desirable and from the recommended minimum number of broodstock [112]. This causes certain alleles and haplotypes to be overrepresented in the new population, leading to a reduction in the effective population size [128]. Besides this, microsatellite markers used indicated that juvenile individuals used for supplementation were genetically different from wild populations [112]. This means a poor representation of the wild gene pool in broodstock as a result—among other factors—of the low number of individuals used as broodstock since, in the particular case of marine invertebrates, they present a very high fecundity associated with a large variance in reproductive success [129], resulting in small Ne in cultured populations.

In Parrondo et al. [112] work, at least 3.5% overall of the total recaptured sea urchins with hatchery origin were found by randomly sampling 100 juveniles from each of the two restocked localities. Comparison of recapture rates is complicated for pilot studies [118], as they may depend on the objective of the experiment, the number of releases and size of individuals at the time of release, sampling effort, as well as the length of the experimental period and areas surveyed, and also the variance between studies. Despite the already commented previous findings, genetic monitoring of the restocked localities showed that they currently do not differ from the rest of the Asturian localities in terms of genetic diversity using both genetic markers, with no evidence of the Ryman-Laikre effect in the restocked populations [112]. Similar results were found in the *P. maximus* breeding program in Brest Bay (France), which had no effect on genetic diversity [130] and no R-L effect was detected [131]. Even if the reduction in allelic diversity and the alteration of allele frequencies were limited, they could accumulate over generations, gradually eroding the genetic variability of *P. lividus*, so long-term monitoring of these populations is proposed as standard practice.

Habitat restoration—taking into account that kelp forests are in serious decline due to, among other factors, the increase in temperature in the Cantabrian sea that has been occurring since the beginning of the 21st century, the increase in the intensity of storms or the limitation of available nutrients due to changes in the frequency of coastal upwelling [132], the construction of shelters against waves, which also seem to be effective in promoting the colonization of kelp [133], as well as the establishment of marine protected areas (MPAs)—which would allow maintaining the supply of larvae—may be other options to be considered to improve sea urchin populations. In some cases, translocation of adult sea urchins could also be considered; however, translocation can have an impact on the "host" population, so it is necessary to manage the stock of animals to

be translocated [133]—many of these measures have been successfully implemented in Japan. The integration of aquaculture-based enhancement with habitat restoration presents a notable opportunity for future research and development [134].

#### *2.3.4 Facing biological invasions and their threat to Asturias exploited fishery resources. The Crepidula case study in the Bay of Biscay*

Biological invasions are a key component of the ecological and biodiversity conservation crisis. One of the main threats caused by introduced species is the alteration of the structure of host communities -both terrestrial and aquatic- and the modification of ecosystem functioning [135, 136]. Although only a small fraction of the many species introduced outside their native range are able to thrive and invade new habitats, their impact can be dramatic [9, 137]. The invasion process unfolds as a multistage operation involving the acquisition of a propagule in its native range, the transport of that propagule to the new range, and the introduction, establishment, and spread of the invader in the new habitat [138].

Accurate analysis and effective modeling of current and future distributions of invasive alien species (IAS) are highly dependent on the availability and accessibility of occurrence data and information on the natural history of the species [139]. Because conventional sampling techniques often have very low probabilities of detecting rare species in aquatic ecosystems [140], such as the initial stages of invasion processes, not being really effective until the population is established—years after the first introduction—[141], tools that favor immediacy are necessary to combat the spread and establishment of invasive species, carrying out strategies of "early detection and rapid response" (EDRR).

Early detection is a vital step for the effective management of invasive species. The species-specific molecular markers for *C. fornicata* presented by Miralles et al. can be used to detect the early stages of invasions due to their sensitivity, low cost, and ease and speed of laboratory testing [142]. The results of that research demonstrate the presence of *C. fornicata* in close proximity to the *M. gigas* culture facilities operating in the Eo estuary. This oyster is very robust, with great physiological tolerance and an enormous reproductive potential, qualities that favor its cultivation and that have allowed it to become naturalized in all continents, making its eradication a complex task when it reaches high densities. In addition, it is an engineering species that generates important structural changes in the ecosystems it colonizes. *M. gigas* is also responsible for the global dissemination of many harmful species and pathogens, as is the case of *C. fornicata* [143]. This is why intensive sampling is necessary, as well as management measures to prevent the spread of *C. fornicata*, as *M. gigas* has been previously found in the region [144] even attached to floating marine debris [145], demonstrating its potential disperser of biological invaders [146–148].

#### **2.4 Marine citizenship for the new generations in Asturias? Education as tools for a sustainable fishing strategy**

There is a need for an education strategy at all levels for sustainable development that provides knowledge, skills, attitudes, and values to enable citizens to make informed decisions to take responsible action for environmental integrity. Environmental education is necessary to foster behavioral changes in the population that lead to a "citizenship of the sea." People who contribute to this "citizenship of the sea" show awareness and concern for the ocean and are motivated to take personal action to contribute to its

#### *Sustainable Management Plans in Fisheries and Genetic Tools: An Overview of the Challenge… DOI: http://dx.doi.org/10.5772/intechopen.105353*

protection [149]. The number of youth conservation movements is increasing around the world, for example, Fridays for Future—as the younger generations are more aware of the environmental issues affecting the planet. Consequently, young people seem to play a key role in the development of successful conservation programs. However, other avenues need to be developed to bridge the gap between positive attitudes and a real commitment to conservation and sustainable management [150].

The use of games as tools to enhance the acquisition of technical knowledge has long been studied as a powerful tool for learning, engaging, and tackling complicated tasks [151]. Sustainable sea is a strategy game developed for educational purposes in which players assume the role of fishermen while learning concepts related to the sustainable management of fishery resources [152]. Despite the small sample size, the board game provides information that can be useful for teaching fisheries management. According to the results of the pre- and post-tests, regardless of educational level, all groups improved their knowledge of specific topics after the activity. Knowledge gained by playing an educational game seems to be more effective because it is acquired through hands-on learning [153]. This not only raises awareness of the marine environment and its issues but also encourages a change in values to take personal responsibility for protecting the ocean [154].

The board game can be used to enhance the learning of technical concepts related to marine conservation, fisheries, and sustainable management of marine resources, being an alternative to conventional methods and a more useful educational resource, if possible, in the current context. In 2020, as the COVID-19 pandemic spread across the globe, most countries announced temporary school closures, affecting more than 91% of students worldwide—in April 2020, nearly 1.6 billion children were out of school - [155]. This board game can be very helpful tool when developing Information, Education, and Communication (IEC) activities by teachers. It can be played both in the classroom and among household members, the latter option being interesting because parents/guardians have greater responsibility for making decisions about household practices and this requires greater attention to be paid to how adults and children respond to environmental messages [156]. Marine issues are partly rooted in individual behavioral choices, which, either directly or indirectly through the global marketplace, have the potential to make a significant impact on the marine environment, such as through food choices – choosing correctly labeled or eco-labeled seafood – waste – reducing plastic use – and products [150, 154]. The development of learning can be seen as an intergenerational and multidirectional process that includes (but is not limited to) the information that children bring to families through educational formats for sustainability [156]. In addition, the game can be adapted to other fishing and marine resource scenarios, which are close to the players, as well as different educational levels. In the case of the board game, taking into account its low reproduction cost, it is an affordable tool for schools of all educational levels, as well as for anyone who may be interested in working on the goals of the 2030 Agenda.

#### **3. Take home messages while moving forward to a regional strategy for the management of exploited invertebrates' marine stocks in Asturias**

The work summarized in this chapter suggests that advancing toward a sustainable fisheries management that guarantees both, the employability and profitability of the sector, as well as the cultural and natural heritage in the region, is a priority

that can only be achieved through R + D + i and educational strategies—which require funding—and the development and implementation of a regional strategy oriented toward sustainable management and exploitation.

#### **3.1 Traceability and control over the products from fishing activities marketed in Asturias are still deficient and must be improved**

Irregularities in labeling and high levels of substitution fraud have been found in the analysis of scallops marketed with different degrees of processing and acquired in different establishments. The most processed products are those with the highest proportion of incorrect labeling. There is a need to carry out a review of the legislation and control methodologies—routine inspections, sanctions, etc.—that guarantee consumer rights, as well as the reliability of the databases on the first sale of fishery products on which fishery statistics are based and, therefore, fishery management, so that they become an efficient tool for the establishment of sustainable development strategies in the region.

#### **3.2 Design and establishing of new coastal marine protected areas (MPAs) in Asturias is necessary**

MPAs favor the conservation of biodiversity; the protection of critical habitats; the increase of fisheries productivity through the regeneration of populations; the increase of knowledge of the marine environment; the refuge and protection of genetic diversity; and the protection of heritage and cultural diversity [157]. The implementation of Marine Protected Areas (MPAs) in Asturias coasts under the umbrella of Ecosystem-Based Management (EBM) and Integrated Coastal Zone Management (ICZM) has been suggested by different fields, as they are subordinated to the wider ecological, social, economic, and political context of the coastal and oceanic zones of which they form part. The sustainable management and, therefore, the preservation of such relevant natural resources of the region as barnacles, sea urchins, and octopus, would benefit significantly from this. Ecosystem-based management (EBM) and Integrated coastal zone management (ICZM) based on knowledge of fish stocks and the implementation in Asturias of possible marine protected areas (MPA) closer to or including the coast is necessary to ensure an efficient larval supply. On the other hand, it seems advisable to establish the figure of technical personnel to assist in fisheries management in the fishery associations, similar to the existing one in Galicia.

#### **3.3 Start as soon as possible new eco-labeling certification processes for Asturias fisheries**

The fishermen's guilds that are part of the co-managed barnacle extraction schemes of central-western Asturias could proceed with an application for MSC ecosustainability certification. Previous studies endorse the high levels of sustainability of this fishery [103], which are managed through TURFs where fishermen actively participate in all aspects of management and share responsibilities with the administration in decision-making. However, among the disadvantages of this certification are the high costs derived from the external audits necessary to carry out its implementation, as well as the successive recertifications. The standards of this type of certification, which incorporate many aspects that were not considered in traditional management, can be incorporated into the management plans of artisanal fishing

*Sustainable Management Plans in Fisheries and Genetic Tools: An Overview of the Challenge… DOI: http://dx.doi.org/10.5772/intechopen.105353*

resources, being the Administration itself the one that requires them to obtain the "privilege" of exploiting a resource. The application of these sustainability standards to management has been very positive in the octopus experience, so they could be adapted to other well-controlled fisheries, such as stalked barnacles or the extraction of *Gelidium* sp.

#### **3.4 Prevent ecosystem damages due to restocking strategies and think about ecosystem restoration**

Mitigation and restoration strategies for the decline of exploited marine populations require genetic control and monitoring programs to confirm that hatchery individual truly represent the wild gene pool and for early detection of possible adverse effects on genetic diversity. Moreover, the mitigation of population decline with autochthonous individuals—as it is being done—is the only possible option, because the use of allochthonous individuals—even those coming from any other population of Atlantic origin—could negatively affect the genetic diversity of wild populations since the new variants could displace the autochthonous ones and affect the adaptability and fitness of local populations. It has become evident that it is extremely important to increase the number of broodstock used to obtain these juveniles. In addition, it is recommended to evaluate the implementation of a habitat restoration plan for sea urchins in Asturias, since this type of combined strategy has proven to be more effective in the recovery of populations.

#### **3.5 Generalize the use and application of genetic tools in the management strategies of the Asturias fisheries**

Genetics offers a diverse collection of versatile and useful tools to inform fisheries management on biologically based issues. However, the application of genetic tools is still incipient in many species of fishery interest. Genetic data need to be integrated into the management of fishery resources in Asturias, as they can address issues of direct relevance to the management of these resources; therefore, the implementation of routine genetic studies in management plans is recommended, always from a collaborative approach with managers and taking full advantage of new genetic technologies. The genomic era and the use of eDNA are still waiting to be effectively implemented in the management of marine invertebrates in Asturias.

