In what follows, I will thus sidestep questions about the feasibility of global citizenship in order to examine how the new technological revolution will lead to innovations in political life and will create Hannah Arendt's public spaces of 'virtual' citizenship. By so doing, I take it for granted that blockchain is a 'game-changer' and that it *could* have significant transformative effects on societies, politics and citizenship. I use the verb 'could' because I do not wish to embrace determinism or to imply the existence of a causal relation between technology and political processes. Blockchain has the potential to transform the way we think about public spaces, citizenship and political participation, but this potential can only be realised if technology is put to uses which can enhance democratic political processes.

My critics might object here that we do not need technological advancements in order to procure new conceptions of public space. Analyses informed by the thinking of philosophers, such as Henri Lefebvre, and geographers, such as Doreen Massey and Edward Soja, have highlighted that spaces are not given but are constructed in different ways by politics and discursive practices. Readers might recall Peter Maier's anthology on the changing boundaries of the political in the late 1980s.1 In it, Maier mapped the blurring of the distinction between the state and civil society, while a few years later, Gilles Deleuze commented on the shifting of borders and the proliferation of political spaces within contemporary societies of control.2

While all this is true, blockchain promises to realise those ideas in unprecedented ways. It also holds the promise of generating huge publics beyond (and across) geographical borders and territorially defined communities and thus of opening up new citizenship spaces. Rainer Bauböck and Peter Spiro have noted this in their contributions. Citizenship relies on the existence of public spaces of communication, of exchange of ideas, arguments and contested viewpoints and of joint decision-making. For a significant period of time, the agoras of the direct democratic experimentation in ancient Athens became remnants of a distant past that had no chance to be replicated in the present and future. Now, virtual agoras 'containing' millions of active and activist individuals can be built onto blockchain.3 The mythical space of a distant past becomes connected with, and re-enacted within, the contemporary world of an embodied digital network that makes citizenship a network good.4

1 Maier, C. C. (ed.) (1987), *The Changing Boundaries of the Political.* Cambridge: Cambridge University Press.

2 Deleuze, G. (1992), 'Postscript on the Societies of Control', MIT Press October 59: 3–7. Available at http://www.jstor.org/stable/778828.

3 Isin, E. & M. Saward (2013), *Enacting European Citizenship*. Cambridge: Cambridge University Press.

4 Kostakopoulou, D. (2008), *The Future Governance of Citizenship*. Cambridge: Cambridge University Press, 107–110.

This is essentially the realisation of Hannah Arendt's conception of 'virtual' public spaces. Virtual 'agoras' built on blockchain will become shared common worlds of continuous flows of speech and action, that is, spaces where people would recognise one another as equals or at least equally entitled to express their views, to 'deal only with one's peers' and to decide on common actions at national, international and global levels.5 As Arendt had eloquently noted, the (public) space of speech and action 'can find its proper location almost any time and anywhere'.6 By transcending topological as well as institutional accounts of the 'public space', blockchain technology not only lends credence to Arendt's conception of public space, but it also promises to open up decentralised public spaces in which all participants can be contributors, deciders and holders of institutional memories. The participants' geographical location does not matter. In an unprecedented bordertranscending move, new spaces of citizenship appear 'almost any time and anywhere' as Arendt had argued. What ties all the blockchain participants together in the virtual public space of citizenship is simply their ongoing concern and active engagement.7 These are, in reality, the characteristics that sustain all communities, be they virtual or not: members are visibly concerned about the common state of affairs and want their claims, needs, and aspirations to be heard.

This development can bring about a complete reconceptualisation of the nature of international society; non-statist ways of defining it will gain prominence. Hedley Bull's envisaged transformation of international society from a society of states to a society of peoples will be progressively realised.8 Cloud agoras will also prompt a rethinking of communitarian ways of defining communities and international society which see society and culture as interlocked. This is because they do not rely on some form of cultural homogeneity or conformity to a majority's ideas and narratives; the rely, instead, on the coming together of strangers9 in order to share their concerns and information, express their interests, make demands on the political system and to articulate proposals for common action. All this is bound to give rise to interesting questions about ways of constructing political order and legitimacy in international relations and politics.

5 Arendt, H. (1958), *The Human Condition*. Chicago: Chicago University Press.

6 Above n. 5, at 198.

7 Kostakopoulou, D. (1996), 'Towards a Theory of Constructive Citizenship in Europe', *Journal of Political Philosophy* 4 (4): 337–358.

8 Bull, H. (1977), *The Anarchical Society*. London: Macmillan.

9 Young, I. M. (1986), 'The Ideal of Community and the Politics of Difference', *Social Theory and Practice* 12 (1): 1–26, at 21–23.

While cloud agoras have the potential of dislocating citizenship from its statist reference point and stimulating citizen involvement by delivering the affirmative requirements for an active citizenry, namely, information sharing, the exchange of ideas and preferences, capability for action and the means of exerting influence and pressure, they will not be able to resolve the 'problem of equality of voice'. Claude Lefort, Nancy Fraser, Jürgen Habermas and others have commented on the inequalities that persist in democratic public spheres. Some voices will be louder and more influential than others and women will always struggle to find time to engage even virtually. Socio-economic disparities measured in terms of education, income and occupation will also allow certain participants to easily convert their possessed resources into political involvement. The cognitive and linguistic skills for political articulations and activity are not uniformly distributed. Nor do they exist independently of individuals' socio-economic setting and geographical location across the globe. Peter Spiro, Lea Yip and Stefania Milan correctly highlight this problem. Cloud agoras therefore will not be able to transcend the difficulties of ensuring full inclusion in the open public grid. They will certainly be more inclusionary that the existing publics, but they will still represent a stratified model of political community or public space(s). They will also have their own 'spinners', exploiters and manipulators of public opinion. I recall Jean Mansbridge's observations about the dark world of domination and manufactured invisibility of actors underpinning deliberative democracy.10

Although it is true that participatory parity cannot be easily achieved even in cloud agoras, it is equally true that the common world of citizenship beyond borders, states and nations could be more activist. And this is good news for democracy in general. It would be relatively easy for millions of blockchain members to mobilise on specific issues and to demand change in law and policy regionally, nationally and globally. It would also be more difficult for decision-making elites to ignore the voices of so many people and to pretend that they do not count or that their claims do not matter. Civic awakenings and political mobilisations in cloud agoras are also likely to exert influence on other public spaces that are more conventional and delineated across national and statist lines. For the boundaries of public spaces, virtual and non-virtual ones, will always be porous and issues will leak from one domain to another. The dawn of global citizenship will thus be a combination of the activation of an international or global society and of a more activist citizenship. Virtual global citizenship promises to be more virtual, in

10 Mansbridge, J. (1995), 'Does Participation Make Better Citizens?', *The Good Society* 5 (2): 1–7.

the republican sense; citizens will continually question aspects of public life, make public disclosures of wrongdoing, take an active part in public affairs and engage in regular, assertive action.

That this is good news for citizenship, democracy and politics in general cannot be denied. The virtual public space of blockchain communities will make citizens think, engage and act more virtually. In other words, the virtual reality of cloud agoras will have an impact on institutions and the participants themselves; it will yield pressures for more open, transparent and accountable institutions and will result in more virtuous, that is, actively engaged, citizens. Whether cloud agoras will prove to be decisive public spaces and strong promoters of democratic processes that make wealth, power and privilege accountable or merely subaltern counter publics will depend on the intentions and actions of their participants. In other words, the answer to the question whether the virtual public space of global citizenship will have a decisive influence on global, regional and national public policymaking is not theoretical or scholarly; it will be a contextual one.

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

## **Global Cryptodemocracy Is Possible and Desirable**

Ehud Shapiro

The fascinating discussion kicked-off by Liav Orgad addresses the interplay between the clouds and earth: How do cloud citizens and cloud communities relate to their earthly counterparts?

Arguments by Orgad, Primavera De Filippi, Francesca Strumia, Peter Spiro and Dora Kostakopoulou espouse the potential benefits of global citizenship, ordained by the clouds, and cloud communities that such global citizens can form, inhabit and govern. Counterarguments by Rainer Bauböck, Robert Post, Michael Blake, Costica Dumbrava, Yussef Al Tamimi, Jelena Dzankic, Lea Ypi and Dimitry Kochenov suggest that what happens in the cloud stays in the cloud, and may not be helpful or relevant to, or at least cannot substitute for, earthly dominions, due to fundamental differences between the two. I will try to counter these counterarguments.

A key introductory point made by Bauböck is that Orgad 'must have some form of global federal democracy in mind', yet that 'his main vision is, however, the emergence of alternative forms of political community at the sub-global level'. It is this main vision of Orgad that much of the weighty and thoughtful criticism is directed at.

To address it, I recall a strategy from mathematics: When faced with a difficult problem, namely a difficult theorem to prove, turn it into an even bigger problem: Define a more general and broader theorem, prove it, and then the original theorem easily follows as a corollary. This seeminglyparadoxical strategy works sometimes since a higher vantage point may offer a clearer view of the crux of the matter. I try to apply this strategy here: I will not address criticisms directed at sub-global political cloud communities directly. Instead, I will paint a vision of a global democracy, enabled by the internet and the emerging technologies of blockchain and cryptocurrencies, explain how subsidiary communities based on shared territory or common interests, as envisioned by Orgad, can emerge and operate within it, and respond to criticism from this broader and more encompassing perspective.

