Every year, the Executive Board and Supervisory Board report on the company's corporate governance in the Annual Report as required by the German Corporate Governance Code (GCGC). The following statements and details are provided as a Declaration on Corporate Governance in the sense of § 289a of the German Commercial Code (HGB).
"Pursuant to § 161 of the German Stock Corporation Act (AktG), the Executive Board and Supervisory Board of MLP SE hereby declare that the company has generally complied with and will comply with the recommendations of the "German Corporate Governance Code" government commission (version dated February 7, 2017) since the last Declaration of Compliance was issued. Only the recommendations in Sections 4.1.5, 4.2.3 Sentence 11, Section 4.2.3 Sentence 12 to 14, Section 5.1.2. Sentence 2, Section 5.1.2 Sentence 8, Section 5.4.1 (2) Sentence 2 clause 4, clause 5 and clause 6 and Section 5.4.1 (5) Sentence 2 clause 1 were not and will not be applied.
According to the recommendations of the German Corporate Governance Code, the Executive Board should take diversity into account when filling managerial positions and, in this respect, aim for an appropriate consideration of women. The Executive Board sets out targets for proportional female representation at the two management levels below Executive Board level.
In the current financial year, the Executive Board has intensified its efforts with respect to diversity when filling managerial positions and, in particular, strives to achieve appropriate consideration of women at managerial levels in the Company. In the past, the Executive Board of MLP SE already took measures directed at reconciling working life and family life. In the financial year 2017, the Executive Board again reviewed the effectiveness of these measures and already adopted an overall concept in November 2013. If appropriate, it will undertake modifications as necessary or initiate further measures in order to achieve appropriate consideration of female candidates at managerial levels in the Company, taking into account the Company’s specific situation. This concept also includes guidelines for diversity-compliant promotion, which will, however, still require final elaboration. Thus, no specifications for concrete selection decisions with respect to filling positions have yet been established. Nevertheless, the Executive Board of MLP SE has only decided upon a percentage of female members amounting to 0 percent at the first management level below the Executive Board as MLP SE is a holding company that has only a very limited number of staff with just a few managers. Beyond this, MLP SE does not have a second level of management below the Executive Board.
The measures are therefore not yet fully compliant with the requirements of Section 4.1.5 of the Code. As was also the case in the financial year 2017, MLP therefore declares it will continue to deviate from this recommendation in 2018.
According to the recommendations of the German Corporate Governance Code, the Supervisory Board should specify the respective targeted level of benefits – also taking into account the length of service on the Executive Board – and consider the correspondingly derived annual and long-term cost to the Company.
The new remuneration system introduced by the Supervisory Board within the context of implementing the stipulations of the Management Board Remuneration Act (VorstAG) provides for a contribution-based commitment to grant benefits when appointing new members of the Executive Board. The level of specific contributions to be made by the Company is laid down for each respective member of the Executive Board. They do not hold the risk of any unexpected knock-on effects for the Company, since the respective member of the Executive Board actually bears the investment risk in relation to the Company. In the course of implementing the new remuneration system, a decision was therefore taken to dispense with the notion of switching over any employer's pension commitments for members of the Executive Board which provide a fixed benefit above a contractually defined age limit to a purely contribution-based system at the time of contract changeover. With specific regard to these employer's pension commitments, which are to remain in place, the Company does not comply in full with this recommendation.
Therefore the Supervisory Board is of the opinion that the implementation progress made to date is not yet sufficient to fulfil the requirements of Section 4.2.3 Sentence 11 of the Code in full. As was also the case in the financial year 2017, MLP therefore declares not to follow this recommendation in the financial year 2018.