#### **3.6 Educating new generations in "sustainability" will be a keystone in the Asturias 2030 fisheries development strategy**

There is an urgent need to bridge the gap between positive attitudes and real engagement of children and youth in ocean conservation, helping to foster real "citizenship of the sea." Gamification can be an efficient alternative learning method that establishes new knowledge, attitudes, and commitment of the new generations with sustainability in the exploitation of marine resources in Asturias.

#### **Acknowledgements**

This work is based on the PhD dissertation *"Genetic tools for the implementation of sustainable management plans in fisheries"* defended by M. Parrondo on December 3, 2021 at the University of Oviedo (Spain). This work was funded by the projects GRUPIN-IDI\_2021\_000040 and ECOSIFOOD (MCI-20-PID2019-108481RB-I00/ AEI/10.13039/501100011033). This is a contribution of the Marine Observatory of Asturias (OMA) and the Biotechnology Institute of Asturias (IUBA).

#### **Author details**

Marina Parrondo Lombardía1 , Lucía García-Florez2 , Eduardo Dopico Rodríguez3 and Yaisel Juan Borrell Pichs1 \*

1 Department of Functional Biology, Genetics, University of Oviedo, Oviedo, Spain

2 Center for Fishing Experimentation, General Directorate of Maritime Fisheries of the Principality of Asturias, Gijón, Spain

3 Department of Education Sciences, University of Oviedo, Oviedo, Spain

\*Address all correspondence to: borrellyaisel@uniovi.es

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Sustainable Management Plans in Fisheries and Genetic Tools: An Overview of the Challenge… DOI: http://dx.doi.org/10.5772/intechopen.105353*

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### **Chapter 6**

## Plasma and Hemocyanin Phenoloxidase Derived from the Hemolymph of Giant Freshwater Prawn *Macrobrachium rosenbergii* (De Man, 1879)

*Mullaivanam Ramasamy Sivakumar and Rangasamy Shanthi*

#### **Abstract**

We attempted to study the immune response in *M. rosenbergii* by melanization reaction produced by plasma phenoloxidase (PO) activity. The substrate affinity of the PO enzyme was determined using different phenolic substrates, and it was found that the diphenols were only oxidized. The enzyme was characterized as catechol oxidase type of PO and L-3,4 dihydroxyphenylalanine (L-DOPA) showed the highest substrate affinity to the enzyme. The biochemical parameters that determined optimum enzyme activity were found to be 2.5 mM L-DOPA at an absorbance of 470 nm, 10 mM Tris–HCl buffer at pH 7.5, temperature at 25°C, and 15 min incubation. Kinetic characteristics of plasma were studied from the *M. rosenbergii.* The hemocyanin was isolated by gel filtration chromatographic technique using Sephadex G-100. The *M. rosenbergii* hemocyanin (*Mr*HC) showed only one band with a molecular weight of 325 kDa on native polyacrylamide gel electrophoresis (PAGE) when stained with Coomassie Brilliant Blue (CBB) and bathocuproine sulfonic acid. The reduction of *Mr*HC protein in SDS-PAGE displayed three subunits with a molecular weight of 74, 76, and 78 kDa, respectively. Determination of optimal condition for PO activity of plasma has also been attempted. The plasma optimal condition taken for the *Mr*HC was tested for its ability to oxidize diphenols such as L-DOPA was shown only PO activity. These results showed that in the presence of PO and peroxidase inhibitors, phenylthiourea (PTU) and tropolone respectively have decreased plasma and *Mr*HC PO activity. This indicates that hemocyanin triggers innate immunity probably through one of its subunits that function as the active moiety.

**Keywords:** innate immunity, phenolic substrates, phenoloxidase, hemocyanin, kinetics, inhibitors

#### **1. Introduction**

Aquaculture is the fastest-growing, food-producing, profitable, and one of the major employment generating sectors in coastal areas and is expected to quintuple in the coming 50 years [1]. The giant freshwater prawn, *Macrobrachium rosenbergii* known as scampi, is an important aquaculture species in tropical and subtropical regions with the immense commercial export market value; however, the production of the prawns is curtailed by diseases caused by opportunistic pathogens in the rearing environment [2]. Resistance to diseases is based on the strategic improvement of the immune system of the animal and requires intensive research on immune components and its function [3]. As for any invertebrate, the innate immune mechanism in prawns includes cellular [4, 5] and humoral components [6–9].

Invertebrates lack adaptive immunity; therefore, they completely depend on innate immune systems for host defense. Melanization, which is a major innate defense system in invertebrates, is controlled by the enzyme phenoloxidase (PO) [10–12]. The active PO is a bifunctional enzyme that catalyzes the *o*-hydroxylation of monophenols to catechols and the oxidation of *o*-diphenols into *o*-quinones [13]. The first reaction involves monophenolase activity, which converts tyrosine to L-DOPA, which is then oxidized to quinone by diphenols activity of PO [13, 14]. The resulting quinones are converted to melanin by a series of intermediate steps involving enzymatic and nonenzymatic reactions [15, 16]. In one of these enzymatic reactions, dopachrome is decarboxylated by dopachrome isomerase (also called dopachrome tautomerase or Dopachrome Conversion Factor) to form dihydroxy indole, which is then converted to melanin [17–19].

The immune response in crustaceans mainly depends on nonspecific immunity, involving the cellular immunity of hemocytes [20] and humoral immunity through phenoloxidase [8] and agglutination [9]. In insects and crustaceans, phenoloxidase usually exists as a nonactive zymogen, prophenoloxidase (proPO), whose activation to the PO form is tightly regulated via an enzymatic cascade because the melanization reaction generates toxic compounds such as quinone species. This cascade is triggered by the presence of several microbial cell wall components such as β-1,3-glucan, lipopolysaccharides, and peptidoglycan [10]. There is a detectable or high amount of PO activity in crustacean plasma [8, 21, 22] that could be derived from proPO released from hemocytes [22] or from hemocyanin [8, 23, 24] for melanization activity, which remains unclear.

The present study attempted to characterize plasma PO activity in terms of substrate specificity, optimum ionic strength, pH, temperature, and incubation time to determine the biochemical and physiological conditions that support enzyme activity. Furthermore, to understand the substrate affinity of the plasma PO enzyme activity, the kinetics of the enzyme's rate of reaction was determined in the Lineweaver-Burk plot. There is evidence to show that the kinetics of the crustaceans phenoloxidases vary among the different components of the hemolymph as well as species [25–28]. Hence, an attempt has been made to optimize the conditions for determining PO activity of plasma including Km and Vmax value of freshwater prawn *M. rosenbergii*. Based on the determination of optimal condition, PO activity in *Mr*HC (325 kDa) has also been attempted.

*Plasma and Hemocyanin Phenoloxidase Derived from the Hemolymph of Giant Freshwater… DOI: http://dx.doi.org/10.5772/intechopen.104268*

#### **2. Materials and methods**

#### **2.1 Experimental freshwater prawns**

Adult intermoult of the giant freshwater prawns, *M. rosenbergii* weighing around 25–30 ± 1.58 g were purchased from Aqua Farm, Kalpakkam, Kanchipuram District, Tamil Nadu, India. The prawns were carefully transported to the laboratory maintained in 500 L FRP tanks (10 no of tanks, per tank 25 animals) containing at room temperature (28°C + 1°C) continuously aerated freshwater, which was changed thrice a week. The prawns were fed with egg white *ad libitum* and were acclimatized to the laboratory conditions for at least 4–5 days before use. Twenty-five percent of water was renewed daily to remove the unfed and fecal materials. The uninjured, intermoult animals were used throughout this study.

#### **2.2 Hemolymph collection and preparation of plasma**

Hemolymph (100 μl) was collected by cardiac puncture using a 23G needle attached to a clean, sterile plastic syringe containing 1.9 ml of ice cold iso-osmotic buffer, TBS-I (Tris 50 mM, NaCl 210 mM, KCl 5 mM, MgCl2 2.5 mM, pH 7.5) mixed and centrifuged in a pre-chilled polypropylene tube (161 x g, 8 min, 4°C) to obtain 1.5 ml of the supernatant as plasma. The exclusion of hemocytes was verified in the collected plasma by observation under phase-contrast microscope. About 50 prawns (each determination, N = 50) were required for collection of 100 μl acellular plasma, following Sivakumar et al. [8].

#### **2.3 Oxidation of phenolic substrates**

We tested the oxidative activity of 0.1 ml plasma was tested by incubating with 1.9 ml of different phenolic substrate solutions (5 mM tyrosine, tyramine, L-DOPA, DL-DOPA, dopamine, catechol, hydroquinone, and pyrogallol) for 20 min at 25°C. The color developed was measured spectrophotometrically (Shimadzu UV-160A spectrophotometer, Kyoto, Japan) at 300–700 nm against a reagent blank in which suitable substrates were substituted for plasma.

#### **2.4 Effect of different concentrations of L-DOPA**

To 0.1 ml of plasma was mixed 1.9 ml of L-DOPA at different concentrations (1–10 mM) and incubated for 20 min at 25°C. The color developed was measured spectrophotometrically at 470 nm against a reagent blank (L-DOPA).

#### **2.5 Effect of ionic strength on oxidation of L-DOPA**

The effect of buffer ionic strength on oxidation of L–DOPA by plasma was assessed by incubating 0.1 ml plasma with 1.9 ml of 2.5 mM L–DOPA prepared in different ionic strength (5–100 mM) at 25°C. After 20 min, the optical density of each of these reaction mixtures was determined spectrophotometrically at 470 nm against a reagent blank (L-DOPA).

#### **2.6 Effect of pH on oxidation of L-DOPA**

The ability of plasma to oxidize L-DOPA at different *p*H was tested by incubating 0.1 ml of plasma with 1.9 ml of a substrate of (2.5 mM L-DOPA) solutions prepared in 10 mM Tris–HCl buffer at different pH (6.0–9.0) for 20 min at 25°C. The color developed was measured spectrophotometrically at 470 nm against a reagent blank (L-DOPA).

#### **2.7 Oxidation of L-DOPA exposed to different temperature**

Effect of different temperature was tested by incubating 0.1 ml of plasma with 1.9 ml of substrate (2.5 mM L-DOPA) solutions prepared in 10 mM Tris–HCl (pH 7.5) buffer at a different temperature ranging from 10 to 90°C for 20 min. The color developed was measured spectrophotometrically at 470 nm against a reagent blank (L-DOPA).

#### **2.8 Effect of various time intervals on L-DOPA**

To 0.1 ml of plasma was mixed 1.9 ml of 2.5 mM L-DOPA (10 mM Tris–HCl; pH 7.5) and incubated for different time intervals (5–30 min) at 25°C. The color developed was measured spectrophotometrically at 470 nm against a reagent blank (L-DOPA).

#### **2.9 Kinetic parameters, km, and Vmax of plasma phenoloxidase enzyme**

To measure the kinetic parameters of plasma PO enzyme, different concentrations of L-DOPA (1.0–10.0 mM) were mixed with 0.1 ml of plasma and incubated for 15 min and absorbance was read at 470 nm. Michaelis–Menten constant was estimated by plotting substrate concentrations [S] and rate of PO activity [V]. Lineweaver-Burk plot was plotted as reciprocal of substrate concentration [1/S] and rate of PO activity [1/V]. The resultant plot is given a line that intercepted X-axis to give −1/Km value and intercepted the Y-axis to give 1/Vmax. The slope Km/Vmax was determined, and the resultant plot was rechecked using Eq. Y = mx + c.