From the outset, key criticisms that apply to subsidiary cloud communities do not apply to a global democracy, whether on or off the cloud (we note in parenthesis the respective critics): It has a clear territory (Bauböck, Post, Blake, Al Tamimi) – Planet Earth; it has diverse membership (Bauböck, Blake) – humanity at large; membership is involuntary (Bauböck, Post, Al Tamimi, Ypi) and by decree – just as earthly states conscript citizens by decree; it has room for political communities 'that differ profoundly in their interests, identities and ideas about the common good' (Bauböck, Al Tamimi); and, due to all the above, it is clearly political (Bauböck, Post, Blake, Al Tamimi). Key remaining criticisms not answered by generalising the vision to incorporate all of humanity are those related to the use of coercion in community governance (Bauböck, Post, Blake, Dumbrava, Al Tamimi), lack of inclusivity (Ypi and Kochenov), and the risks of new technology (Dumbrava), which I will answer now in turn.

For our envisioned global democracy to be worthy of its name, it must uphold democratic values1 , including *sovereignty*, *equality*, *freedom of assembly*, *the subsidiarity principle*, *transparency*, and the conservation of the natural and imprescriptible human rights: *liberty, property, safety and resistance against oppression*. 2

A fundamental advantage of blockchain technology is that it is the only technology to date that can uphold *sovereignty*: The multitudes participating in the operation of the blockchain are its sovereign; no member, third party or outside entity has omnipotent 'super user' or 'administrator' capabilities over the system, and no-one can pull the plug on it: it will survive as long as there are interconnected participants who are able and willing to continue its operation.3 Hence, the answer to Stefania Milan's question, 'do we really need the blockchain to enable the emergence of cloud communities?', is: Yes, if we want cloud communities to be sovereign and not subservient.

The situation is not as rosy with *equality*. Governance trepidations of the 'cloud communities' of the leading cryptocurrencies, Bitcoin and Ethereum, which consist of their developers, miners and owners, resulted in community

1 Shapiro, E. (2017), 'Foundations of e-Democracy', *Computers and Society*. Available at https://arxiv.org/abs/1710.02873

2 Ibid.

3 I acknowledge Milan's point that such interconnectedness (but not the computers being connected!) would most-probably be commercially-owned, and that it is essential that such interconnectedness be neutral and unhindered, even if owned and controlled by private or government interests. Given that, global citizens can be the true sovereign of the global democratic blockchain outlined below.

breakups termed 'hard forks'. Hence, second- and third-generation cryptocurrencies attempt to address their self-governance from first principles.4 However, they offer only plutocratic solutions,5 espousing 'one coin – one vote' instead of the 'one person – one vote' principle necessary for *equality*. It may be ironic, given the thrust of our discussion, that the only approach available today to realise equality on the blockchain is to piggyback on identities issued by earthly governments. Besides defeating the purpose of freeing cloud communities from the grasp of their earthly counterparts, this approach cannot mix and match governments or identity-granting authorities, lest people with multiple government-issued identities have multiple votes in the cloud; and it excludes people, such as refugees, who may be hard pressed to present a government-issued identity.

Realising truthful, unique and persistent global digital identities for all, a precondition for making an egalitarian blockchain, is a major open challenge.6 But, for the sake of the vision we wish to paint, please suspend disbelief and assume that: (i) a worthy method for granting global digital identities to all has been devised, allowing any individual to claim a global identity (which functions as the 'attested individual identities' Kochenov aspires for); call the rightful owners of such global identities *global citizens*; (ii) unhindered internet access has been globally recognised as a basic civil right and is provided, directly or via a proxy, to any individual wishing to become a global citizen. While disbelief regarding the first assumption could be discharged in a decade, the second one will take longer. However, stating the goal of universal access as a basic civil right, taking concrete steps to implement it effectively, and making interim amends to compensate for its temporary lack, are all essential for our vision to be legitimate (and to address the justified criticisms of exclusion by Ypi and Kochenov). With this in mind, let us explore the vision of bringing about a global democracy of global citizens.

4 Bitshares (2018), *Technology*. Available at https://bitshares.org/technology/; Tezos (2018), *Governance*. Available at https://www.tezos.com/governance

5 Buterin, V. (2018), 'Governance, Part 2: Plutocracy Is Still Bad', *Vitalik Buterin's Website*, 28 March, available at https://vitalik.ca/general/2018/03/28/ plutocracy.html

6 Disclosure: My team at Weizmann aims to address this global challenge. Note that it will not be solved just by achieving broader coverage of local government-issued IDs ('Identification for Development', available at http://www. worldbank.org/en/programs/id4d).

As much as disbelief is suspended, a method for granting global digital identities will never be perfect. Hence, the global democracy will have to grapple with fraud (fake, duplicate and stolen identities, Sybil attacks), extortion (the \$5 wrench attack) and negligence (lost/forgotten password). Resolving such matters with due process would require a court. Such a court would need to rule according to a constitution. And the operation of the court (populated most likely by a combination of people and machines) will have to be financed. So we have hardly left the doorstep in our journey towards a global cloud democracy, and already discovered that in order to realise *equality* we need a global court, a global constitution, and a global currency.

That the global democracy needs a currency immediately suggests a cryptocurrency. But, how can we entrust the future of humanity to the hands of an environmentally-harmful7 , plutocratic regime? The answer is of fundamental importance: Current cryptocurrencies were architected on the premise that participants are anonymous and trustless, and resorted to the deliberately wasteful (Milan) proof-of-work protocol to cope with trustlessness. If indeed we have a mechanism for granting truthful and unique global digital identities that is reasonably resilient to attacks (e.g. at most one third of the global identities are compromised at any time)8 then the global democracy can deploy an egalitarian and planet-friendly cryptocurrency with a democratic governance regime; let's call such a cryptocurrency a *democratic cryptocurrency*.

Let's take stock: We have a democratic cryptocurrency governed by sovereign global citizens that are subject to a global court that rules according to a global constitution and is financed by the democratic cryptocurrency. This may sound a bit circular, but that's exactly how earthly states finance their operation. For example, the democratic cryptoeconomy can be fuelled by a universal basic income to all global citizens.9 Income, wealth and trans-

7 Present-day cryptocurrencies are unsustainable, even environmentally-harmful, since the proof-of-work protocols that underlie, for example, Bitcoin and Ethereum are unfathomably energy-wasteful on purpose: The ongoing operation of Bitcoin alone consumes as of today more energy than does the entire state of Israel, with its more than 8 million inhabitants (Bitcoin Energy Consumption Index, available at https://digiconomist.net/ bitcoin-energy-consumption)

8 Algorand (2018), *Algorand Website.* https://www.algorand.com/

9 Flynn, J. (2018), 'The Cryptoeconomics of Funding a Universal Basic Income', *Technophile Musings*, 21 March, available at https://jamespflynn. com/2018/03/21/the-cryptoeconomics-of-funding-a-universal-basic-income/

actions could be taxed, progressively if the global democracy decides so. Tax revenues would be disbursed to finance the operation of the global democracy, in particular the court and the underlying computational infrastructure ('mining'), as well as other purposes, according to a democraticallyformed budget.10 To prevent speculative manipulation of the exchange rate of the democratic cryptocurrency, a global central bank may be established, with authority to purchase and sell foreign (crypto)currency to hinder such manipulations; the bank can similarly set an interest rate. The constitution will have to be updated as the global democracy develops, and subsidiary legislation will have to be adopted. So, in just a few short paragraphs we have come to realise that the global citizens of a global cloud democracy that has its own cryptocurrency and cryptoeconomy will have to recreate almost all the functions of earthly states; let's call this resulting specific vision a *global cryptodemocracy*, to distinguish it from the more general and abstract idea of a global democracy. If successful, it would show that a technology built with an 'underlying philosophy of distributed consensus, open source, transparency and community' can be both 'highly disruptive' *and* 'serve similar purposes as those of states' (Milan); and it could achieve that without a reliance on the private sector and corporate capital that would necessitate paying undue attention to their interests and lobbying (Milan).

Additional key criticisms concern the ability of our global cryptodemocracy to protect human rights (Bauböck, Blake, Kochenov), collect taxes (Bauböck, Post) and in general enforce the rule of the law, given that physical coercion is possible on earth but not in the clouds (Bauböck, Post, Blake, Dumbrava, Al Tamimi). To redress crimes against global identities, we propose that global identities be realised as programmable software agents, aka 'smart contracts', programmed to obey certified court orders. Thus, coercion is achieved through design and programmability, without violence: If the court determines a global identity to be fake, then it can directly order it to terminate; if determined to be a duplicate, then it can be ordered to merge into another identity, and if stolen then to change its owner. Regarding Milan's observation that 'activism today is characterised by […] a tendency to privilege flexible, multiple identities', we cannot hold the stick at both ends: aspire for egalitarian rule of law in a global democracy, and undermine it with flexible (and hence unaccountable) and multiple (and hence unfairly privileged) identities.

10 Shapiro, E. & N. Talmon (2017), 'A Condorcet-Consistent Democratic Budgeting Algorithm', *Computer Science and Game Theory*, available at https://arxiv.org/abs/1709.05839

We propose to integrate the global citizen's global identity with her democratic cryptocurrency wallet into one entity, termed *global persona*. A global persona is the global citizen's proxy in the cloud: it is entrusted with the global citizen's identity information and crypto-assets, and it performs financial transactions and civic duties in the global cryptodemocracy on behalf of the global citizen it represents. Being unique and persistent makes a global persona accountable for the global citizen it represents. Hence, in addition to the court orders described above, a court may also issue fines against a global persona, payable immediately from her wallet, or deducted from her future (universal basic) income. As the global persona is programmed to obey court orders, no force is needed to collect such fines either. Income, wealth and transaction taxes can be similarly collected without the use of force, by programming global personas to obey the (democratically instituted) tax rules that are in effect. Of course, the court must be open to appeals on any decision and transaction.