According to the recommendations of the German Corporate Governance Code, the Company should, when concluding Executive Board member contracts, ensure that payments, including fringe benefits, to an Executive Board member following premature cessation of Executive Board duties without serious cause, do not exceed the value of two years’ remuneration (severance payment cap). If the contract of service is terminated due to serious cause on the part of the Executive Board member, no payments will be made to the Executive Board member. The calculation of the severance payment cap should be based on the total remuneration of the previous financial year and, if appropriate, also on the expected total remuneration of the current financial year. In the event of premature termination of Executive Board membership due to a change of control, any commitment for payments should not exceed 150% of the severance payment cap.
During the course of 2011 MLP completed its amendment to the employment contracts of the members of the Executive Board to incorporate a new remuneration system and, particularly in 2014, aligned them with the requirements of the German Banking Act (KWG) and the Remuneration Ordinance for Institutions. Since the first-mentioned changeover, MLP has complied with the aforementioned recommendations.
However, there is no provision for a severance payment in the event of contract termination by mutual consent. Rules concerning a mutually-sought termination of contract can, in a contractual law sense, in any case only serve as a guideline from which, however, the parties could at any time agree to deviate. For this reason, any provisions of this nature would be no more than a formal act.
As was also the case in 2017, MLP will therefore not comply with this recommendation in 2018.
Based on the recommendations of the German Corporate Governance Code, the Supervisory Board should also pay attention to diversity in the composition of the Executive Board and in particular aim for an appropriate consideration of women. Accordingly, the Supervisory Board should specify target numbers for the degree of female representation on the Executive Board.
The Supervisory Board at MLP SE strives to further intensify its future efforts with respect to diversity and, in particular, an appropriate consideration of women when appointing members of the Executive Board. The Supervisory Board gives specific consideration to applications from suitable female candidates in its selection procedures. In the financial year 2014 the Supervisory Board continued to review this aspect and will undertake further measures in order to build on the Group-wide overall concept already passed by the Executive Board for the implementation of Section 4.1.5 of the Code (observance of diversity for managerial positions) and also achieve an appropriate consideration of women within the Executive Board of the Company, taking into account the Company’s specific situation. Nevertheless the Supervisory Board of MLP SE intends to continue to base its selection decision with respect to appointments primarily on the individual and professional qualifications of prospective candidates. For this reason, the Supervisory Board has specified a target figure for female representation on the Executive Board – which incidentally currently consists of just three persons – of 0 percent.
The Supervisory Board is of the opinion that the implementation progress made to date is not yet sufficient to fulfil the requirements of Section 5.1.2 Sentence 2 of the Code in full. As was also the case in the financial year 2017, MLP therefore declares not to follow this recommendation in the financial year 2018.
According to the recommendations of the German Corporate Governance Code, an age limit should be set for members of the Executive Board.
MLP did not follow this recommendation in 2017. There is no set age limit for members of the Executive Board at MLP. The appointment of members of the Executive Board should be based solely on their knowledge, skills and specialist experience. As was the case in 2017, MLP will therefore not comply with this recommendation in 2018.
According to the recommendations of the German Corporate Governance Code, the Supervisory Board is to take into account a pre-defined age limit, a standard limit for the length of service and diversity with regard to its composition while also taking into consideration the Company's specific situation, international operations, any potential conflicts of interest and the number of independent members of the Supervisory Board in the sense of Section 5.4.2.
No age limit or standard limit for the length of service is specified for members of the Supervisory Board. In light of the knowledge, expertise and specialist experience stipulated in Section 5.4.1 Sentence 1 of the Code, it seems inappropriate to specify an age limit and standard length of service for members of the Supervisory Board. As was the case in 2017, MLP will therefore not comply with this recommendation in 2018.