#### **2.10 Partial purification of hemocyanin**

To 50 ml of plasma was centrifuged and dialyzed (MW exclusion limit <14,000 kDa and > 12,000 kDa) extensively against TBS-II (Tris 10 mM, NaCl 200 mM, CaCl2 10 mM; pH 7.5). Then the dialyzed plasma was ultracentrifugation at 200,000 xg for 180 min at 4°C (Beckman LE-80; Beckman Coulter, Brea, CA, USA). After ultracentrifugation, the supernatant was decanted and the pellet, which is made of hemocyanin, was collected and dissolved in TBS-II and used freshly for further purification.

#### **2.11 Purification of** *Mr***HC**

To purify the *Mr*HC*,* a Sephadex G-100 (Sigma-Aldrich; bead diameter: 40–120 μm) column (36 x 1.6 cm; XK 16, Pharmacia, Uppsala, Sweden) was prepared using gel filtration chromatography technique and thoroughly equilibrated with TBS-II. The hemocyanin collected by ultracentrifugation from the plasma was passed through the Sephadex G-100 column at a flow rate15 ml.h−1. The purified fractions

*Plasma and Hemocyanin Phenoloxidase Derived from the Hemolymph of Giant Freshwater… DOI: http://dx.doi.org/10.5772/intechopen.104268*

were continuously monitored for absorbance at 280 nm and 1 ml fractions were collected. The collected protein samples were stored at −80°C for further analyses.

#### **2.12 Determination of protein**

The protein content in the plasma and purified *Mr*HC (325 kDa) samples were determined according to Bradford [29] using bovine serum albumin as the standard. All chemicals used in the study were purchased from Sigma-Aldrich, St. Louis, MO, USA.

#### **2.13 Electrophoretic analysis**

The protein profiles of plasma and purified *Mr*Hc were analyzed in discontinuous polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions [30]. This was performed using 3% stacking gel (pH 6.7) and a 7% separating gel in Tris-glycine buffer (pH 8.9). Electrophoresis was performed at a constant current of 3 mA/sample at 10°C for 2 h on a slab gel measuring 8 x 8 cm. The gels were stained with Coomassie brilliant blue (CBB) R-250 (GE Health Care Biosciences, Tamil Nadu, India) or bathocuproine sulfonic acid following the methods of Maurer [30] and Bruyninckx et al. [31].

The molecular masses of the purified *Mr*Hc were estimated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with 5 and 10% polyacrylamide as the stacking and separating gel, respectively, following the method of Laemmli [32]. The purified *Mr*Hc subunits were visualized by staining the gel with CBB R-250. The molecular weight of the purified *Mr*Hc subunits was calculated using molecular marker proteins (GE Health Care Biosciences, Tamil Nadu, India).

#### **2.14 Oxidation of diphenolic substrates by purified** *Mr***HC**

We tested the oxidative activity of 40 μl purified *Mr*HC by incubating with 160 μl of different phenolic substrate solutions (2.5 mM L-DOPA, DL-DOPA, dopamine, and catechol) for 15 min at 25°C. The color developed was measured spectrophotometrically at 300–700 nm against a reagent blank in which Tris–HCl buffer (10 mM, pH 7.5) was substituted for purified *Mr*HC.

#### **2.15 Phenoloxidase activity**

The PO activity of plasma (0.1 ml) or purified *Mr*HC (40 μl) was incubating with 1.9 ml or 160 μl of phenolic substrate solution (2.5 mM L–DOPA) for 15 min at 25°C. After incubation, the color developed was measured spectrophotometrically (Shimadzu UV-160A spectrophotometer, Japan) or using ELISA plate reader (BioTek, PowerWave XS, USA) at an absorbance of 470 nm against a reagent blank of substrate solution (L–DOPA).

#### **2.16 Effect of inhibitors on PO activity**

In this experiment, 0.1 ml of plasma or 40 μl of purified *Mr*HC was mixed with an equal volume of inhibitors. 3 mM Phenylthiourea (PTU) or 16 mM tropolone containing 16 mM H2O2 was preincubated for 10 min at 25°C. An aliquot of 0.2 ml or 80 μl of these reaction mixtures from control or experiments was incubated with 1.8 ml or 120 μl of 2.5 mM L-DOPA for 15 min at 25°C. After incubation, the color

developed was measured spectrophotometrically or ELISA plate reader at an absorbance of 470 nm against a reagent blank of substrate solution.

#### **2.17 Statistical analysis**

The data were expressed as mean ± SD of triplicate experiments from five determinations. Statistical analyses were done using SPSS software (version 20; SPSS, New York, USA). The variation between experimental and control was evaluated by one-way analysis of variance (ANOVA) and significance was assessed at 0.01 probability (\*\**p* < 0.01).

#### **3. Results**

#### **3.1 Effect PO activity with various substrates**

The plasma separated from the hemolymph of the freshwater prawn *M. rosenbergii* was tested for PO activity with various phenolic substrates. Among the diphenolic substrates, the plasma showed the highest activity with L–DOPA (470 nm) when compared with DL-DOPA (440 nm), dopamine (440 nm), and catechol (470 nm) as shown in **Figure 1**. However, the monophenols including tyramine and L-tyrosine or polyphenols such as hydroquinone and pyrogallol failed to show any oxidation by plasma. Since the highest oxidative activity was obtained with L-DOPA, this substrate was used to detect PO activity in all subsequent experiments performed in this study.

#### **3.2 Effect of substrate concentration on PO activity**

The plasma PO activity was tested with different concentrations of L-DOPA (1.0–10.0 mM), and the PO activity was found to be higher with L-DOPA at a

#### **Figure 1.**

*PO activity of plasma with different phenolic substrates (5 mM) in Tris buffer (Tris–HCl 50 mM, pH 7.5) incubated at 25°C for 20 min and absorbance at 300–700 nm. The PO activity in optical density obtained at absorbancy maxima of respective substrates. Data represents mean of triplicate repeats of five determinations (mean ± SD) in the same way in all further experiments.*

*Plasma and Hemocyanin Phenoloxidase Derived from the Hemolymph of Giant Freshwater… DOI: http://dx.doi.org/10.5772/intechopen.104268*

concentration of 2.5 mM than that of 1 mM or higher concentrations (5.0, 7.5, and 10.0 mM) as shown in **Figure 2**. This experiment clearly suggested that the optimum concentration for testing PO activity in plasma was 2.5 mM of L-DOPA.

#### **3.3 Effect of ionic strength**

The PO activity of plasma was tested with Tris–HCl buffer (pH 7.5) of different ionic strengths (5–100 mM), and the highest PO activity was found with 10 mM Tris– HCl buffer when compared with other ionic strengths tested as shown in **Figure 3**. This result recommended that the optimum concentration for testing PO activity in plasma was 10 mM of Tris–HCl buffer.

**Figure 2.** *Effect of different concentrations of substrate (L-DOPA) by plasma phenoloxidase activity of freshwater prawn M. rosenbergii.*

#### **Figure 3.**

*Effect of ionic strength of Tris–HCl buffer on oxidation of L-DOPA (2.5 mM) by plasma phenoloxidase activity of freshwater prawn M. rosenbergii.*

#### **3.4 Effect of optimum pH**

The PO activity of plasma was assessed by oxidation of L-DOPA at various pH values ranging from 6.0 to 9.0, pH above 7.5 showed the brown color formation of dopachrome. The PO activity was decreased at pH 6.0–7.0 and 8.0–9.0; thus, pH 7.5 was taken as the optimum pH for the study of plasma PO activity (**Figure 4**).

#### **3.5 Effect of optimum temperature**

The PO activity of plasma was demonstrated by performing oxidation of 2.5 mM L-DOPA in the presence of 10 mM Tris–HCl at a pH 7.5. The reaction mixture was incubated for 20 min at different temperatures ranging from 10 to 90°C. The PO activity was stable and attained a peak at 25°C, which was taken as an optimum temperature for PO activity. At temperature below or above 25°C, a decline in PO activity was observed (**Figure 5**).

#### **3.6 Effect of time intervals**

The PO activity of plasma was evaluated by performing oxidation of 2.5 mM L-DOPA in the presence of 10 mM Tris–HCl at a pH 7.5 and temperature 25°C at various incubation periods ranging from 5 to 30 min. The maximum PO activity was at 15 min, which was determined as the optimum incubation time (**Figure 6**).

#### **3.7 Kinetic behavior**

The kinetic characteristics of plasma PO activity were determined from the rate of the reaction, which was calculated from the oxidation of L-DOPA at different concentrations (1.0–10.0 mM) in 15 min. The Michaelis–Menten constant Km was calculated to be 0.75, and maximum velocity (Vmax) was found to be 0.58 as shown in **Figure 7A**. Application of Km and Vmax yielded Lineweaver-Burk plot with a line slope of 1.2, which on extrapolation intercepted at −1.3 that was plotted as −1/Km and on Y-axis 1/Vmax was derived at 1.7 on X-axis (**Figure 7B**).

#### **Figure 4.**

*Effect of pH on oxidation of L-DOPA (2.5 mM), Tris–HCl buffer (10 mM) by plasma phenoloxidase activity of freshwater prawn M. rosenbergii.*

*Plasma and Hemocyanin Phenoloxidase Derived from the Hemolymph of Giant Freshwater… DOI: http://dx.doi.org/10.5772/intechopen.104268*

#### **Figure 5.**

*Effect of temperature on oxidation of L-DOPA (2.5 mM), Tris–HCl buffer (10 mM), pH 7.5 by plasma phenoloxidase activity of freshwater prawn M. rosenbergii.*

**Figure 6.** *Effect of incubation time on oxidation of L-DOPA (2.5 mM), Tris–HCl buffer (10 mM, pH 7.5) by plasma phenoloxidase activity of freshwater prawn M. rosenbergii.*

#### **3.8 Purification of hemocyanin from the plasma of** *M. rosenbergii*

The hemocyanin was loaded on the Sephadex G-100 column for gel filtration chromatographic separation, and the purified *Mr*HC peak fractions were collected at an absorbance of 280 nm (Fiure 8A). Hemocyanin protein was identified on PAGE–7% by CBB staining as distinct single bands of molecular weight 325 kDa (**Figure 8B**; lane 2). Staining with BCSA affirmed that the proteins contained copper and represented the copper containing proteins of hemocyanin (**Figure 8B**; lane 3). The chromatographic separation with electrophoretic observations of the separated proteins clearly indicated the occurrence of hemocyanin in *M. rosenbergii* as single separate copper-containing protein. As shown in **Figure 8C**, the purified *Mr*HC protein after reduction in SDS-PAGE (10%) cleaves into three subunits of 74, 76, and 78 kDa molecular mass, respectively (lane 2).

#### **Figure 7.**

*(A) Kinetic properties of PO activity in plasma of M. rosenbergii at different substrate concentrations of L-DOPA as shown in Michaelis–Menten curve. (B) The Km and Vmax values are calculated using Lineweaver-Burk plot of PO activity in plasma of M. rosnbergii with L-DOPA as substrate.*

#### **3.9 Phenoloxidase activity with diphenolic substrates in** *Mr***HC**

The purified *Mr*HC (325 kDa) was tested for PO activity with diphenolic substrates. Among the substrates, the purified *Mr*HC (325 kDa) showed activity only with L-DOPA while with the other diphenols, such as DL-DOPA, dopamine, and catechol, it failed to show any oxidation activity. Since the highest oxidative activity was detected with L-DOPA (**Figure 9**), this substrate was used for the determination of phenoloxidase activity and inhibition study.