A key remaining criticism relates to relying on and overseeing the technologies that will underlie our envisioned global cryptodemocracy (Post, Dumbrava). The criticism is valid, but is mostly equally valid of any technology on which humanity depends today, and there are many. Perhaps one key technological vulnerability is related to the democratic process itself, ensuring that elections and more generally voting on the blockchain at least stand up to earthly standards.11 Regarding overseeing blockchain technology, blockchain governance is indeed an issue of active research and experimentation, with the recognition that a change of underlying technology of a blockchain is as akin to, and as grave as, a change of constitution in a democracy. The global cryptodemocracy would employ the constitutional approach to its core technology, allowing constitutional change by its sovereign global citizens via a democratic process. Such a process must dampen the immediacy of internet communication, lest mob dynamics may rule, by employing hysteresis measures such as special majority requirements.12 Recovery mechanisms would also be established, and invoked, by democratic decision.

Let us now consider Orgad's vision of multiple cloud communities with a shared concern or ascriptive, thematic or geographic memberships 'whose

11 European External Action Service (2018), *Compendium of International Standards for Elections*. Available at https://eeas.europa.eu/sites/eeas/files/ compendium-en-n-pdf.pdf

12 Shapiro, E. (2018), 'Foundations of e-Democracy', *Computers and Society*. Available at https://arxiv.org/abs/1710.02873

aim is political decision-making and in which individuals take part in a process of governance and the creation of law.'

First, we note that all these communities can be subsidiary communities of the global cryptodemocracy, potentially with multiple levels of hierarchy (e.g. subsidiary animal rights or Bahá'í communities, with their own subsidiary communities based on country of residence); that the ability to form them is a manifestation of *freedom of assembly* in the clouds; and that allowing them to conduct their affairs without outside intervention is in line with the *subsidiarity principle*.

Second, such communities, within the context of a functioning global cryptodemocracy, may have at least one clear political goal: To draft and promote, within the parent global cryptodemocracy, policy and legislation that pertain to the rights and goals of their (possibly minority) community members. Recall the second article of the 1789 Declaration of the Rights of Man and Citizen: 'The goal of any political association is the conservation of the natural and imprescriptible rights of man. These rights are *liberty, property, safety and resistance against oppression*'. To uphold these, the conduct of all subsidiary cloud communities must be *transparent* in order to ensure that no subsidiary community aims to harm the liberty, property or safety of other communities or global citizens.

Third, within these rich and multi-faceted cloud communities, a virtual punishment with a global scope against one's global persona, e.g. temporary suspension or even just a public reprimand, applied to all subsidiary cloud communities, would be severe indeed. Hence, the higher the value of the subsidiary cloud communities to peoples' lives, the mightier the coercive power of the global cryptodemocracy.

While we have implicitly assumed an egalitarian, democratic decisionmaking process at the core of global cryptodemocracy and in its subsidiary communities that will choose to adopt it, we have not specified this process. Such a mechanism faces many challenges, including 'tyranny of structurelessness', 'tyranny of emotions', decision-making by 'microconsensus' within small cliques (Milan) and many others. The question of how to best reach a democratic decision has been investigated sporadically for centuries (e.g. by Llull, Condorcet, Borda), and intensively for the last 70 years within Social Choice theory. Much theory was developed, much confusion was sowed, and confidence in democracy has eroded, mainly due to Arrow's impossibility theorem and its follow-on work. I will just hint that adding a taste for reality to social choice theory can undo much of this damage and restore trust in democratic decision making, on and off the cloud.13

I have aimed to show that a vision of a global cryptodemocracy, with a rich set of subsidiary cloud communities, is realisable and have tried to address many of the criticisms raised in this debate. But, even if a global cryptodemocracy is realisable, and successfully addresses criticism, is it desirable? My personal answer is positive for two reasons: First, I believe that, since the days of Kant and even before,14 the proponents of a world government own the moral high ground, and the weakness of their position was practical: Until now, for a world government to materialise, local governments have to volunteer to cease some of their power; and giving up of power is not known to happen voluntarily. Fortunately, earthly democracies are sufficiently free so that the formation of a global cryptodemocracy does not require their consent. True, dictatorial regimes may prevent their citizens from participating, but this would, eventually, be at their own peril, as the interests of their people will not be represented as well. And true, the full power of a global cryptodemocracy will not be realised until proponents of global democracy become majorities in the majority of their respective earthly states. Yet, embryonic as it may be, the global cryptodemocracy vision presented here may very well be the only concrete proposal towards the ultimate realisation of a global democratic government based on currently available technologies.

And this relates to my second reason. I believe that for representative democracies to rebounce from their worldwide decline, they should undergo a major revision and adopt the practices of one of the oldest and most successful democracies in the world, namely the Swiss federal direct democracy. Given that those in power never give it up voluntarily, and that direct democracy disempowers representatives, such a major shift cannot happen without a major outside force in its favor. And new technology can offer such a force. In particular, political e-parties, formed as subsidiary cloud communities of the global cryptodemocracy, sharing the same technology and networking to share winning practices and methods, may be able to win earthly elections and change earthly democracies for the better. This in turn

13 Shapiro, E. & N. Talmon (2018), 'Incorporating Reality into Social Choice', *Computers and Society*, available at https://arxiv.org/abs/1710.10117

14 Global Challenges Foundation (2018), *Global governance models in history.* Stockholm: Global Challenges Foundation, available at https://api.globalchallenges.org/static/files/GG%20models%20in%20history%20EN.pdf

may result in such earthly democracies officially supporting15 the global cryptodemocracy in its rise into a *bona fide* egalitarian democratic world government of all global citizens.

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

15 For example, a state may create government-attested global personas for all its citizens, place them in the escrow of the state notary, and assign them to citizens upon their presentation of a government-issued ID. This would immediately turn all state citizens into global citizens. A state citizen who already owns a global persona will have to merge it with the received government-attested global persona, lest she would be guilty of owning duplicate global personas.

## **The Future of Citizenship: Global and Digital – A Rejoinder**

#### Liav Orgad

This has been an insightful discussion that touches upon some of the most fundamental concepts in political theory – communities, states, citizenship, and sovereignty. New technologies challenge the meaning and essence of these terms and blur the lines between physical and digital, local and global. The nature of the transformation is still a puzzle, but sooner rather than later the 'Fourth Industrial Revolution' will reach the institution of citizenship. The possible effects are promising but, as this GLOBALCIT debate shows, scary too.

My celebration of the potential of blockchain technologies to advance the idea of global citizenship lost in the GLOBALCIT digital agora, at least if we count 'votes.' There are four firm supporters (Primavera De Filippi, Francesca Strumia, Dora Kostakopoulou, Ehud Shapiro), five strong objectors (Robert Post, Michael Blake, Peter Spiro, Lea Ypi, Dimitry Kochenov), and five people who are somewhere in between, acknowledging the potential yet expressing concerns (Rainer Bauböck, Costica Dumbrava, Yussef Al Tamimi, Jelena Dzankic, Stefania Milan). The objections are wide – theoretical and practical, empirical and normative, methodological and conceptual. The idea of blockchain-based global citizenship, which can lead to the development of cloud communities that seek to take part in international decision making, is seen as 'techno-utopianism' (Milan), 'escapism' (Kochenov), and 'exclusion[ary]' (Ypi), a risk to 'territorial democracy' (Bauböck) that may bring a 'world without law' (Post) and 'legitimate coercion' that is so essential for the protection of human rights (Blake).

My kick-off had several premises. When discussing the need for an international legal persona for all human beings, I indicated three fundamental problems: human rights concerns (1.1 billion people do not have an official identification), lack of self-governance (individuals have no direct voice in

The research is supported by the European Research Council (ERC) Starting Grant (# 716350).

international law-making), and unequal representation (the principle of 'onestate, one-vote' leads to disparities in individual voting power). I identified three developments – the rise of global interconnectedness, identity, and responsibility – that, taken together, can end up with the creation of an international legal persona and digital identity (as a form of 'global citizenship'), thereby mitigating some of these problems. I also indicated one possible outcome of global citizenship – the emergence of (top-down and bottom-up) decentralised 'cloud communities' in which global citizens, sharing a common bond, can be politically organised and collaborate with the purpose of influencing international decision making and, eventually, becoming part of it. The authors in this debate have not addressed the premises, yet challenged my observations (e.g., global interconnectedness) and my conclusion – the potential of global digital citizenship to do more good than harm. I cannot do justice to all the subtle replies, so let me first express my gratefulness to the participants – this has been enriching experience, although it has not changed my optimistic view – and briefly address below some issues that I see as central.

#### **Cloud computing**

A large percentage of humanity is already engaged with some forms of cloud computing on a daily basis. Whenever you use Google Drive, Apple iCloud, and Dropbox, you spend time 'in the cloud.' Whenever you use audio and video streaming, online storage, and mobile services, you are 'in the cloud.' Government services, research data, medical records, and consumer services are available 'in the cloud.' Social networks too are 'in the cloud.' I have never physically met most of the authors who contributed to this debate, but I meet them on a daily basis on Facebook. The reason why we call these digital structures 'cloud' is not due to the lack of territory – the hardware is located somewhere – but because territory is largely *irrelevant* for the user and the service.

Cloud computing does not create, in and of itself, a 'community' (Post, Spiro), let alone a political community (Blake). Facebook is a social network, not a political community. It is commercial and dictatorial – members have no common bond and cannot create law or engage in governance – and it does not guarantee a truthful unique identity. Yet, in recent years there have been attempts to create cloud-based 'communities' by using blockchain and other technologies. This started as private initiatives, such as Bitnation, but spread into government initiatives, as illustrated by Estonia's e-residency. True, e-Estonia is far from creating a 'community'; Estonia's e-residents do not interact with one another or cooperate for political purposes. They are a group of clients more than a sovereign. It is also true that the notion of DBVNs (Decentralized Borderless Voluntary Nations), where anyone can build a 'community' in a Pangea jurisdiction – an IKEA-style do-ityourself nation – is unrealistic and undesirable. Still, the idea of a political community in which territory is largely irrelevant for certain political functions is worth considering. Thus far, it has been regarded as radical because it was promoted by anarchists and like-minded people looking for disruptive technologies to replace the nation-state. But as technology becomes more developed, it is just a matter of time until the idea will crystallise.