MLP did not follow this recommendation in the current financial year. In its meetings over the last few financial years and again in 2017, the Supervisory Board of MLP AG and, since September 21, 2017, the Supervisory Board of MLP SE has addressed the topic of setting a concrete target for the composition of the Supervisory Board, paying particular attention to diversity, and approved an expertise profile. The Supervisory Board has set itself the target that in the presence of candidates of equal professional and personal suitability, it would seek to fill at least 16.5 percent of the Supervisory Board positions with suitable female members. However, the Supervisory Board does not currently regard the setting of a specific time frame for the implementation of this objective to be appropriate, primarily due to the small number of members of the Supervisory Board as anchored in the Articles of Association. It is therefore also not yet possible to report on any specific implementation steps towards such objectives in the Corporate Governance Report. Nevertheless, as early as in the financial year 2015, the Supervisory Board presented a resolution proposal to the shareholders at the Annual General Meeting to approve the appointment of a woman to the Supervisory Board of the Company. The resolution was subsequently adopted and the target figure was thus achieved.
At the same time, in the financial year 2018 – as in 2017– MLP will thus deviate from this recommendation as the Supervisory Board has not set any concrete target figures but rather sees a subsequent appointment against the background of the candidate’s respective qualification.
As per the recommendations of the German Corporate Governance Code, all candidate proposals are to be submitted to the Annual General Meeting together with a CV that provides information on the relevant knowledge, expertise and experience of the respective candidate.
MLP will deviate from this. MLP discloses all legally required information with its candidate proposals to the Annual General Meeting. Indeed, MLP has already published the CVs of members of the Supervisory Board. However, sufficient clarification has yet to be provided in the legal discussion as to whether the ruling to include a CV with all candidate proposals submitted to the Annual General Meeting applies only to the resolution on election of shareholders' representatives or also employees' representatives and which information specifically needs to be included in the CVs in order to provide information on the respective candidate's knowledge, expertise and experience. In addition to this, including CVs with the candidate proposals on the agenda increases the risk of disputes regarding the election of members to the Supervisory Board. From MLP's perspective, posting the corresponding CVs on the homepage should therefore be seen as sufficient.
MLP therefore declares that it will deviate from this recommendation in future, and consequently also in the financial year 2018.
In December 2017, the Executive and Supervisory Boards issued the above Declaration of Compliance pursuant to § 161 of the German Stock Corporation Act (AktG) and made it permanently available to shareholders via the company's website. The Declaration of Compliance in the version of December 19, 2017 can also be viewed online at www.mlp-se.com.
You can also find more information on the topic of corporate governance at MLP on the aforementioned homepage.
MLP SE is a European stock corporation that is based in Germany and subject to the European SE provisions, the German SE Implementation Act (SEAG) and German stock corporation law. The company was created on September 21, 2017 as a result of the change in corporate form of MLP AG and the corresponding entry in the Mannheim commercial register. As an SE, the company has a dual management and control structure, comprising an Executive Board and a Supervisory Board. The third corporate body is the Annual General Meeting.
As the management body of an "Aktiengesellschaft" (public limited company), the Executive Board runs the business and is tied to the interests and business principles of the company within the scope of corporation law. The responsibilities and duties of the Executive Board are laid down in the German Stock Corporation Act (AktG), in MLP SE's Articles of Association, as well as in a set of rules of procedures and the schedule of responsibilities of the Executive Board attached to it.
The members of the Executive Board hold joint responsibility for the entire management. Decisions made by the Executive Board are reached during Executive Board meetings held at regular intervals. Resolutions are drafted as ordinary resolutions with majority votes and recorded accordingly.
Resolutions of the Supervisory Board are made during meetings convened by the Chairman of the Supervisory Board or on his behalf and require a majority vote. The Supervisory Board is informed of particularly important or urgent projects outside of the regular meetings. Where necessary, resolutions can also take the form of circular resolutions or be passed via telephone. A transcript of each meeting is drafted.