#### **3.10 Effect of PO inhibitors on oxidation of L-DOPA by plasma and purified** *Mr***HC**

Pretreatment of plasma or purified *Mr*HC with PTU (3 mM) decreased the oxidation of 2.5 mM L-DOPA compared with control, and the reduction was found to be

*Plasma and Hemocyanin Phenoloxidase Derived from the Hemolymph of Giant Freshwater… DOI: http://dx.doi.org/10.5772/intechopen.104268*

#### **Figure 8.**

*(A) Gel filtration chromatographic profile of hemocyanin sample was applied on to the pre-equilibrated column of Sephadex G-100. The elution was performed at a flow rate 15 ml.h−1. The fractions were continuously monitored for absorbance at 280 nm. (B) Electrophoretic analysis (PAGE—7%) of purified MrHC stained with CBB and BCSA after gel filtration chromatography from freshwater prawn M. rosenbergii. Lane 1: Molecular weight markers; lane 2: Purified MrHC (325 kDa) CBB stained; lane 3: Purified MrHC (325 kDa) bathocuproine sulfonic acid stained under UV light for copper-protein. (C) Electrophoretic profile of purified MrHC (325 kDa) protein was run under reduced conditions of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE 10%) and stained with CBB. Lane 1 molecular weight protein markers; lane 2 purified MrHC (74, 76 and 78 kDa).*

#### **Figure 9.**

*PO activity of purified MrHC (325 kDa) with diphenolic substrates (2.5 mM) in Tris buffer (10 mM, pH 7.5) incubated at 25°C for 15 min and absorbance at 300–700 nm. The PO activity in optical density obtained at absorbancy maxima of respective substrates. Data represents mean ± SD of 5 determinations using purified MrHC (325 kDa) sample from different preparations.*

#### **Figure 10.**

*(A) Phenoloxidase activity in plasma and (B) purified MrHc (325 kDa) of M. rosenbergii and conformation of PO activity using inhibitors (PTU and tropolone). Asterisk indicates significant variation determined from the value obtained for a treatment to untreated control by one way ANOVA at \*\*p < 0.01. Data represents mean of triplicate repeats of five determinations (mean ± SD).*

*Plasma and Hemocyanin Phenoloxidase Derived from the Hemolymph of Giant Freshwater… DOI: http://dx.doi.org/10.5772/intechopen.104268*

about 91.55% and 55.80%, respectively. However, pretreatment of plasma or purified *Mr*HC with tropolone (16 mM), another strong inhibitor of PO activity, also showed a significant reduction in the oxidation of L-DOPA when compared with control, and the reduction in the phenoloxidase with plasma and *Mr*Hc (77.88% and 88.95%) was statistically significant (*p* < 0.01) as shown in **Figures 10A** and **B**.

In summary, for the plasma or purified *Mr*HC (325 kDa), the optimal conditions for measuring PO activity on L-DOPA (2.5 mM) were 10 mM Tris–HCl, pH 7.5 at 25°C for 15 min at 470 nm.

#### **4. Discussion**

The hemocyanin showed phenoloxidase (PO) activity in *M. rosenbergii* and appears to be enhanced with activators such as proteases, SDS, and microbes [8] and agglutination activity [9]. The PO enzymes and hemocyanin molecules belong to the same class of copper proteins, and this explains its PO function [33]. Considering the complexity of crustacean immune defenses, our study attempted to explain the variance in the immune function of PO activity in the plasma and hemocyanin of *M. rosenbergii*. In most crustacean species active PO is a bifunctional enzyme that catalyzes *o*-hydroxylation of monophenols to diphenols and then oxidizes *o*-diphenols into *o*-quinone [15, 26]. The substrate affinity of plasma PO activity was attempted with monophenols including tyrosine and tyramine, diphenols such as L-DOPA, DL-DOPA, dopamine, catechol, or polyphenols such as hydroquinone and pyrogallol. The results clearly suggested that the plasma showed the highest substrate affinity with diphenols, and among the diphenols, L-DOPA was found to show the highest PO activity suggesting catechol oxidase activity.

Biochemical studies were undertaken to describe the optimum condition of the plasma PO activity. The enzyme reaction was observed with different concentrations of L-DOPA. There was a steady increase in the enzyme activity from 1 mM to 2.5 mM concentration of L-DOPA after which an increase in substrate concentration did not enhance the enzyme activity proving substrate inhibition as the cause of the decline in enzyme activity. The previous reported substrate-specific phenoloxidase activity of hemocytes derived from *Penaeus monodon* and *M. rosenbergii* using 1.6 mg/ml of L-DOPA [4] and *M. rosenbergii* injected with Gram-positive *Lactococcus garvieae* and Gram-negative *Aeromonas veronii* was monitored for changes in phenoloxidase (PO) activity using 10 mM L–DOPA [34].

Since PO is an enzyme, its activity depends on the steady state of the active sites, which are necessary for substrate binding and subsequent activity. The optimum ionic interactions were studied by taking the plasma in different ionic strength of Tris–HCl buffer, and PO activity was determined. The optimum ionic strength of 10 mM Tris–HCl that showed highest PO activity was used as a buffer for the study. To continue on ionic interactions, the optimum pH of the buffer required for plasma PO activity was also determined. The optimum pH was observed at pH 7.5 (brown color formation of dopachrome), which was same as that of purified *Charybdis japonica* PO [27] against L-DOPA and *Penaeus chinensis* [35], but different from that of brown shrimp *Penaeus californiensis* that showed optimum pH at 8.0 [36], *Penaeus setiferus* at pH 7.5 [37]. The differences in optimum pH may be correlated with the species specificity.

Temperature is an important factor that can either enhance enzyme activity or decline it. As the enzyme is a protein catalyst, a steady state of an active site binding to substrate depends on the intactness of the active site, which can be disrupted by temperature. In the present study, the optimum temperature of plasma PO activity in *M. rosenbergii* was determined by incubating plasma at various temperatures ranging from 10 to 90°C. The optimum temperature was found to be 25°C. The hemocyte of *M. rosenbergii* showed optimum PO activity at 37°C [4]*.* The differences in temperature optima in plasma and hemocytes suggest a difference in PO characteristics.

However, in different crustaceans, several authors found maximum activities of PO activity in a temperature range of 40–45°C [27, 35, 37–41] while reported maxima at 30°C and 55°C for shrimp *P. paulensis*, lobster *Homarus americanus,* and tiger prawn *P. monodon*, respectively [42–44]. The difference in temperature optima among different species of crustacea can be attributed to species specificity and existing physiological conditions. Time is an important factor that can either enhance PO enzyme activity or decline it. In the present study, the oxidation of the substrate (L-DOPA) was tested at various time intervals from 5 to 30 minutes, and the activity was observed to be high at 15 min of incubation. The hemocyte of *M. rosenbergii* showed optimum PO activity at 1 minute [4] while 40 minutes was recorded in *C. japonica* [27]. The differences in time interval optima in plasma and hemocytes suggest a difference in PO characteristics.

The enzyme kinetics of the plasma PO activity was determined using Michaelis– Menten curve by plotting various concentrations of L-DOPA (1–10 mM), and the rate of reaction was determined in 15 min (1/V). The initial rate of reaction increased up to a maximum reaction velocity after which it stabilized and then declined. The Km value determined for substrate enzyme affinity was 0.75 mM, and this suggested a strong affinity between the enzyme and L-DOPA and the Vmax was calculated as 0.58. Lineweaver-Burk plot showed a slope of 1.2 with a correlation coefficient of R<sup>2</sup> = 0.996. This indicated that the enzyme had active sites to maintain a steady increase in the rate of reaction. The kinetic and biochemical characteristics of the plasma PO activity demonstrate a distinct PO activity among the crustaceans [27, 45].

Our study also included the determination of PO activity concerning substrate affinity and inhibition using the optimized conditions as determined in plasma for hemocyanin (325 kDa) separated from the hemolymph of *M. rosenbergii.* This study on substrate affinity of purified *Mr*HC was undertaken with diphenols such as L-DOPA, DL-DOPA, dopamine, and catechol and was specifically PO activity with L-DOPA only. It failed to show any binding affinity with any other diphenolic groups, and this indicated its distinct catechol oxidase nature [46].

Comparative inhibition studies with the PTU and tropolone were made to confirm the PO activity in the plasma and purified *Mr*HC. The typical *o*-diphenoloxidase inhibitor, phenylthiourea, inhibited the enzyme activity drastically in the plasma and the tropolone inhibited the phenoloxidase activity in purified *Mr*HC. The inhibition studies revealed that the plasma and the purified *Mr*HC showed phenoloxidase activity. These results are following phenoloxidase from *P. californiensis* [36], *P. chinensis* [35], *C. japonica* (Liu et al. 2006) [27], and *Limulus polyphemus* [47]. Phenylthiourea (PTU), known as a chelating reagent of copper [47, 48], effectively inhibited the activity of plasma phenoloxidase activity and also that of purified *Mr*HC, suggesting that phenoloxidase from *M. rosenbergii* prawn has copper in its active site. Furthermore, the observed oxidation of L-DOPA was not due to peroxidase since tropolone that inhibited PO activity in the plasma and purified *Mr*HC did not act as a substrate for peroxidase in the presence of H2O2 [49, 50].

*Plasma and Hemocyanin Phenoloxidase Derived from the Hemolymph of Giant Freshwater… DOI: http://dx.doi.org/10.5772/intechopen.104268*

#### **5. Conclusion**

In the present study, we conclude that the immunological function of phenoloxidase observed in plasma and *Mr*HC (326 kDa) of freshwater prawn *M. rosenbergii* appears to enhance resistance against various diseases, and investigation of PO activity in plasma and hemocyanin protein revealed catechol oxidase type. However, for the plasma and purified *Mr*HC (325 kDa), the optimal conditions for measuring PO activity on L-DOPA (2.5 mM) were 10 mM Tris–HCl, pH 7.5 at 25°C for 15 min at 470 nm. This clearly indicates the significance of humoral immune components in boosting immune response. This finding provides evidence that the plasma and *Mr*HC of *M. rosenbergii* are a potent immune system with an ability to enzymatically function as humoral PO.

#### **Funding**

Not applicable.

#### **Conflict of interest**

The authors declare no conflict of interest.

### **Declarations**

I confirm that the manuscript, or its contents in some other form, has not been published previously by any of the authors and/or is not under consideration for publication in another journal at the time of submission.

### **Abbreviations**


#### **Author details**

Mullaivanam Ramasamy Sivakumar1 and Rangasamy Shanthi2 \*

1 Laboratory of Pathobiology, Department of Zoology, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India

2 Laboratory of Nutrition and Crustacean Biology, Department of Zoology, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India

\*Address all correspondence to: crusshanthi@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Plasma and Hemocyanin Phenoloxidase Derived from the Hemolymph of Giant Freshwater… DOI: http://dx.doi.org/10.5772/intechopen.104268*

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### **Chapter 7**

## Isolated Crayfish Stretch Receptor Neuron Electrophysiology May Explain a Longstanding Mystery of Human Brain Functioning: Eureka Moment

*Toru Yazawa*

#### **Abstract**

Neural network of our brain is complex, but single-neuron physiology is still important to understand the higher brain function. While conducting electrophysiological experiments using the isolated crayfish stretch receptor neuron, a phenomenon which may explain a longstanding mystery of human brain functioning, Eureka moment, was found. In this article, we demonstrate electro-physiologically GABAergic inhibitory synapses contribute for "switching" and propose a novel idea that can explain how sudden switching occurs in the brain.

**Keywords:** crayfish, stretch receptor neuron, GABA, principal cell, inhibitory interneuron, shunting inhibition, higher level, science of the mental

#### **1. Introduction**

"It's no secret that the brain's complexity is vast, and although scientists have come closer to understanding how it functions, there is still a long way to go [1]." Historically speaking [2], observed detailed architecture of the brain cells with silver staining (Golgi's method). Eccles [3] and Kuffler [4] illuminated the working of the nervous system on a large scale. Buzsáki [5] explained why the rhythms of the brain are important, and Stuart and Spruston [6] emphasized the critical role of dendrites in information processing in the brain.