#### **Political community**

Even if the idea crystallises, can we really call cloud networks a 'political community,' or would they be like a 'community of video gamers,' to use Spiro's analogy, or just an addition to global civil society (Bauböck, Post, Milan)? The essence of the community I envision is indeed political, having members who share a common bond (say, the protection of animal rights) and seek to become part of national (and mainly international) decision making. There are similarities between cloud communities and global civil society (Bauböck, Post, Milan) as they are both voluntary, political in nature, civil (in the sense of non-governmental), and usually non-profit. But there are some differences. The global civil society is not composed of sovereign political entities where decision making is based on a 'one person, one vote' principle; global civil society organisations are acting *on behalf of* a group, while decentralised cloud communities can form themselves democratic collectives acting on a global scale.

Do cloud communities merit being called 'political communities'? It depends on the nature of such a community and how it will be developed. At least three components should come together: 1) members should have a self-perception as belonging to a collective entity, a shared consciousness of forming a political community; 2) members should have political relations and act with a collective responsibility; 3) members should be capable of acting collectively with regard to some functions. Take immigrants, for example. If all international migrants– more than 250 million people in 2017– joined a virtual community, it would be the world's fifth largest 'country' (after China, India, USA, and Indonesia). It could act as a self-governed collective at the international level, negotiating with states and UN agencies, collecting taxes, and promoting immigrant rights worldwide – all based not on representatives or NGOs, but on direct decision-making by its members.

#### **Digital coercion**

What about coercion – how can there be a political community without a recourse to force (Bauböck, Post, Blake, Dumbrava)? Normatively, the coercive force of law can be independent of the state or its territory; it requires authority. Such authority exists also in a blockchain-based community with one main difference – it is decentralised. If, for example, the 'migrant cloud community' decides collectively to stop migration to a certain country that does not respect migrants' rights or to buy products from certain retailers, and a migrant who is a member violates the rules, s/he can be sanctioned (through fines, suspension, limited access to rights/data, or termination of membership). As long as membership provides some benefits, particularly the ability to influence and shape decisions that affect the member's life, these sanctions are not minor or trivial.

*Technologically*, since membership is virtual, coercion is realised via software. As Shapiro notes, one's virtual identity (or 'global persona') is programmed to obey the community's decisions ('coercion is achieved through design and programmability, without violence'). In fact, state laws represent 'weak coercion'; there are papers that set rules (e.g., a prohibition of murder or crossing a red light) and one decides whether to follow the rules or violate them, in this case there are punishments and sanctions. Internet protocols are one step further. They are a form of 'strong coercion'; internet codes (e.g., restrictions and blockings) are stronger than papers because the law of the software is more difficult to violate – it is not in the discretion of an individual but requires knowledge and effort. A digital society represents a form of 'absolute coercion.' Transaction monitoring (e.g., voting, tax, or registry) is governed by blockchain rules that one cannot violate.

*Socially*, 'punishment' in a digital society is of a different type. A person cannot be sent to jail, but her reputation can be discredited. In the digital era, reputation capital is a valuable asset and a factor for providing services and products (think of Airbnb, Uber, eBay). In other words, online reputation has a real-world value. As Al Tamimi observes, 'A punishment in terms of such social devaluation imposed by the cloud state is conceivably more painful and restricting to the individual than traditional methods of punishment, such as fines or jail.'

#### **Functional sovereignty**

The territorial dimension of states has been seen central to citizenship (Bauböck, Post, Blake). Indeed, territory is considered the state's most characteristic feature; states are, by definition (Article 1, Montevideo Convention, 19331 ), territorial units. Territory is considered necessary for assuming most of the normative functions of the state – for instance, as a source of security and identity,

1 Available at http://avalon.law.yale.edu/20th\_century/intam03.asp

and for managing natural resources. Against this background, the concept of a deterritorialised state – or cloud communities that would replace the state and fulfil all of its functions – is politically inconceivable. But this does not entail that none of the state's essential functions can be reconceptualised. Cloud communities are not a state-replacement, but an improvement – they seek to add a circle to the already dynamic and multi-layered rich dimensions of citizenship. They are not supposed to act in the physical world – and thus have no sovereignty on issues like murder (Post) – but to govern the transaction of values or data that exist in the digital world (voting, registries, certificates, etc.). However, as cloud communities become politically more important, what happens there will not remain confined to the cloud but influence real-world political decision-making.

The idea of 'concentric circles' of citizenship – to use Cicero metaphor – with each circle having a different normative function, is not foreign to the theory of sovereignty. There are three options: cloud communities can be seen as sub-sovereign entities, semi/quasi-sovereign entities, or functional sovereign entities. Let me focus on the third option – functional sovereignty. Under this approach, sovereignty is divided by functions, with each being governed by a different entity. Think of federal systems, a condominium of states, mandate/trusteeship, autonomy (e.g., Quebec or Puerto Rico), or municipalities (where certain functions are governed by local sovereignty). Divisible sovereignty can be exercised over territories – e.g. Andorra, which was a condominium before independence in 1993 and still had two heads of state (the French president and a Catalan bishop) – or peoples. Sovereignty can be divided between political entities, as in federations or in the European Union, or between political and nonpolitical entities – think of religion (in Israeli law, for example, religious law is sovereign in family issues). The idea of functional sovereignty, as coined by Willem Riphagen in 1975,2 enables entity *A* to have sovereignty over social welfare, entity *B* to be the sovereign on financial issues, and entity *C* to enjoy sovereignty over security – all in the same territory. It also makes it possible for different political authorities to exercise functional sovereignty over different peoples in the same space. The switch is from a jurisdiction over territories to a jurisdiction over functions, peoples and services. As this is not a new concept, we can understand how it could be applied to blockchain-based cloud communities as well.

The normative functions of cloud communities remain an open question in the debate. My focus has been on global topics – global warming, the

2 Riphagen, W. 1975. 'Some Reflections on Functional Sovereignty', *Netherlands Yearbook of International Law* 6: 121–165.

environment, and other issues of global sustainability – but it is for the states to decide which functions to delegate to self-sovereign communities. Ultimately, states would set the boundaries and decide the sensitive areas in which sovereignty cannot/should not be divided or shared.

#### **Coda**

We can construct theoretical models of digital citizenship but, as this debate has shown, there are plenty of uncertainties – political, technological, and psychological ones – before it can become actually operative. I agree with Milan that 'much work is needed . . . before we can proclaim the blockchain revolution.' In particular, I share the concern about global inequality generated by ideas of cloud communities due to lack of internet access (Dzankic, Ypi, Kochenov) – this gap, however, has tremendously (and rapidly) narrowed and in 104 states more than 80 per cent of the youth population (aged 15–24) are now online. The situation will further improve if a right to internet access is universally recognised. And I cannot but share Bauböck's worries about the tyranny of the majority in the cloud – addressing it is a matter of constitutional design of voting mechanisms (note, however, that there will be judicial review, decisions that require supermajority, and perhaps even veto rights in the digital world as well). Discussing these (and others) concerns will keep theorists and policy makers busy in the years to come. While the focus of this debate is on global citizenship and virtual communities, I see it as a broader invitation to reflect on the nexus between new technologies and the future of citizenship.

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

## **Correction to: You Can't Lose What You Haven't Got: Citizenship Acquisition and Loss in Africa**

Bronwen Manby

#### **Correction to: Chapter 36 in: R. Bauböck (ed.),** *Debating Transformations of National Citizenship***, IMISCOE Research Series, https://doi.org/10.1007/978-3-319-92719-0\_36**

The footnote given by the author was inadvertently missed in the previously published original version. The footnote is added as "\*This text was written in November 2014 and reflects events current at that time. Some important later developments are not reflected, including, most importantly, the readmission of Morocco to the African Union in January 2017 and the decision of the African Court on Human and Peoples' Rights in the *Anudo* case against Tanzania issued in March 2018."

The author's request, during proofing, to add a footnote to the text, was unfortunately overlooked and is missing in the current text.

The updated online version of this chapter can be found at https://doi.org/10.1007/978-3-319-92719-0\_36


**2.** *In vitro* **multiplication of plants from genus** *Thymus***,** *Mentha***,** *Calendula* **and** *Catharanthus*

The multiplication by *in vitro* culture, means micropropagation, is a very important methodology to obtain a great number of homogeneous plants in a short period of time. *In vitro* culture is an important system in order to optimize and increase the secondary metabolites production. With this technique, *explants* from different species could be micropropagated under optimized condition of culture media, temperature and photoperiod. In this study, different species of plants with antifungal activity were micropropagated, and the fungicide activity of *in vitro* plants compared with the field plants.

In all the studies presented, the culture media were solidified with 0.7% of agar, and pH was adjusted to 5.6-5.8. Culture media were autoclaved at 121ºC for 15 min. The cultures were maintained in a growth chamber at 24 ± 1 ºC on a 16/8-h photoperiod (73 mol m-2 s-1).

Data were subjected to analysis of variance (ANOVA) using STATVIEW 5.0 program, treatment means separated using Fisher´s Least Significant Difference (LSD) test at P = 0.05.

#### **2.1 Micropropagation of** *Thymus*

Thyme is a perennial herb, a 20 to 50 cm shrub, of the Lamiaceae family, an aromatic plant native to the Mediterranean region (Miguel et al., 1999). The genus *Thymus* is exceptionally rich in species, and due to the diversity and plasticity of these plants, their geographical range is very wide. In Portugal are known, at least, 11 *Thymus* species (Afonso & McMurtrie, 1991). *Thymus* plants are of much interest owing to their use in different applications, in medicine because of their antiseptic properties, in the cosmetic industry or as a food additive for their organoleptic properties (Duke et al., 2002; Torras et al., 2007). *Thymus* species differ with regard to their morphological features and metabolism, which influences their chemical constitution. Within individual species there are chemical variations that are characterized by different plant oil compositions, usually without any morphological differences (Smolik et al., 2009). Increasingly, plant breeding has taken advantage of developments in molecular biology in order to genotype the species of interest in a way that considerably accelerates their selection. These types of approaches consist of choosing desired genotypes on the basis of molecular markers, or having prior knowledge of the genes that determine the formation of a particular trait in a plant (Pradeep-Reedy et al., 2002). There are many publications related to the antibacterial and antifungal activities of thyme essential oil (Urbanczyk et al., 2002; Priestley et al., 2003; Rasooli & Mirmostafa., 2003). It has been used as weed germination inhibitor (Angelini et al., 2003), and the different extracts from thyme leaves have shown the presence of a large number of flavonoids and vitamin E, compounds of great interest in the food industry due to their antioxidant activities (Sotomayor et al., 2004). There is also interest in using thyme essential oil for delaying the autoxidation of food lipids (Youdim et al., 2002).

#### **2.1.1 Methodology**

120 Fungicides for Plant and Animal Diseases

level facilitate strategies for the effective manipulation of secondary products in plants

To overcome the problem of antifungal resistance in human pathogens, plants with antimicrobial properties have been extensively studied for a possible application in food microbiology and as alternative treatments for diseases or to prevent bacterial and fungal growth. Many studies have proven very good fungicide effect of plants (Zabka et al., 2011). The details of plants screened, their families, vernacular names and their therapeutic uses

**name Therapeutic use**

(Pina-Vaz et al., 2004)

(Edris et al., 2003)

(Hänsel et al., 1992)

(Jaleel et al., 2009)

Antiseptic, antispasmodic, expectorant, vasoconstrictor

Skin problems, fevers, anti-inflammatory, anti-viral, anti-bacterial and fungicide

Anticancer, antidiabetic, laryngitis, rheumatism, dysmenorrhea

and *Thymus zygis* Lamiaceae Thyme Antiseptic, antispasmodic, antitussive

periwinkle

**2.** *In vitro* **multiplication of plants from genus** *Thymus***,** *Mentha***,** *Calendula*

The multiplication by *in vitro* culture, means micropropagation, is a very important methodology to obtain a great number of homogeneous plants in a short period of time. *In vitro* culture is an important system in order to optimize and increase the secondary metabolites production. With this technique, *explants* from different species could be micropropagated under optimized condition of culture media, temperature and photoperiod. In this study, different species of plants with antifungal activity were micropropagated, and

In all the studies presented, the culture media were solidified with 0.7% of agar, and pH was adjusted to 5.6-5.8. Culture media were autoclaved at 121ºC for 15 min. The cultures were maintained in a growth chamber at 24 ± 1 ºC on a 16/8-h photoperiod (73 mol m-2 s-1).

Data were subjected to analysis of variance (ANOVA) using STATVIEW 5.0 program, treatment means separated using Fisher´s Least Significant Difference (LSD) test at P = 0.05.

Thyme is a perennial herb, a 20 to 50 cm shrub, of the Lamiaceae family, an aromatic plant native to the Mediterranean region (Miguel et al., 1999). The genus *Thymus* is exceptionally rich in species, and due to the diversity and plasticity of these plants, their geographical range is very wide. In Portugal are known, at least, 11 *Thymus* species (Afonso & McMurtrie,

(Kumar & Gupta, 2007).

are given in Table 1.

*Thymus mastichina*

**and** *Catharanthus*

**2.1 Micropropagation of** *Thymus*

**Plant species Family Common**

*Mentha rotundifolia* Lamiaceae Applemint

*Calendula sp.* Asteraceae Marigold

*Catharanthus roseus* Apocynaceae Madagascar

Table 1. Ethnomedical information of the studied species.

the fungicide activity of *in vitro* plants compared with the field plants.

Branches of *Thymus* plants, species *Thymus mastichina* L. and *Thymus zygis* L., with 10 to 20 cm lenght were collected in University of Trás-os-Montes e Alto Douro (UTAD) Botanical Garden, in Vila Real, Portugal. The *explants* disinfected with commercial bleach 60% (v/v) and washed three times with sterile water, were fragmented into nodal segments and placed in different culture media. Basal culture media MS (Murashige & Skoog, 1962) were evaluated without growth regulators or supplemented with a cytokinin, 1 mg/L BAP (6-benzylaminopurine), alone or combined with an auxin, 0.2 mg/L NAA (α-naphthalene-acetic acid). Two carbon sources, sucrose and glucose, at three concentrations (2, 3 or 4 % w/v) were also tested. Number and length of shoots have been evaluated after four weeks in culture.

#### **2.1.2 Results and discussion**

The two *Thymus* species studied showed a good response to *in vitro* culture conditions (Fig. 1). After four weeks in culture, *T. mastichina* L. presented higher length of shoots (11.2 mm vs 6.0 mm), while *T. zygis* L. showed bigger shoot number per *explant* (1.9 vs 1.8). The culture media revealed a statistical significant effect (P<0.05) for the parameters evaluated, shoot number and length.

In what concern to carbon source, sucrose seems to be the best carbon source to thyme micropropagation. For both species, MS medium containing 3 % sucrose produced the highest shoots length (Fig. 2), similar results were obtained by Bandeira et al. (2007) in *T. vulgaris* L.. With 3 % sucrose concentration the highest shoots length (15.9 mm) was observed in *T. mastichina* L., in MS medium supplied with 1 mg/L BAP. One of the explanations for this is the presence of endogen auxin that releases the addition NAA. The biggest shoot number (9.7) appeared in *T. zygis* L. in MS medium with 2 % sucrose and 1 mg/L BAP. However, considering all the media, the ones with glucose revealed higher values in shoot number when compare with the sucrose ones, 3.6 *vs* 3.3 (though, the difference was not statistically significant P>0.05). Cunha and Fernandes-Ferreira (1999) and Harada e Murai (1996) obtain similar results with *Linum usitatissium* L. and *Prunus mume*, respectively.

*In Vitro* Multiplication of Aromatic and Medicinal Plants and Fungicide Activity 123

The genus *Mentha* belongs to the family Lamiaceae (Labiatae) consisting of about 25 to 30 species mainly found in temperate regions of Eurasia, Australia , South Africa and North America (Shinwari et al., 2011). Mints grow 10 - 120 centimetres tall and can spread over an indeterminate sized area. Mints are used either in the herb form or as an essential oil for flavouring, perfume production and medicinal purposes (Edris et al., 2003). The *Mentha* plant produces secondary metabolites such as alkaloids, flavanoids, phenols, gummy polysaccharides. Terpens and quinines are used in food and pharmaceutical, cosmetics and pesticide industries (Khanuja et al., 2000). Some members of this genus are also used as herbal teas and condiments both in fresh and dried form due to their distinct aroma. Morphological markers (such as plant height, leaf shape, colour, etc.) are among the oldest markers used in the evaluation of genetic variability, namely in *Mentha*. However, they are not sufficiently specific and informative because different gene expression in different environments causes wide variability of phenotypic characters in individuals. In some cases congruence between morphology and molecular phylogenetics were reported (Shinwari et al., 2011 as cited in Shinwari, 1995). Genetic diversity refers to the variation at the level of individual gene and provides a mechanism for the plants to adapt in ever changing

The existence of different chemotypes, based on qualitative differences within a taxon, is a common feature in most *Mentha* species and hybrids. As a result, the mint plants produce a number of commercially valuable essential oils, viz. spearmint oil, peppermint oil,

Nodal segments of *Mentha rotundifolia* plants were collected in the greenhouse of Botanical Garden of UTAD (Vila Real, Portugal). The *explants* were disinfected first with ethanol 70% (v/v) then with commercial bleach 40% (v/v) and washed three times with sterile water, then were fragmented into nodal segments and placed in different culture media. MS culture media with different concentrations of BAP (1.0 or 2.0 mg/L) and NAA (0.1 or 0.2

The number and length of shoots and roots, and the presence and diameter of *calli* were evaluated during the eight weeks of *in vitro* culture. After that acclimatization was done in a

The micropropagation of Menthe had, among others, the purpose to evaluate the effect of cytokinin and auxin concentrations in the plant development. After four weeks of *in vitro* culture there was the development of plants in all culture media (Fig. 3). The results obtained in MS + 1 mg/L BAP medium with different concentrations of auxin (0.1 or 0.2 mg/L) revealed that the lowest concentration of auxin induces higher values of shoots length (27.0 mm), and the highest concentration causes higher values of the roots length (16.9 mm)(Table 2). These results are due to the fact that a low ratio of auxin / cytokinin stimulates the development of stem sprouts, and a higher ratio to promote root development (Torres et al., 1998). Pascal et al. (1991) found that the addition of a synthetic

**2.2 Micropropagation of** *Mentha*

pennyroyal oil, etc. (Edris et al., 2003).

environment.

**2.2.1 Methodology**

mg/L) were used.

mixture of peat and perlite (1:1).

**2.2.2 Results and discussion**

Fig. 1. Micropropagation of *Thymus* plants. A – *Explant* in the establishment medium culture; B – *In vitro* plants of *T. zygis*; C – *In vitro* plants of *T. mastichina*.