Buzsáki and da Silva [7] mention that "high frequency oscillations constitute a novel trend in neurophysiology." Thus, oscillations, including fluctuation, synchronization, and coupling, of assembled neurons are distinct physiological events occurring in the brain. However, there seems to be no satisfactory explanation for the science of the mind: "Eureka moment," and "Rubin's vase" or the "rabbit–duck illusion" which are bi-stable switching with ambiguous images or reversible figures. The switching is subjective, autonomic, or voluntary transformation occurring in adult brain.

We should go back again to the basics, observing a single-isolated neuron, asking whether "switching" occurs. Neuron is physical machinery composed of three parts: the dendrite, the cell body, and the axon. The commonality must be evolutionary consequence of convergent adaptation, from crustacean to human.

It is known that gamma-amino butyric acid (GABA) is the most important and dominant inhibitory neurotransmitter substance in the brain of human and crustacean too. However, its biochemical characteristics, inhibitory nature, and evidences for its synaptic release have been not established in human brain. But rather, crustacean neurons were used to show a significance of GABA molecule as the most important inhibitory neurotransmitter. Using crustacean nerve cells in 1950s, a German neurobiologist Florey, and Kuffler and his lab researchers; e.g., Otsuka and Kravicz [8, 9], contributed for identifying GABA as an inhibitory substance [10].

In mammalian brain, isolation of a single principal cell such as "pyramidal cell" is possible but not practical idea. In the brain slice preparation, it is inevitable that undetectable actions might affect neuron's response when artificially stimulating target neuron(s) in the slice. Dissociated and isolated culture neuron are not adequate specimen because it is not matured naturally.

We would like to use the crustacean stretch receptor neuron, which is the mechanosensory receptor neuron (RN) of the muscle receptor organ (MRO). The RN is one of the most thoroughly understood neurons studied by many researchers [11–31]. Using this material as a model of the principal neuron (such as the pyramidal cells in the cortex), we examine whether "switching" is observable at a single-cell level. We consider that sudden change of electric current flows in the neurons might be occurring at "Eureka moment."

It is known that, in mammalian brain, the principal neurons receive abundant inhibitory inputs from the interneurons such as Ramón y Cajal's "basket" neurons. This inhibition is mediated by chloride ions [4]. MRO neurons (i.e., RNs) also receive GABAergic inhibition which is also mediated by chloride ions [4].

In the present study, we focus on the inhibitory transmission around the principal neurons. More precisely, we examine the role of GABAergic inhibition in the microcircuit. We will discuss the functional significance of GABAergic neurons for explaining neurophysiological mechanisms of "Eureka moment."

#### **2. Single-neuron experiments**

#### **2.1 Cell body matters**

**Figure 1** shows five examples for the loci of soma within the architecture of neuron. A shows the soma of RN in the MRO. This neuron extends its axon to the brain (abdominal ganglia). In the ganglia, the axon connects to the inhibitory interneuron (black neuron in **Figure 1A**). RN receives recurrent inhibitory influence (pink arrow in **Figure 1A**). B shows the soma of the principal neuron of vertebrate. Pyramidal cell is known to send axon collateral to the inhibitory interneuron (i.e., Cajal's basket cell, black neuron in **Figure 1B**). C shows the spinal sensory cell. D and E show representative neurons of crustaceans, mollusks, and insects. There are distinct two types in the brain: one is A and B, the other is C, D, and E. The latter case (C, D, and E) is understandable, but the former case is not. The soma of A and B sit on the main route. It is seemingly an obstacle. It might be disturbing the information flow. The soma loci (A and B) might have undiscovered physico-physiological functions contributing for "neural computation" or "information-processing" in the highly cognitive brain.

*Isolated Crayfish Stretch Receptor Neuron Electrophysiology May Explain a Longstanding… DOI: http://dx.doi.org/10.5772/intechopen.109732*

#### **Figure 1.**

*Diagrammatic representation of neurons demonstrating the soma loci. A, crustacean MRO neuron. B, vertebrate principal neuron. C, vertebrate sensory neuron in the spinal cord. D and E, neurons of invertebrates such as crustaceans, mollusks, and insects. Arrow heads, afferent inputs. Arrows, direction of electrical signal flow. Pink arrows, recurrent inhibitory microcircuit.*

Intriguingly and surprisingly, the soma A and B are covered by highly packed synaptic knobs on surface (see Figures 1–4, page 7 of ref. [3]). Most of synapses on the soma are inhibitory [3]. What is the function of the soma? Is it not obstacle? What is the function of inhibitory synapses densely distributing on the somal and axonal membranes? We found answers in the present study. We found an important role of soma-axonal inhibitory synapses. It might be working everywhere across our brain.

#### **2.2 Cell isolation for electrophysiological experiments**

#### *2.2.1 Nerve dissection: Microscopic technique*

**Figure 2** shows dissection procedure of abdominal MRO in the present study. **Figure 2A** shows the locus of MRO, at the fourth segment (blue), **Figure 2B** shows MRO from rostral segments, and **Figure 2C** shows MRO from caudal segments. Note that caudal MROs are stick together. **Figure 2D–F** illustrates dissection procedure, separating two MROs off using a forceps and a fine needle. In **Figure 2E**, RNs may not seriously be injured at this state. But in **Figure 2F**, desheathing manipulation of the soma-axon portion of RNs (red) is conducted although it is venturous procedure. As a consequence, caudal MRO is suitable for obtaining a successful desheathed preparation.

**Figure 3** shows light-microscopic view of MRO in the present study. The photographs were taken by a handy camera through an eyepiece. Left picture in **Figure 3** corresponds to the diagrammatic representation shown in **Figure 2C**. Arrows point axons of two RNs. F denotes one of the clamp forceps clamping at the cut ends of the receptor muscles. Right picture in **Figure 3** corresponds to the diagrammatic representation shown in **Figure 2F**. Axon diameter is less than 10 μm.

#### *2.2.2 Electrophysiology: Methods*

The fast and slowly adapting stretch receptor organs with their receptor neurons, muscles, and efferent nerves attached were dissected out from the abdomen (**Figure 2**). Fundamental electrophysiological methods are previously mentioned [32]. Briefly, A

#### **Figure 2.**

*Dissection procedure of abdominal MRO used in the present study. A, crayfish abdomen. B and C, isolated MROs from the abdomen. D–F, further dissection procedures after isolation.*

#### **Figure 3.**

*Light-microscopic view of MRO. Left, an isolated MRO-RNs. Two axons of the slowly adapting and fast adapting RNs are pointed by arrows. Right, the slowly adapting neuron alone is isolated. One can see that an axon is exposed.*

perfusion chamber contains 0.6 ml of van Harreveld solution [33], flowing speed 0.1 ml/s, flowing direction from dendrite to axon (see **Figure 4** inset), at room temperature, 18–22°C. Glass microelectrode is filled with 4 M potassium acetate, for recording and passing current through the membrane. For the iontophoretic application of GABA, a double-barreled electrode (see **Figure 4** inset) system was applied, one barrel filled with 1 M GABA, pH adjusted to 4.0 with HCl, and the other filled with 1 M NaCl. GABA was ejected as cations when a positive pulse was applied. For the prevention of possible leakage of GABA, negative current of 5 nA was continuously applied to the GABA electrode. The ejection current was superimposed on this breaking current. Stimulation of nerve was done by a suction electrode. The membrane potential and the iontophoretic current for GABA application were displayed on an oscilloscope and photographed. Facilities at the laboratory of Kazuo Ikeda (deceased 2016), Neuroscience, City Of Hope, Duarte, CA, USA, were used in 1985–1986. Crayfish *Procambarus clarkii* specimens were captured locally at Ontario, CA, USA.

*Isolated Crayfish Stretch Receptor Neuron Electrophysiology May Explain a Longstanding… DOI: http://dx.doi.org/10.5772/intechopen.109732*

#### **Figure 4.**

*GABA responses recorded intracellularly in the present study. Inset shows diagrammatically our experimental methods in general for the present article. GABA was applied to a spot on the soma iontophoretically by a 30 nA, 50 ms pulse. Uppermost trace shows the GABA current for A, B, and C. In A, the tip of GABA, electrode rested just at the surface of soma. In B, the GABA electrode was pulled back by 10 μm, and in C, by 20 μm.*

#### *2.2.3 Intracellular recording: Technical difficulty*

We tried to reproduce recordings appeared in Kuffler's reports [15, 16, 21–23]. As shown in **Figure 5**, we successfully reproduced action potentials (APs) which were recorded from the soma membrane (**Figure 5B**). Inhibitory postsynaptic potentials (IPSPs) were also reproducible (**Figure 5B**). However, success rate is not high. Penetration of glass microelectrodes into the soma was an obstacle. For example, we obtained valuable data from only three neurons out of 37 neurons, during a two-week

#### **Figure 5.**

*We confirmed action potentials and inhibitory postsynaptic potentials in our preparation in the present study. Unpublished data.*


**Table 1.** *Low success rate: A two-week five-day experiment.*

#### *Isolated Crayfish Stretch Receptor Neuron Electrophysiology May Explain a Longstanding… DOI: http://dx.doi.org/10.5772/intechopen.109732*

five-day experiment, for example (see **Table 1**). **Table 1** summarizes what neuron we used and what end result we obtained, hit, or miss.

**Figure 5A** shows diagrammatical illustration of stimulation and recording methods in the present study. A glass microelectrode (Rec) is penetrated to the soma of neuron to observe membrane potential of slowly adapting neuron. S-Ax (arrow-1) denotes the point where antidromic stimulation of axon of slowly adapting receptor neuron (shown in blue-purple) was applied. Electrical pulse 0.05 ms duration was used at just above threshold intensity. S-Ax (arrow-2) denotes the point where stimulation of the inhibitory nerve (black) was applied. This inhibitory stimulation was done following the "axon reflex method" described by Kuffler and Eyzaguirre [23]. Using the just above threshold intensity is important, for preventing that excessive electric currents could stimulate other unrelated neuronal elements in the tissue.

**Figure 5B** shows antidromically initiated APs recorded at soma evoked by stimulation at S-Ax (arrow-1 in **Figure 5A**), and IPSPs evoked by stimulation at S-Ax (arrow-2 in **Figure 5A**). Time difference between arrow-1 and arrow-2 is fixed, approximately 20 ms, and several oscilloscope sweeps are superimposed.

**Figure 5C** shows superimposed weeps of APs and IPSPs. The time of antidromic stimulation was gradually shifted but IPSP stimulation time was fixed. IPSP blocks invasion of axonal spikes (failure of initiation of somal active spikes), but passive axonal spike is seen. B and C are different experiments with different cells thus, our experiments showing AP and IPSP are reproducible in the present study.

#### **3. GABAergic inhibition**

#### **3.1 GABA iontophoretic application**

Dendrites, soma, and axon areas receive a number of inhibitory synapse contacts, in both the MRO and the vertebrate principal neurons [3–6, 18–20]. This issue has not been well examined by physiological methods, because vertebrates, including human, neurons are impossible to isolate at a healthy-normal-operating state. Therefore, we considered that crustacean MRO neurons are beneficial. We examined synaptic function by iontophoretically applying small dose of GABA to localized area of the dendrites, soma, or axon (**Figures 4** and **6**). To do this experiment properly, it is imperative that the applied GABA is localized to a small spot so that there is no chance that other area will be stimulated by the application. Thus, the smallest detectable dose was determined, and the size of the affected area was estimated (**Figure 4**).