Fig. 2. Effect of culture media (MS) in shoot number and length in *T. zygis* (A and B) and *T. mastichina* (C and D). GLU- glucose, SUC- sucrose, 20- 2%, 30- 3% 40- 4%. Error bars show the 95% confidence interval.

#### **2.2 Micropropagation of** *Mentha*

122 Fungicides for Plant and Animal Diseases

Fig. 1. Micropropagation of *Thymus* plants. A – *Explant* in the establishment medium

A B C

Fig. 2. Effect of culture media (MS) in shoot number and length in *T. zygis* (A and B) and *T. mastichina* (C and D). GLU- glucose, SUC- sucrose, 20- 2%, 30- 3% 40- 4%. Error bars show

the 95% confidence interval.

culture; B – *In vitro* plants of *T. zygis*; C – *In vitro* plants of *T. mastichina*.

The genus *Mentha* belongs to the family Lamiaceae (Labiatae) consisting of about 25 to 30 species mainly found in temperate regions of Eurasia, Australia , South Africa and North America (Shinwari et al., 2011). Mints grow 10 - 120 centimetres tall and can spread over an indeterminate sized area. Mints are used either in the herb form or as an essential oil for flavouring, perfume production and medicinal purposes (Edris et al., 2003). The *Mentha* plant produces secondary metabolites such as alkaloids, flavanoids, phenols, gummy polysaccharides. Terpens and quinines are used in food and pharmaceutical, cosmetics and pesticide industries (Khanuja et al., 2000). Some members of this genus are also used as herbal teas and condiments both in fresh and dried form due to their distinct aroma. Morphological markers (such as plant height, leaf shape, colour, etc.) are among the oldest markers used in the evaluation of genetic variability, namely in *Mentha*. However, they are not sufficiently specific and informative because different gene expression in different environments causes wide variability of phenotypic characters in individuals. In some cases congruence between morphology and molecular phylogenetics were reported (Shinwari et al., 2011 as cited in Shinwari, 1995). Genetic diversity refers to the variation at the level of individual gene and provides a mechanism for the plants to adapt in ever changing environment.

The existence of different chemotypes, based on qualitative differences within a taxon, is a common feature in most *Mentha* species and hybrids. As a result, the mint plants produce a number of commercially valuable essential oils, viz. spearmint oil, peppermint oil, pennyroyal oil, etc. (Edris et al., 2003).

#### **2.2.1 Methodology**

Nodal segments of *Mentha rotundifolia* plants were collected in the greenhouse of Botanical Garden of UTAD (Vila Real, Portugal). The *explants* were disinfected first with ethanol 70% (v/v) then with commercial bleach 40% (v/v) and washed three times with sterile water, then were fragmented into nodal segments and placed in different culture media. MS culture media with different concentrations of BAP (1.0 or 2.0 mg/L) and NAA (0.1 or 0.2 mg/L) were used.

The number and length of shoots and roots, and the presence and diameter of *calli* were evaluated during the eight weeks of *in vitro* culture. After that acclimatization was done in a mixture of peat and perlite (1:1).

#### **2.2.2 Results and discussion**

The micropropagation of Menthe had, among others, the purpose to evaluate the effect of cytokinin and auxin concentrations in the plant development. After four weeks of *in vitro* culture there was the development of plants in all culture media (Fig. 3). The results obtained in MS + 1 mg/L BAP medium with different concentrations of auxin (0.1 or 0.2 mg/L) revealed that the lowest concentration of auxin induces higher values of shoots length (27.0 mm), and the highest concentration causes higher values of the roots length (16.9 mm)(Table 2). These results are due to the fact that a low ratio of auxin / cytokinin stimulates the development of stem sprouts, and a higher ratio to promote root development (Torres et al., 1998). Pascal et al. (1991) found that the addition of a synthetic

*In Vitro* Multiplication of Aromatic and Medicinal Plants and Fungicide Activity 125

as ornamental and medicinal plants. These plants are known to contain saponins, triterpenic alcohols and their fatty acid esters, carotenoids, flavonoids, coumarins, essential oils, hydrocarbons and fatty acids (Hänsel et al., 1992). *Calendula* is known by its medicinal properties - mainly anti-inflammatory, anti-viral, anti-bacterian and fungicide. Considering secondary metabolites, *C. arvensis* is very similar to *C. officinalis* and therefore, the majority of the traditional or folk medicinal uses are similar for both species, including for the cure of skin problems, fevers, chronic infections, wounds, bites and stings (Grieve, 1984; Chevallier, 1996). *Calendula* sp. micropropagation was achieved for the first time using several types of *explants* (Çoçu et al., 2004). Callogenesis can be useful to obtain suspension cell cultures and regeneration of plants *via* indirect organogenesis. *Calli* induction can be obtained with the addition of growth regulators, namely auxins. However, some genotypes are able to induce callus formation even in its absence. In *C. officinalis*, 2,4-D and IAA associated with cytokinins promoted an efficient callus development (Grzelak & Janiszowska, 2002). As secondary metabolites are accumulated in cell cultures from numerous species, much work is focused on their production in shoot or root cultures formed by dedifferentiation of callus (Banthorpe, 1994). Metabolites extracts can be obtained from *in vitro* material, namely *calli* (Grzelak & Janiszowska, 2002), plantlets (Schmeda-Hirschmann, 2005) and flowers

Nodal segments obtained from plants growing in greenhouse conditions were washed in running water, surface-sterilized with a 25% (v/v) sodium hypochlorite solution for 15 min. and finally rinsed 3 times with sterile distilled water (10 min./wash). The *explants* were inoculated in MS media supplemented with BAP (1 or 2 mg/L) alone or combined with 0.1 mg/L of NAA. All the media were supplemented with 30 g/L sucrose. After 4 weeks, the results were analyzed by several parameters like the number and length of shoots and induction and development of *calli*. The *calli* obtained were transferred to MS culture

In general *calli* induction was detected in all the media. Callogenesis occurred simultaneously with organogenesis and, after 4 weeks of *in vitro* culture, flower induction was obtained (Fig. 4). It was not observed a significant effect of the culture medium on the number and length of shoots. The highest number of shoots was obtained in the medium with 2 mg/L BAP and the larger length was obtained in the medium with 1

Considering all the media tested, the average rate of multiplication of *C. arvensis* was 4.21 shoots/*explant*. ÇoÇu et al*.* (2004), using different culture media to micropropagate *C. officinalis* obtained similar values. The best results were obtained in the culture medium with 2.0 mg/L of BAP (7.8 shoots/*explant*) (Fig. 5). The presence of the auxin NAA (in combination with the cytokinin BAP), did not induce a positive effect in the number of shoots (Fig. 5). The media with 2.0 mg/L BAP and 0.1 mg/L NAA registered 4.75 shoots/*explant*. The medium with 1 mg/L BAP produced a higher number of shoots/*explant* in the presence of NAA, but not statistically different (P>0.05) of the

medium added with 3 mg/L 2,4-D, for organogenic development.

(Hamburger et al., 2003).

**2.3.2 Results and discussion**

mg/L BAP.

medium without it.

**2.3.1 Methodology**

auxin (IBA) induces an increase in mulberry shoots. Analyzing the effect of BAP concentration, culture media MS + 1 mg/L BAP + 0.2 mg/L NAA and MS + 2 mg/L BAP + 0.2 mg/L NAA, it is clear that the medium with the lowest BAP concentration led to higher values for all parameters, except for *calli* induction that are similar in both medium (0.7 mm diameter). Similar results were obtained by Erig et al. (2002) with black mulberry tree by checking that an increase in BAP concentration resulted in a decrease in length of shoots.

The results showed that the composition of the culture medium influences the response of Menthe to micropropagation. Overall, MS medium without growth regulators showed better results in terms of the number of shoots (2.0 / *explant*), although shoots length was higher in MS medium with 1 mg/L BAP and 0.1 mg/L NAA (27.0 mm). The plants were successfully acclimatized with a rate of 100%.

Fig. 3. Micropropagation of *M. rotundifolia*, plants after four weeks in different culture media. A – MS; B – MS + 1 mg/L BAP + 0.1 mg/L NAA; C – MS + 1 mg/L BAP + 0.2 mg/L NAA; D – MS + 2 mg/L BAP + 0.2 mg/L NAA.

Table 2. Number and length of shoots and roots and percentage and diameter of *calli* per explant, during eight weeks of *in vitro* culture.

#### **2.3 Micropropagation of** *Calendula*

The genus *Calendula* belongs to Asteraceae family, also known as Compositae*,* and includes several species, namely *Calendula arvensis* and *Calendula officinalis*, which are commonly used

as ornamental and medicinal plants. These plants are known to contain saponins, triterpenic alcohols and their fatty acid esters, carotenoids, flavonoids, coumarins, essential oils, hydrocarbons and fatty acids (Hänsel et al., 1992). *Calendula* is known by its medicinal properties - mainly anti-inflammatory, anti-viral, anti-bacterian and fungicide. Considering secondary metabolites, *C. arvensis* is very similar to *C. officinalis* and therefore, the majority of the traditional or folk medicinal uses are similar for both species, including for the cure of skin problems, fevers, chronic infections, wounds, bites and stings (Grieve, 1984; Chevallier, 1996). *Calendula* sp. micropropagation was achieved for the first time using several types of *explants* (Çoçu et al., 2004). Callogenesis can be useful to obtain suspension cell cultures and regeneration of plants *via* indirect organogenesis. *Calli* induction can be obtained with the addition of growth regulators, namely auxins. However, some genotypes are able to induce callus formation even in its absence. In *C. officinalis*, 2,4-D and IAA associated with cytokinins promoted an efficient callus development (Grzelak & Janiszowska, 2002). As secondary metabolites are accumulated in cell cultures from numerous species, much work is focused on their production in shoot or root cultures formed by dedifferentiation of callus (Banthorpe, 1994). Metabolites extracts can be obtained from *in vitro* material, namely *calli* (Grzelak & Janiszowska, 2002), plantlets (Schmeda-Hirschmann, 2005) and flowers (Hamburger et al., 2003).

#### **2.3.1 Methodology**

124 Fungicides for Plant and Animal Diseases

auxin (IBA) induces an increase in mulberry shoots. Analyzing the effect of BAP concentration, culture media MS + 1 mg/L BAP + 0.2 mg/L NAA and MS + 2 mg/L BAP + 0.2 mg/L NAA, it is clear that the medium with the lowest BAP concentration led to higher values for all parameters, except for *calli* induction that are similar in both medium (0.7 mm diameter). Similar results were obtained by Erig et al. (2002) with black mulberry tree by checking that an increase in BAP concentration resulted in a decrease in length of shoots.

The results showed that the composition of the culture medium influences the response of Menthe to micropropagation. Overall, MS medium without growth regulators showed better results in terms of the number of shoots (2.0 / *explant*), although shoots length was higher in MS medium with 1 mg/L BAP and 0.1 mg/L NAA (27.0 mm). The plants were

Fig. 3. Micropropagation of *M. rotundifolia*, plants after four weeks in different culture media. A – MS; B – MS + 1 mg/L BAP + 0.1 mg/L NAA; C – MS + 1 mg/L BAP + 0.2 mg/L

**A B CD**

**MS + 1BAP+0.2NAA**

**MS + 2BAP+0.2NAA**

**1BAP+0.1NAA**

Shoot number 2.0 1.9 1.8 1.5

Root number 3.2 2.6 2.5 0.4

(mm) 18.0 27.0 22.2 11.8

(mm) 18.9 14.5 16.9 4.3

*calli* (%) 0.0 15 15 6

(mm) 0.0 0.7 0.8 0.7

Table 2. Number and length of shoots and roots and percentage and diameter of *calli* per

The genus *Calendula* belongs to Asteraceae family, also known as Compositae*,* and includes several species, namely *Calendula arvensis* and *Calendula officinalis*, which are commonly used

successfully acclimatized with a rate of 100%.

NAA; D – MS + 2 mg/L BAP + 0.2 mg/L NAA.

**media MS MS +**

explant, during eight weeks of *in vitro* culture.

**2.3 Micropropagation of** *Calendula*

**Culture**

Shoot length

Root length

*Explants* with

*Calli* diameter

Nodal segments obtained from plants growing in greenhouse conditions were washed in running water, surface-sterilized with a 25% (v/v) sodium hypochlorite solution for 15 min. and finally rinsed 3 times with sterile distilled water (10 min./wash). The *explants* were inoculated in MS media supplemented with BAP (1 or 2 mg/L) alone or combined with 0.1 mg/L of NAA. All the media were supplemented with 30 g/L sucrose. After 4 weeks, the results were analyzed by several parameters like the number and length of shoots and induction and development of *calli*. The *calli* obtained were transferred to MS culture medium added with 3 mg/L 2,4-D, for organogenic development.

#### **2.3.2 Results and discussion**

In general *calli* induction was detected in all the media. Callogenesis occurred simultaneously with organogenesis and, after 4 weeks of *in vitro* culture, flower induction was obtained (Fig. 4). It was not observed a significant effect of the culture medium on the number and length of shoots. The highest number of shoots was obtained in the medium with 2 mg/L BAP and the larger length was obtained in the medium with 1 mg/L BAP.

Considering all the media tested, the average rate of multiplication of *C. arvensis* was 4.21 shoots/*explant*. ÇoÇu et al*.* (2004), using different culture media to micropropagate *C. officinalis* obtained similar values. The best results were obtained in the culture medium with 2.0 mg/L of BAP (7.8 shoots/*explant*) (Fig. 5). The presence of the auxin NAA (in combination with the cytokinin BAP), did not induce a positive effect in the number of shoots (Fig. 5). The media with 2.0 mg/L BAP and 0.1 mg/L NAA registered 4.75 shoots/*explant*. The medium with 1 mg/L BAP produced a higher number of shoots/*explant* in the presence of NAA, but not statistically different (P>0.05) of the medium without it.

*In Vitro* Multiplication of Aromatic and Medicinal Plants and Fungicide Activity 127

*roseus* (L.) G. Don is one of the most important medicinal plants due the accumulation in the leaves of two indolalcaloids vinblastine and vincristine, which were the first natural anticancer agents to be clinically used. The high pharmaceutical value of these secondary metabolites made of *C. roseus* one of the most studied medicinal species. A few protocols for micropropagation of *C. roseus* were reported in the last years (Junaid et al., 2007;

Nodal segments of *Catharanthus roseus* were collected from plants that were potted. *Explants* were disinfected with a 40% (v/v) sodium hypochlorite solution for 15 min. and finally rinsed 3 times with sterile distilled water (10 min./wash). After that, *explants* were inoculated in MS media alone or supplemented with BAP (1 or 1.5 mg/L) alone or combined with 0.2 mg/L of IBA (indole butyric acid) or NAA. All the media were supplemented with 30 g/L sucrose. To evaluate the effect of culture medium in the development of *explant*s was recorded, over eight weeks, the number and length of their

In this study a protocol was established for the micropropagation of *Catharantus roseus* (Fig. 6), MS culture medium was used because Pietrosiuk *et al* (2007) and Dhandapani *et al* (2008)

**B**

Fig. 6. Different phases of the micropropagation of *C*. *roseus*. A – *Explants* after two weeks of in vitro culture; B - Plantlet of *C*. *roseus*, at six weeks of *in vitro* culture; C – Plantlet of *C*.

**C**

shoots and roots, the number of internodes and the presence and diameter of *calli*.

**A**

Dhandapani et al., 2008; Ilah et al., 2009).

**2.4.1 Methodology**

**2.4.2 Results and discussion**

described good results with this media.

*roseus* with flower, at eight weeks of *in vitro* culture.

Fig. 4. *C*. *arvensis* plants after four weeks of *in vitro* culture showing flower development.

The weak response of NAA was also observed by ÇoÇu et al. (2004) with hypocotyl and cotyledon *explants* of C. *officinalis* concluding that the addition of KIN and NAA to culture media reduced the frequency of shoot organogenesis. Considering the length of the shoots, they reached an average of 23.44 mm at the 4th week of *in vitro* culture.

Culture medium

Fig. 5. Effect of different culture media on the number and length of shoots of *C. arvensis*.

#### **2.4 Micropropagation of** *Catharanthus*

*Catharanthus roseus*, an important medicinal plant of Apocynaceae family, is an herbaceous sub-shrub, also known as Madagascar periwinkle and *Vinca rosea* (synonym). Worldwide has been extensively studied by different groups and has been identified to be a source of numerous active principles of therapeutic importance. It is cultivated mainly for its alkaloids (Taylor et al., 1975), with anticancer activities (Jaleel et al., 2009). *Catharanthus*  *roseus* (L.) G. Don is one of the most important medicinal plants due the accumulation in the leaves of two indolalcaloids vinblastine and vincristine, which were the first natural anticancer agents to be clinically used. The high pharmaceutical value of these secondary metabolites made of *C. roseus* one of the most studied medicinal species. A few protocols for micropropagation of *C. roseus* were reported in the last years (Junaid et al., 2007; Dhandapani et al., 2008; Ilah et al., 2009).


**1.1 Malaria** *(Plasmodium spp)*

Different species of *Plasmodium spp*. can infect humans causing malaria disease; the most common, *Plasmodium falciparum*, is responsible for the majority of deaths. In contrast, *Plasmodium vivax* is responsible for the majority of cases. The symptoms of malaria range from asymptomatic parasitemia to severe disease, including cerebral malaria and death. Pregnant women and children under the age of five are particularly vulnerable to the disease. A combination of infected red blood cell sequestration in the microvasculature, endothelial activation, procoagulant action, and, most importantly, pro-inflammatory responses are thought to be the cause of the pathology. This disease is a huge public health burden, with an estimated 241 million cases reported in 2020 in 85 malaria-endemic countries (including the territory of French Guiana), resulting in 405,000 deaths [1–3].

Female Anopheles mosquitoes transmit the parasites, which have a complex life cycle that alternates between sexual and asexual phases. The infection begins with the bite of the mosquito, which injects parasites into the host in the form of sporozoites, which then travel to the liver. After replicating in liver cells, they mature into merozoites and are released into the bloodstream to invade host erythrocytes. Although the high parasite burden (up to 30,000 merozoites) stresses the host cell, infected hepatocytes do not undergo stress-mediated apoptosis, implying that the parasite interferes with this process in the host cell, In the erythrocyte, the parasites develop into immature gametocytes or ring-stage trophozoites, which are followed by mature trophozoites, schizonts, and merozoites. The immune system attacks these parasites; sporozoites in the liver find hepatic macrophages known as Kupffer cells, while parasites in the blood can find circulating monocytes and neutrophils [4].

#### **1.2 Chagas disease** *(Trypanosoma cruzi)*

*Trypanosoma cruzi is the causative agent of Chagas disease, also known as American trypanosomiasis. This is a public health problem in Latin America where it affects approximately 7 million people worldwide, and 100 million people are at risk of contracting it. Furthermore, it has become increasingly common in the United States of America, Canada, and many European and Western Pacific countries in recent decades* [5, 6]*.*

The life cycle of the protozoan parasite *Trypanosoma cruzi* involves both vertebrate and invertebrate hosts. Vectorial transmission to vertebrate hosts occurs via the bite of insect triatomine vectors (from the Reduviidae subfamily known as "kissing bugs"), which shed metacyclic trypomastigotes in their feces after feeding allowing the entry of trypomastigotes through skin wounds and mucosal membranes. Other infection routes are the oral ingestion of food contaminated with triatomine feces, such as fruit juices, blood transfusion or organ transplant, laboratory accidents, and congenital transmission from the mother to child during pregnancy. The last form became the most important nowadays and explains the presence of new cases in non-endemic countries as mentioned above.