In **Figure 4**, we found that a single 30 nA current pulse of 50 ms duration produces a 1 mV response when the GABA electrode is located just on the surface of the somal membrane of the slowly adapting receptor. When the electrode was pulled back from the membrane, the latency of the response increased (see A and B in **Figure 4**) and the amplitude of the response diminished until it had disappeared completely with a 20 μm withdrawal from the membrane. This suggests that the GABA ejected by this current pulse is probably effective only in an area about 20 μm from the electrode.

When GABA current was increased, larger response with a similar time course was obtained (not shown). For the maximum GABA current employed in this experiment, the distance from the neuron surface to the tip of GABA electrode was 50 ± 10 μm to make the GABA response that disappears. Thus, even when the strongest ejection current (50 nA) was applied, synapses located more than 50 μm from the tip of the electrode were unaffected by the applied GABAs in this experiment.

**Figure 6.**

*Response to GABA applied locally on the various spots of the dendrite (right), and soma and axon (left). A 27 nA, 50 ms pulse was used for all GABA applications (uppermost trace). Application site was indicated in the diagram.*

It is known that somal and axonal areas receive synapse contacts [18–20]. In **Figure 6**, we conducted the experiment surveying the GABA-sensitive area on the somal and axonal surfaces of the slowly adapting receptor neuron (**Figure 6** left panel). Hyperpolarizing responses can be seen when a small dose of GABA was iontophoretically applied to the soma and axon. The numbers in a diagram denote the distance from axon hillock. The experiment was repeated with 14 different preparations. Somal and axonal responses were obtained in every case.

A similar experiment was also done with an undesheathed preparation (likewise **Figure 2E**). When GABA was applied from the outside of the axon sheath, the response was almost null, even with the highest dose (50 nA, 1 sec).

With the same technique, the responses to GABA applied to the dendritic area were investigated with undesheathed preparations in the slowly adapting receptor neuron (**Figure 6** right panel). The amplitudes of the responses in the dendritic area were variable, depending on the location of the GABA electrode. The variability may well be related to the structure of the dendrite, but the fine structure of the dendrite in this recording condition was not clearly observable under a low-magnification microscope.

The electric currents using GABA application might cause direct effects on neuronal membrane. Thus, the possibility that the observed response was caused by the ejecting current, but not by GABA, was tested using a double-barreled electrode (see inset **Figure 4**) in which one barrel was filled with 1 M GABA and the other with 1 M NaCl (**Figure 7**). In this experiment, GABA was first applied to the dendrite, and then, the same current was passed through the NaCl barrel. As can be seen in **Figure 7A** and **B**, the neuron responded only to GABA.

It is known that neurotransmitter substances affect both pre- and postsynaptic terminals [4, 25]. There is a possibility that the response could be mediated through a presynaptic terminal. If the iontophoretically applied GABA was affecting inhibitory *Isolated Crayfish Stretch Receptor Neuron Electrophysiology May Explain a Longstanding… DOI: http://dx.doi.org/10.5772/intechopen.109732*

#### **Figure 7.**

*A, response to GABA and B, that to NaCl applied to the same site in the dendritic area of the slowly adapting receptor neuron of the 3rd abdominal segment using double-barreled electrode (see inset in* **Figure 4***). The same 12.5 nA iontophoretic current of 50 ms duration was used. C, D, response to GABA in a saline containing 3 mM CoCl2.C, soma and D, axon (70 μm away from axon hillock).*

#### **Figure 8.**

*IPSPs recorded extracellularly from a site on the axon between 90 μm and 180 μm from the soma of the slowly adapting receptor neuron of the 3rd abdominal segment. Inset shows diagrammatically experimental method (see also* **Figure 5** *inset for the method of inhibitory stimulation). In both A and B, two CRO traces were superimposed. In the record with faster sweep speed (in B), an impulse of the inhibitory axon preceding the IPSP is seen (B is 2.5 times faster than A).*

terminals, which in turn released transmitter to the receptor neurons, an inhibitory potential would result. This possibility was tested by applying GABA in the presence of 3 mM CoCl2 (see a standard text book, such as ref. [4]). The receptor neuron is responded to iontophoretically applied GABA in a 3 mM CoCl2-containing solution in a similar manner to that in the standard van Harreveld solution (**Figure 7C** and **D**). It was, thus, confirmed that the applied GABA directly affects the postsynaptic membrane of the receptor neuron.

#### **3.2 IPSP at the axon**

The above results show that the GABA receptor exists not only on the dendrites, but also on the soma and the axon. Further evidence can be obtained by the focal recording of extracellular IPSPs upon stimulation of the inhibitory neuron. As shown in **Figure 8**, the local IPSP was recorded from the axon of slowly adapting receptor neuron at a portion of the axon 90–180 μm away from the soma. In the record (**Figure 8B**), the impulse of the inhibitory axon preceding the IPSP can be seen.

#### **4. Function of the somal and axonal inhibitions**

The stretch receptor neurons in crustaceans are one of the most extensively studied neurons [11–31]. The input signal, i.e., sensory-muscle extension, stimulates the dendrites and induces the generator potential at the dendritic membrane [4]. The depolarizing current conducts to the axon causing the initiation of the action potential [4]. The greater of the generator potential, the receptor neuron produces the higher frequency of discharge of action potential [4]. The inhibitory neuron can reduce the amount of the generator depolarization. This is the long-established understanding of neuronal function [4].

However, the above results in the present study revealed that crayfish stretch receptor neuron is innervated by the inhibitory neuron not only on its dendrite but also on its "soma" and part of its "axon." The presence of GABA-mediated inhibitory synapse is conclusively proven in the present study.

In our brain, the principal neuron (e.g., the pyramidal cell in the cerebral cortex) is innervated by the inhibitory neuron [3]. The most notable inhibitory interneurons that innervate the principal neuron are two cells: the basket cell and axo-axonic (or chandelier) cell [3–5]. The former innervates on its soma, and the latter innervates on its axon (i.e., initial segment or axon hillock). It is believed that the principal neuron plays important roles in advanced cognitive functions [6, 7].

Questions arise. What is the function of these inhibitory synapses distributing on the somal and axonal membranes? Do they work to attenuate the excitatory input? The functional role of the inhibitory synapses on the axon, axon hillock, or initial segment, should be reexamined.

#### **4.1 Novel inhibitory function**

#### *4.1.1 Blockade of antidromic invasion of impulses into the soma by GABA applied to the axon*

Axonal GABA receptors are demonstrated in **Figure 6** (see also [14]). However, the functional significance of axonal GABAergic synapse is not demonstrated physiologically [4, 5]. It is understandable that dendritic and somal inhibitory GABAergic synapses can inhibit neuronal activity. But, what is the functional significance of axonal GABAergic synapses? Why the axon receives GABAergic synapses?

We investigated the function of inhibitory synapses on the axon by observing nerve impulses with an intracellular electrode inserted into the soma (**Figure 9**). The cut end of the axon was stimulated while applying GABA iontophoretically onto a part of the axon 40 μm away from the soma. In the example shown in **Figure 9**, while the axon was being stimulated with a frequency of 2 Hz, GABA was being applied with a single ejecting current of 50 nA and 500 ms, a portion of the axon 40 μm away from the soma. Impulses designated A to F were recorded successively. GABA was applied for 50 msec beginning slightly before record B and ending prior to record C. In C and D, the somal regenerative impulses are blocked, leaving the axonal impulses, which electrically spread into the soma. The GABA effect lasted for about 1 sec. Thus, it is demonstrated that the invasion of the impulse into the soma was blocked, so that only the axonal impulse which is occurring prior to the point of inhibition by GABA is observed. We also observed that the blockage of the somal invasion required a large dose of GABA when applied to a single spot on the axon. In comparison to the dose of GABA which causes a detectable membrane hyperpolarization or depolarization (see **Figure 4**), the dose necessary for the blockage was more than 10 times higher. However, it can be assumed that this dose does not mean that the conduction block requires a higher dose of GABA to induce a larger conductance increase at a spot on the axon but rather indicates that a larger area of the axon must be inhibited to block

*Isolated Crayfish Stretch Receptor Neuron Electrophysiology May Explain a Longstanding… DOI: http://dx.doi.org/10.5772/intechopen.109732*

#### **Figure 9.**

*Blockade of antidromic invasion of impulses into the soma by iontophoretic application of GABA onto the axon. Inset shows diagrammatically present experimental method. Antidromic impulses were elicited by stimulating (0.05 ms duration square pulse, blue-color suction electrode) the cut end of the axon of the slowly adapting stretch receptor neuron of the 3rd abdominal segment: Every 500 ms. GABA was applied with a pulse of 50 nA and 500 ms at a portion of the axon 40 nm away from the soma at the time indicated in the figure. Impulses were recorded successively on moving film. A shows the control before application of GABA. The GABA pulse was applied just before the impulse in B, and ended before C. One can see that somal invasion was blocked in C and D and recovered in E and F. In E, the conductance increase caused by GABA is seen as a faster time course of the impulse.*

#### **Figure 10.**

*Spatial effect of inhibition on the blockage of antidromic impulses. Inset diagrammatically presents experimental method. A: 9 nA, 500 ms pulse at 0 μm, No blockage. B: 9 nA, 500 ms pulse at 60 μm, No blockage. C: 9 nA, 500 ms simultaneous pulses at 0, 60 μm. Three impulses failed to invade. D: 12 nA, 500 ms simultaneous pulses at 0, 60 μm, five impulses failed to invade.*

an impulse, i.e., the effectiveness is related to the spatial coverage rather than the concentration at a particular spot. Blockage of the conducting impulse may require a certain length of axon to be inactivated (i.e., shunt); otherwise, the impulse may jump the inactivated area. This possibility was tested below (see **Figure 10**).

In **Figure 10**, antidromic impulses were elicited with a frequency of 3 Hz by stimulating the axon of a slowly adapting receptor neuron of the 3rd abdominal segment. GABA was applied with two electrodes, one placed at the axon hillock (0 μm), the other 60 μm down the axon (see inset of **Figure 10**). Two glass capillary microelectrodes of the same resistance when filled with 1 M GABA were selected. Two electrodes in parallel were connected to a 1000 M ohm resister through which the ejecting current was applied.

**Figure 10** shows that single-GABA electrode did not block antidromic spikes (see **Figure 10A** and **B**). But, two GABA electrodes, 60 μm apart each other, blocked antidromic spikes (see **Figure 10C** and **D**). Experiments shown in **Figure 10** demonstrate that blockage of the conducting impulse requires a certain length of axon to be inactivated (i.e., shunt); otherwise, the impulse may jump the inactivated area.

#### *4.1.2 Effect of invading impulses on the stretch-induced impulse*

Experiments shown in **Figure 10** demonstrated that antidromic-impulse invasion can be blocked when axonal GABAergic synapses are activated. Action potentials of neuron arise generally at the axon initial segment of neuron [4, 6]. Then, the action potentials travel toward two directions: one for orthodromic direction and the other for antidromic direction [4, 6]. It is obvious that orthodromic impulses convey neural information to the next cells. But functional significance of antidromic impulses is still controversial (see ref. [6, 34]).