Trypomastigotes can infect a wide range of nucleated cells, including macrophages, cardiac muscle cells, and nervous system glial cells, exploiting phagocytic or non-phagocytic mechanisms depending on the class of cell involved. After a brief residence in a parasitophorous vacuole, parasites go through the cytoplasm and differentiate into amastigotes. After several divisions (binary fission), amastigotes transform back into trypomastigotes, which are released from the host cell and can infect neighboring cells or reach the bloodstream and infect different organs, particularly the heart.

*Trigonoscuta cruzi* infection in humans is characterized by a brief acute phase with nonspecific symptoms and a long chronic phase in which most individuals do not exhibit pathology. In contrast, some infected people (around 10 to 30% of cases) *Close Encounters: Pathogenic Protists-Host Cell Interactions DOI: http://dx.doi.org/10.5772/intechopen.111398*

develop specific pathology cardiomyopathy and mega syndromes of the digestive system, which cause significant morbidity and may lead to mortality [5–7]. The development of Chagas pathology is complex and multifactorial, involving parasite immune evasion strategies, genetically programmed deficiencies in host immunological homeostasis, and autoreactive events marked by the presence of autoantibodies. *T. cruzi* genetic material has been identified in tissues destroyed during chronic infection, showing that the parasite plays an active role in pathogenesis [8, 9]. In fact, despite a vigorous immune response, the host fails to clear the parasites from the tissues, allowing the infection to remain indefinitely.

#### **1.3 Leishmaniasis** *(Leishmania spp)*

To cause leishmaniasis, *Leishmania* parasites infect and develop into phagocytic cells [10]. Clinical symptoms of the disease range from skin or mucocutaneous disorders to visceral infections, which are caused by different parasite strains and the delicate balance of parasite proliferation, the patient's immune response, and the consequent degenerative alterations. Consequently, *L. major*, *L. tropica*, and *L. mexicana* produce mainly the cutaneous forms, *L. braziliensis* causes the mucocutaneous illness, and *L. donovani* causes the most severe visceral disease (called kala-azar which means black fever in Hindi language). Infections caused by *Leishmania spp.* are a major public health concern across the world. This illness is seen in 88 different nations. More than 350 million individuals worldwide are at risk of leishmaniasis [11, 12], with 12 million already infected.

The life cycle of *Leishmania* is rather straightforward, with two basic stages: motile flagellate promastigotes residing in the stomach of the sandfly vector and immotile amastigotes within the phagolysosomal vesicles of vertebrate host macrophages.

A variety of sandfly species from two primary genera, *Phlebotomus* and *Lutzomyia*, transmit the illness to the host. Female infected sandflies spread the illness by injecting the promastigote form into the skin during a blood meal. After being inoculated into the upper dermis, metacyclic promastigotes are phagocytosed by skin-resident macrophages and dendritic cells and largely localized to phagolysosomes [10]. The internal development of Leishmania metacyclic promastigotes into amastigotes devoid of exterior flagella takes 12 to 24 hours. Amastigotes reproduce and survive intracellularly inside the phagolysosomal compartment, acting as a reservoir for transmission [13]. Moreover, polymorphonuclear neutrophils are attracted to the site of infection to clear promastigotes [14]. Explaining the significant inflammatory response produced after roughly 3 weeks [15]. As a sandfly feeds on the blood of an infected vertebrate host, it consumes amastigotes-containing monocytes and macrophages. Amastigotes are discharged into the sandfly's midgut, where they evolve into flagellated promastigotes through a process known as metacyclogenesis. Metacyclic promastigotes enter the throat and oral cavity, where they will be transmitted during the next blood meal.

#### **1.4 Toxoplasmosis** *(Toxoplasma gondii)*

*Toxoplasma gondii* is an obligate intracellular parasite of the order *Coccidia* with felines as the unique definitive hosts. It is a zoonotic illness that regularly affects a range of wild and domestic animals, with humans serving as unwitting hosts.

The protozoan parasite *T. gondii* infects 25 to 30% of the world's human population, with significant prevalences in South America and tropical African countries. As of 2020, the World Health Organization reported around 240 million illnesses and 600,000 deaths [16] (World malaria report 2021). Infected fetuses (congenital toxoplasmosis) and immunocompromised people are the most vulnerable to this illness. More than 80% of cases of primary acquired infection in immunocompetent people in Europe or North America are asymptomatic. In other instances, patients may develop fever or cervical lymphadenopathy, which may be accompanied by myalgia, asthenia, or other nonspecific clinical symptoms. Toxoplasmosis is extremely dangerous in immunocompromised patients, and toxoplasmic encephalitis, the most common manifestation of the disease in these patients can cause a variety of symptoms ranging from headache, lethargy, lack of coordination, or ataxia to hemiparesis, loss of memory, dementia, or focal major motor seizures, usually associated with fever. The lungs, eyes, and heart are also often damaged, leading to myocarditis, while *Toxoplasma* has been isolated from other organs such as the liver, pancreas, bone marrow, bladder, lymph nodes, kidney, spleen, and skin. Toxoplasmic retinochoroiditis is a less prevalent complication.

Congenital infection is typically the outcome of a primary infection acquired by the mother during pregnancy. The incidence of vertical transmission and the severity of fetal harm is determined by the stage of pregnancy at which the mother becomes infected. It is more dangerous when the infection develops in the early trimester of pregnancy, resulting in significant abnormalities or termination. The parasite's replication causes necrosis and severe inflammation, resulting in serious abnormalities in the brain and eye organs. Mental retardation, convulsions, microcephaly, hydrocephalus, hearing, and psychomotor impairment are all serious consequences. Microphthalmia, cataracts, increased intraocular pressure, strabismus, optic neuritis, and retinal necrosis can also be detected, as can uveitis and retinochoroiditis, which can lead to blindness. Retinochoroiditis is a typical characteristic that can be present regardless of the period of maternal infection [17].

Intermediate hosts become infected by the consumption of sporulated oocytes present in contaminated meat. In the intestinal epithelial cells, *T. gondii* develops in rapidly growing tachyzoites which travel throughout the body. In the infected cells, parasites proliferate in parasitophorous vacuoles. In response to immunological pressure, the parasites encyst as bradyzoites, a slow-growing form. Tissue cysts are most commonly found in long-lived cells like muscular, endothelium, or neural cells.

When members of the cat family consume bradyzoites, they undergo sexual development within intestinal epithelial cells, ending in the discharge of oocysts that undergo meiosis in the environment to generate eight haploid sporozoites. The consumption of oocysts by a wide range of hosts results in acute infection. Humans become infected by consuming oocysts that can contaminate food or drink, or by eating undercooked meat with tissue cysts [7].

To survive in the host cell, *T. gondii* typically resides in a vacuole, which inhibits lysosomal degradation and promotes parasite reproduction.

### **2. Phagocytosis**

The first person to describe the absorption of particles by cells was Élie Metchnikoff (1845–1916), who also highlighted the significance of this process for the host's reaction to damage and infection. Phagocytosis is a sophisticated mechanism for ingesting and eliminating infections that also plays a crucial role in the elimination of apoptotic cells, which is essential for maintaining tissue homeostasis. Target particle identification, signaling to start the internalization machinery, phagosome formation, and phagolysosome maturation are the four key stages of phagocytosis [18].

The key aspects of the early events of phagocytosis of protist parasites under study will be discussed in the following section.

#### **2.1 Recognition and phagocytosis of** *Plasmodium spp*

Microorganisms express molecules known as pathogen-associated molecular patterns (PAMPs), which are only expressed by pathogens and not by host cells. Glycosylphosphatidylinositol (GPI) anchors, nucleic acids, and Hemozoin are all Plasmodium PAMPs [2]. Pattern recognition receptors (PRRs) such as CD36, toll-like receptors (TLRs), and complement receptor 3 identify these PAMPs and trigger the parasite uptake.

Phagocytes, particularly monocytes, and macrophages, may also perform opsonic phagocytosis of *Plasmodium spp*. Certain opsonins, notably antibodies, have been found in functional investigations to increase successful phagocytosis. Protective immunity in malaria has been linked to the IgG1 and IgG3 subclasses. MSP (the merozoite surface proteins) 2 and 3, MSP-Duffy binding-like proteins 1 and 2, and glutamate-rich proteins have been discovered as targets of these opsonizing antibodies in merozoites [19].

Immune system cells have immunoglobulin (Ig) binding receptors, FcgR I receptors, FcgRII and FcgRIIII, and complement receptors CR1 and CR3. These factors, when combined, can aid in the phagocytic absorption of antigens opsonized with components such as IgG or C3b [1].

The complement receptor CR1 recognizes and phagocytoses ring-parasitized red blood cells opsonized by IgG and complement. Parasites cause changes in the membrane proteins of hosts' erythrocytes, exposing antigenic regions identified by autoantibodies. For example, band 3 protein is clustered and oxidized, and it is also underglycosylated [20]. Protein 1 (PfEMP1), which is expressed on the membrane of *Plasmodium falciparum*-infected erythrocytes, is also a significant target of opsonizing antibodies, with antibodies recognizing distinct domains of this protein [20].

When activated, neutrophils can produce reactive oxygen species (ROS), which are highly poisonous chemicals that can kill parasites by inflicting oxidative damage.