In order to test the possible interaction between invading antidromic impulses and stretch-induced impulses, antidromic stimulation was given while the slowly adapting receptor neuron was stretched and held at constant tension. In **Figure 11A**, the impulse above the horizontal bar is antidromic impulses invading the soma. The stretch-induced impulses are seen to be accompanied by the generator potentials. As can be seen here, the frequency of the stretch-induced impulses becomes reduced for a certain period after the antidromic stimulation. In **Figure 11B**, the somal invasion was blocked for the entire period. Thus, all of the impulses seen here are axonal impulses recorded at the soma electrically. As can be seen, the antidromic stimulation

#### **Figure 11.**

*Effect of antidromic stimulation on the stretch-induced impulses. A. with antidromic invasion. While a slowly adapting receptor neuron of the 3rd abdominal ganglion is firing with constant frequency with somal impulses unblocked, repetitive impulses caused by antidromic stimulation invaded the soma (12 impulses at 7 Hz above the bar). Note the frequency of stretch-induced impulses decreased after the antidromic stimulation, even though the tension was held constant. Time scale of 1 sec applies to A. B. without antidromic invasion. After blocking invasion, the effect was observed similarly. Note the frequency of stretch-induced impulses is unchanged before and after the antidromic stimulation (5 impulses above the bar). Time scale of 0.1 sec applies to B.*

*Isolated Crayfish Stretch Receptor Neuron Electrophysiology May Explain a Longstanding… DOI: http://dx.doi.org/10.5772/intechopen.109732*

failed to affect the frequency of the stretch-induced impulses. This set of experiments demonstrates that when the somal-dendritic area is invaded by regenerative impulses, the frequency of the stretch-induced impulses is reduced. However, if the invasion is blocked, the frequency remains unchanged.

#### *4.1.3 Effect of antidromic invasion of stretch-induced impulses on the stretch-induced frequency*

When orthodromic impulses are set up by stretch, the excitation occurring at the spike initiation site also sends impulses back to the soma [4, 15, 22, 23]. This antidromic invasion must have an effect on the stretch-induced frequency. In other words, unless antidromic invasion is blocked, the output frequency will be lower than the frequency dictated by the generator potential, which is directly coupled with tension.

In order to demonstrate the effect of these returning impulses (antidromic invasion) on their own generator potential, constant tension was given to a slowly adapting receptor to elicit orthodromic impulses of constant frequency, and then GABA was applied to the axon in order to block antidromic invasion.

As can be seen in **Figure 12**, while the receptor neuron was firing at a constant frequency, GABA was applied to a spot on the axon 180 μm away from the soma. Antidromic invasion was blocked during the effective period indicated by the horizontal bar (16 axonal spikes without active somal membrane activities). It is clearly demonstrated that the frequency of firing during this period increased and stayed at a higher constant level. The frequency is returned to the original level after the cessation of the GABA action (**Figure 12**).

This result indicates that when antidromic invasion is allowed, the output frequency is lowered from the level originally set by the fixed tension. Only when antidromic invasion is blocked, does the output frequency precisely reflect the level of tension.

A question arises. How much difference is there between the precise level and the lowered (distorted) level? The frequencies of "higher constant level" and "original

#### **Figure 12.**

*Effect of blocking antidromic invasion of the stretch-induced impulses on stretch-induced frequency. While a slowly adapting receptor neuron of the 3rd abdominal segment was firing at constant frequency with its somal impulses unblocked, GABA was applied with a 50 nA, 2.5 sec pulse (top trace). During the period of GABA action (above the bar, colored yellow), antidromic invasion was blocked. Note the increased frequency of orthodromic impulses even though the tension was held constant. After GABA action, the frequency returned to the original level. Inset (right), a photograph from different specimens with a slower sweep speed. The receptor muscle was kept at a constant tension. Note constant frequency of impulse firing. A brief (30 ms duration, strong current) GABA application to the axon induced the blockage of the antidromic impulses. During the blockage period, it is clearly seen that hyperpolarizing after potential, which is mediated by potassium ions, is disappeared due to disappearance of the somal spikes.*

constant level" are measurable from the oscilloscope-photographed data (9 cells). Seventeen measurements show following results, but it seems that data should not be statistically taken account because technical condition (dissection technique, electrode penetration technique, GABA electrode variation, etc.) varies.

Even though it is measurable in the present studies (all unpublished results). In **Figure 12**, high-level spiking discharge was at 2.47 Hz and low level at 1.64 Hz; thus, the invasion lowered the true information down to a 60 percent level, which is "false-distorted" information but meaningful information (see discussion). The most lowered case was 39 percent. The next significantly lowered case was 48 percent (3.33 Hz vs. 1.61 Hz). In **Figure 12** inset (right), the antidromic invasion lowers the information quality to the accuracy of 66 percent.

All other data obtained from the oscilloscope photographs are as follows: In the present studies, we obtained following calculated numbers: 60, 70, 71, 71, 59, 68, 57, 64, 57, 68, 61, 70, 77, and 78(in percent). In conclusion, the blockage can push the output frequency up to approximately 1.5 times.

#### **5. Discussion**

#### **5.1 Crustacean stretch receptor cell**

When the stretch receptor is uninhibited, antidromic impulse invades the soma. The present results demonstrate that the invasion is blocked when a part of axon is inhibited by the iontophoretic application of GABA. The effect of inhibitory postsynaptic potentials on the antidromically invading impulse was studied by Kuffler and Eyzaguire [23] by stimulating the inhibitory nerve while observing the response at the soma. They reported only a 2 percent decrease in the amplitude of the impulse and no blockage (see Figure 14 of ref. [23]). The inhibitory nerve synapses are not only on the soma and the axon, but also on the dendrites. Thus, stimulating the inhibitory nerve cannot differentiate the function of synapses localized on a particular part of the neuron. This is the reason why iontophoretic application of GABA was used in the present study in place of inhibitory nerve stimulation.

The failure to block the invasion of impulse to the soma in the experiment reported by Kuffler and Eyzaguirre [23] is understandable by looking at the record shown in that paper. The soma is depolarized by a large IPSP (see Figure 14 of ref. [23]), thus, providing a facilitative effect on the invading impulse. The effect of increased conductance must be overcome by this depolarizing potential change. When GABA was applied to a spot on the axon, the dose required for the blockage was as high as the equivalent of 2.5 × 10−8 coulombs in the example shown in **Figure 4**. When GABA was applied with two electrodes 60 μm apart along the axon, the necessary dose for blockage was lowered to the equivalent of 4.5 × 10−9 coulombs. In this case, the applied GABA had to diffuse to cover many synapses along the axon. It seems reasonable that the dose required for blockage by applying GABA at one spot was about five times higher than that required for the application with two electrodes separated from each other by a reasonable distance to effectively cover a certain length of axon. The length of axon necessary to effectively block the conducting impulse was, thus, estimated to be about 100 μm. In **Figure 4**, it was demonstrated that the dose of iontophoretically applied GABA on the axon necessary to cause a hyperpolarizing response of 1 mV was about 7.5 × 10−10 coulombs. If the transport number is assumed to be 0.5, this would correspond to 3.7 × 10−15 moles of GABA. In the neuromuscular junction of the same

*Isolated Crayfish Stretch Receptor Neuron Electrophysiology May Explain a Longstanding… DOI: http://dx.doi.org/10.5772/intechopen.109732*

species of crayfish, Takeuchi and Takeuchi [35] reported that iontophoretic application of 4.2 × 10−15 moles of GABA elicited a depolarizing response of 0.5 mV. Of course, the polarity and the amplitude of the GABA response is dependent on the membrane potential relative to the equilibrium potential of the carrier ion, and deviation of these potentials from preparation to preparation but suggest that the sensitivity of axonal GABA receptors is similar to that of the muscle.

From the present experiment, the estimation of the axon length to be kept under inhibition in order to effectively block the conducting impulses was about 100 μm. The present experiment (**Figure 6**) showed that a part of the axon about 200–300 μm from the soma was found covered with inhibitory synapses and sensitive to GABA. It indicates that the distribution of inhibitory synapses along this length of axon provides a safety factor of 2–3 times for the blockage of conducting impulses.

The requirement that a certain length of axon must be under inhibition in order to block conducting impulses may explain why the orthodromic impulse initiation site of the neuron is at a location on the axon about 300 μm away from the soma. When constant tension is applied to the dendrites of the slowly adapting neuron by stretching the receptor muscle, the receptor neuron fires repetitively with a constant frequency determined by a steady generator potential level which is linearly related to the tension. These orthodromic impulses are initiated at a spike initiation site on the axon about 300 μm away from the soma, which is outside of the axonal area covered by inhibitory synapses. In the stretch receptor neuron of the lobster, the impulse initiation site is reported to be 500 μm away from the soma [16]. The impulses not only propagate toward the CNS (orthodromic direction), but also propagate back to the soma (antidromic). Thus, the antidromic impulse invading the soma is recordable with an intracellular electrode inserted into the soma.

In the experiment shown in **Figure 12**, the receptor neuron was kept under constant tension to induce firing of constant frequency. When GABA was applied to the axon at a spot 180 μm away from the soma, the invasion of impulses to the soma was blocked, leaving axonal impulse only. During this blocking period, the frequency of stretch-induced impulses increased, although the stretch had been kept constant. This implies that the higher frequency under the blockage of antidromic invasion of impulses is the frequency directly related to the generator potential level set by the steady tension. The frequency without blockage of antidromic invasion, on the other hand, is a frequency affected by the antidromic impulses. In other words, when antidromic invasion occurs, the frequency of impulses becomes lower than that directly set by the tension.

When the neuron is firing as a result of a given tension, the persisting generator potential keeps the membrane at a depolarized level set by the tension. If an impulse invades the soma in this condition, the repolarizing phase of the impulse creates a hyperpolarizing after potential which is superimposed on the generator potential resulting in a lowering of the level of the generator potential. This will naturally reduce the frequency of firing. The effect of lowering the generator potential level by invading impulses must be dependent on the frequency, because the mechanism is apparently due to the summation of hyperpolarizing after potentials. Thus, the higher the level of the generator potential, the larger the hyperpolarizing after potential. Therefore, the effect of the invading impulse on the frequency is more stressed when the receptor is firing at a higher frequency. It is predicted that the frequency-tension relationship will be skewed from linearity at the high-frequency end.

Only if antidromic invasion of its own impulses is blocked then the frequencytension relationship can become linear. This must be the role of the inhibitory synapse distributing on the soma and on the axon, between the soma and the spike initiation site. Orthodromic impulses traveling toward the CNS are not blocked by GABA applied to the soma or axon. Thus, under this inhibition, the CNS receives information without being modulated by antidromic invasion—information of high fidelity.

#### **5.2 Vertebrate brain cell**

Inhibitory synapses on the soma and axon hillock have been found in many other neurons (e.g., pyramidal cell). Most of physiological paper has interpreted the function of these synapses as being to attenuate the excitatory input—classical postsynaptic inhibition—the dendrites were originally thought to act as simple receivers (p. 1718 in ref. [6]). The present study has demonstrated that the function of the inhibitory synapse on the soma and the initial segment of the axon is not the classical one, but rather is to protect the dendritic area where input is received from disturbance coming from the backfiring of the soma caused by antidromic invasion of its own impulses. This thereby provides high fidelity for the input-output relationship of the neuron.

#### *5.2.1 Axo-axonic cell or chandelier cell*

As shown in the review [36], GABAergic chandelier cells (axo-axonic cells first documented by Ramón y Cajal) have a unique arborization (see Figure 2 of ref. [36]). One axo-axonic cell innervates 26 pyramidal cell's axon initial segment. Schneider-Menzel et al. [37] reported that 153 pyramidal cells receive chandelier input, although the synapse number is highly variable across the population and is correlated with structure features of the target neuron. This indicates that an interneuron connects to multiple principal cells. Furthermore, pyramidal cells are known to be electrically coupled. Single neurons and populations work together [38]. It is understandable that biophysically homogenous population [39] of neurons, assembly of neurons, can synchronously function as the switching device. We assume that this switch works as powerful (i.e., working simultaneously across the large brain area) converter of specialized cognitive machinery. "Eureka moment" and "rabbit-duck illusion" might use this switch, because we sometimes behave momentary extremely concentrated mode to think about just one thing like thoughtful Archimedes did.

#### *5.2.2 GABA in the brain neural network*

A review [40] described: "neural networks in the brain include principal neurons and GABAergic interneurons (e.g., basket cell). The latter is vital for normal brain function because they regulate the activity of principal neurons. PV (parvalbumin) interneurons (i.e., basket cell) are involved in gamma-frequency oscillations, and they also play a role in complex network operations, including expansion of dynamic activity range, pattern separation, modulation of place and grid field shapes, phase perception and gain modulation of sensory response. PV interneurons also play key role in numerous brain diseases. These include epilepsy and also complex psychiatric diseases such as schizophrenia. Thus, PV interneurons may become important therapeutic targets in the future."

The review continues: "However, much needs to be learned about the basic function of these interneurons before clinical neuroscientists will have a chance to successfully use PV neurons for therapeutic purpose." The present finding on the crustacean

*Isolated Crayfish Stretch Receptor Neuron Electrophysiology May Explain a Longstanding… DOI: http://dx.doi.org/10.5772/intechopen.109732*

stretch receptor neurons provides a novel insight how PV interneurons shape complex network function.

Electrical stimulation to a special point of brain slice is usually not feasible because electric current cannot precisely stimulate the target, producing non-specific influences on many other unrelated neurons. Using GABA application to a point, Gómez et al. [41] studied somatodendritic inhibitory effects of GABAergic transmission. The GABAergic shunt mechanism supporting cognition could be proved in the brain one day. "The blockage of backfire at the axon initial segment" requires verification in the intact vertebrate brain.

#### *5.2.3 GABA and chloride ion*

Inhibitory postsynaptic potential (IPSP) at GABAergic synapse is mediated by chloride ionic currents. The reversal potential of IPSP (*E*i) is close to the resting membrane potential (*E*m). *E*m was −60 mV with a good condition in the present study. If *E*i is assumed to be −65 mV for example, IPSP must show a hyperpolarizing potential. As shown in **Figure 5C**, hyperpolarizing IPSP blocked antidromic invasion (cf. Figure 14 in ref. [23]). If *E*m is close to *E*i, GABA causes little change in membrane potential but a significant decrease in input resistance because chloride channels are open. This shunt inhibition is the mechanism of blockade of antidromic invasion. If *E*i is kept near *E*m, inhibitory nerve can induce only shunting effects without altering the membrane potential.

GABAergic signaling is controlled by the intracellular concentration of chloride ions. The chloride-extruding K+ -Cl− cotransporter (KCC2) and the Na+ -K+ -2Cl− cotransporter (NKCC1) which facilitates the accumulation of Cl− in neurons, maintain intracellular concentration of chloride ions. Thus, GABAergic signaling is controlled by both KCC2 and NKCC1. It is understandable that functional defect of them leads to the alteration in chloride homeostasis in CNS cells. GABA-chloride relationship is important for brain healthiness. Blockage of backfiring under the chloride ionic mechanism could be a therapeutic target in pathophysiology and pharmacology: the Renshaw cell in the spinal cord (this is not GABAergic but chloride dependent inhibitory), the basket cell, and chandelier cell in the brain might be candidate targets.

Abnormal inhibitory neurotransmission causes brain disorders [42, 43]. Gonzalez-Burgos et al. reported that cognitive deficits in schizophrenia may result from a GABA synapse dysfunction that disturbs neural synchrony [43]. The blockage of the antidromic invasion by GABA is not a very scientific fiction idea but physiological phenomena because it is measureable. Legitimacy of this phenomenon requires investigation in the intact brain.

Our brain can quickly shift between different conditional schemes, executing complex cognitive processes. Perhaps our brain has the switching tools to represent concepts, inner world of thought and desire, images and idea, self and consciousness, and so on. "Some interneurons might be to determine the timing of action-potential firing during rhythmic activity" (see the review by Spruston, in page 212 of ref. [44]).

#### *5.2.4 Insight accompanied by "Aha" experience or "Eureka moment"*

People solve problems with a unique process called insight, accompanied by "Aha" experience [45] and revealed a sudden burst of high-frequency gamma-band neural activity at a time of insight solution (see Figures 4–6 of ref. [45]. Both alpha-band and gamma-band power (scalp electro-encephalogram recording with power spectrum

analysis) quickly shifted from a low state to a high state or vice versa at an insight moment. It took approximately 500 ms for this switching. Thus, the switching speed is ca. 500 ms.

In a study where impulse firing rate was studied [46], the switching speed of 260–610 ms was calculated (Pair #1 in Figure 5 in ref. [46] was used for this calculation), although the authors focus was not the speed of switching. In a computer model of neuron and circuit [47], the switching speed of 1.4 s was calculated (Figure 7B in ref. [47] was used for this calculation).

In **Figure 12**, the firing frequency before GABA is 1.64 Hz (antidromic spikes are invading) and that during GABA is 2.47 Hz (during the blockage). It took 500 ms for the switching. If the neuron fires much high frequency, this switching speed would be shorter.

This consideration indicates that brain wave recording in human [45], firing rate recording in rat [46], and crayfish recording (the present study **Figure 12**) do not show inconsistency.

#### *5.2.5 Why principal neuron has a large oval soma?*

**Figure 13** diagrammatically illustrates principal neuron and inhibitory nerve. The dendrites integrate afferent inputs [6, 34]. Integrated currents initiate action potential at the axon. Action potential conducts both orthodromic and antidromic directions (green arrows). Electric currents necessary to initiate antidromic spikes are shown in red arrows. Inhibitory interneuron (black) makes synaptic contacts on the axon (Ax-Ax), on the soma (Soma), on proximal site of dendritic tree (p Dendrite), and on the distant site away from the soma (d Dendrite). Only one dendrite trunk is shown but it represents all dendrites, such as apical and basal dendrites of the pyramidal cell.

The diameter of the axon is 5 μm and the diameter of the oval soma is roughly 50 μm: 10 times difference in diameter between the axon and the soma. Safety factor for the action-potential generation at somal membrane significantly decreases due to less current per area. Consequently, active spike invasion tends to fail. On the other hand, "shunting switch" works efficiently at the soma area and at wider dendrite area too (see ref. [34] for dendritic integration). This consideration is the answer to the question "why principal neuron has a large oval soma?" Once again, axonal firing is relatively uneasy to stop it, because it jumps like the salutatory conduction due to a

*Isolated Crayfish Stretch Receptor Neuron Electrophysiology May Explain a Longstanding… DOI: http://dx.doi.org/10.5772/intechopen.109732*

high safety factor of the axon. On the other hand, somal firing is relatively easy to stop, because less electric current per area flows into.

Actually, sodium channels distribute not evenly (see ref. [34]). Dendrite soma can generate Ca++ spikes [34]. At the dendrite, EPSP, IPSP, and presynaptic IPSP occur [34]. The dendrite is like the battle field of various currents shooting. Thus, things are not simple, but "shunting switch" scenario is untested but potential hypothesis in vertebrate brain.

Individual inhibitory interneuron has own target [3, 34]. Their target to execute shunting is genetically and behaviorally determined during maturation. Each neuron "decides" where to settle, whom to connect, what to do, finally contributes forming LTP synapses, Hebbian synapse, and/or working memory synapses, all for the sake of neuroplasticity.

Each interneuron, selectively and independently, synapses on the soma, on the "p Dendrite" or "d Dendrite" and so on. Among them, axo-axonic synapse (Ax-Ax in **Figure 13**) seems to be most powerful. It can stop all antidromic invasions. The axo-axonic synapse alignment can afford precise output control (i.e., bi-stable, on/off or 0/1 digital control).

In conclusion, in the present study, we revealed powerful switching mechanism from crustacean experiments. The switching phenomenon might be possible to contribute to various mind-mental functioning such as "Eureka," "Rubin's vase," "rabbit– duck illusion," and more such as "selective attention" [48] or "cocktail-party effect." "The science of the brain" and "the science of the mental" [49] move closer to each other. The present study was partly appeared at the IUPS international congress [50].

#### *5.2.6 GABA in brain: Concluding remarks*

Crayfish are great commercial importance as a human food delicacy [51]. The US east coast (Maine) fisheries supply 80% of the world lobster [52]. Its abundance and common have probably contributed to neurobiology progress, as Bullock [53] noted that the crustacean animals were unquestionably a giant stride forward in the understanding of substratum of nervous system function. Historically, Alexandrovicz [11, 12] first introduced lobster neuron for neurobiology research. Kuffler [21], who worked at Harvard and at Woods Hall Marine Laboratory (both closer to the US east coast), used lobster stretch receptor to bring the brain science forward, and then neurobiology has indeed advanced [54].

A small molecule, GABA, was first identified in plant extracts [55]. GABA was then found in mammalian brain tissues [56]. The report [56] showed that GABA is formed naturally from glutamic acid in the brain. However, it takes many years before GABA is recognized as an important inhibitory neurotransmitter in brain [10, 57]. At first, unknown inhibitory substance "an inhibitory factor (factor I)," was described [58], and then isolated and identified as GABA [59]. Finally, GABA formation in nerve cells and GABA release from nerve cells were experimentally demonstrated using "crustacean" nerve cells [8, 9, 22, 23].

The present study might strengthen the brain's GABA history aforementioned, that is, there is a similarity of GABAergic nervous system between invertebrate (i.e., inhibitory neurons innervating stretch receptor neurons) and vertebrate (e.g., basket interneurons innervating pyramidal neurons). In the future, "GABAergic neural switching function" that we report here, could be confirmed by electrophysiology experiments, for example, the multi-electrode recording while studying intact brain [60]. We believe that the major brain function, e.g., "how to select and match

preexisting memory with events in the world [60]" could be executed by GABAergic "switching" mechanisms, which is revealed by the present study. This has worked out with heading along the way paved by Kuffler.

To conclude, a small molecule naturally occurred evolutionally very earlier (in the cells of microorganisms, plants and animals) as a non-protein amino acid [55, 61]. Meanwhile, the same molecule was utilized as a key signaling molecule in the brain of both lower and higher animals. It is beyond controversy that life on earth uses a lot of common basic molecules, like DNA, ATP, etc. to survive. In analogy with these molecules, evolutionally speaking, GABA is a fundamental substance too. Mostly, GABA works inhibitory, and thus, it helps very quietly healthy life everywhere in brain. If alterations in GABA function occur, sickness surely occurs, like epilepsy [57, 62]. This report reports the significance of such a humble substance working in our life. Healthy life is sweet indeed.

#### **Acknowledgements**

I am thankful to the Editor Dr. Noor Saher for her revisions and to Dr. G. Diarte-Plata for helpful comments. I am also thankful to T. Tsuruta, Nippon Unisoft Co., Ltd. Tokyo, for decades-long encouragements for my neurobiology study. This work is supported from the JSPS grant 21 K12688 to TY.

#### **Author details**

Toru Yazawa Department of Biological Science, Tokyo Metropolitan University, Tokyo, Japan

\*Address all correspondence to: yazawa-tohru@tmu.ac.jp

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Isolated Crayfish Stretch Receptor Neuron Electrophysiology May Explain a Longstanding… DOI: http://dx.doi.org/10.5772/intechopen.109732*

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### *Edited by Noor Us Saher*

Fish and fisheries play critical roles in ocean health, community well-being, food security, and economic stability. This book provides a comprehensive overview of fisheries. It includes seven chapters that address such topics as crustacean taxonomy, crustaceans and water quality of the coastal areas of Nigeria, characteristics of freshwater and brackish water fish in the Mekong Delta river system, feeding diversity of finfish, and much more. This volume provides future directions for improvement and advancement in fisheries research.

Published in London, UK © 2023 IntechOpen © Gokcemim / iStock

Pertinent and Traditional Approaches Towards Fishery

Pertinent and Traditional

Approaches Towards Fishery

*Edited by Noor Us Saher*