MLP SE's Articles of Association provide for a Supervisory Board that comprises six members, two of whom are employee representatives. Both the size and the tripartite composition of the Supervisory Board are thereby stipulated in MLP SE's Articles of Association. The members of the Supervisory Board at a dualistically structured SE are generally appointed by the Annual General Meeting (Art. 40 (2) of the SE Regulation). MLP deviates from this with regard to the employees' representatives. The appointment of the employees' representatives on the Supervisory Board is based on a participation agreement that has been agreed between the company and a negotiation committee established for this purpose. Accordingly, employees' representatives are elected directly through elections in the company. The current acting members of the first Supervisory Board at MLP SE are appointed until conclusion of the Annual General Meeting that approves the actions for the first financial year of MLP SE. The members of the Supervisory Board are currently Ms. Tina Müller, Dr. Peter Lütke-Bornefeld (Chairman of the Supervisory Board), Dr. h.c. Manfred Lautenschläger, Dr. Claus-Michael Dill, Mr. Burkhard Schlingermann (employees' representative) and Mr. Alexander Beer (employees' representative).
Based on the recommendations of the GCGC, the Supervisory Board is to stipulate concrete objectives regarding its composition, which, whilst considering the company's specific situation, take into account the company's international operations, any potential conflicts of interest, the number of independent members of the Supervisory Board, an age limit to be specified for members of the Supervisory Board and diversity.
The Supervisory Board has set itself concrete targets for its composition. One item worthy of particular note here is a requirements profile for Supervisory Board candidates, which was passed in the financial year and which summarises the knowledge, skills, professional experience and personal aptitude characteristics necessary for candidates to be considered. In addition to this, appraisals were made regarding diversity and the number of independent members.
As per the new revisions to § 100 (5) of the German Stock Corporation Act (AktG) that became effective in 2016, members of the Supervisory Board no longer need to meet the personal independence prerequisites due to legal obligations. Instead, the Supervisory Board should comprise what it deems to be an appropriate number of independent members as per Section 5.4.2 of the German Corporate Governance Code (DCGK). Pursuant to § 5.4.1 (4) Sentence 3 of the German Corporate Governance Code (DCGK), however, this Corporate Governance report should also provide information on what the Supervisory Board deems to be an appropriate number of shareholders as independent members of the Supervisory Board and the names of these members. For this reason, the Supervisory Board continues to adhere to the requirement for independence. The Supervisory Board considers itself as already consisting of a suitable number of independent members who have no business or personal relationship with the company or members of the Executive Board which could form grounds for a conflict of interest. Accordingly, in its meeting on December 19, 2017 the Supervisory Board confirmed that Dr. Lütke-Bornefeld, Dr. Claus-Michael Dill, Mrs. Tina Müller are independent members of the Supervisory Board of the shareholders. The Supervisory Board established that Dr. h. c. Lautenschläger is not an independent member of the Supervisory Board under this provision.
In terms of independence, the Supervisory Board works to a strict standard to eliminate any doubt regarding its judgement. Since Dr. h. c. Lautenschläger is a significant shareholder in our company, his independence is at least seen as potentially compromised.
The Supervisory Board has also set itself the additional goal of filling at least 16.5% of Supervisory Board member positions with suitable female members, in the presence of candidates of equal professional and personal suitability. However, the Supervisory Board does not currently regard the setting of a specific time frame for the implementation of this objective to be appropriate, primarily due to the small number of members of the Supervisory Board as anchored in the Articles of Association. Nevertheless, this quota was already reached over the course of the year 2015 with the election of Ms. Müller to the company's Supervisory Board. The Supervisory Board also reviewed the knowledge and experience required of the Supervisory Board members and of the Chairman of the Audit Committee under the German Corporate Governance Code with regard to reporting and internal control procedures. The Chairman of the MLP SE Audit Committee fully complies with these requirements.
In 2017 the Supervisory Board also reviewed the efficiency of its own activities. Particular attention was paid to the efficiency of the procedures in the Supervisory Board, the information flow between the committees and the Supervisory Board and the timeliness and sufficient content of reporting by the Executive Board to the Supervisory Board. Further measures aimed at increasing efficiency were drafted in the course of intensive discussions.
The Supervisory Board of MLP SE has set up committees in order to improve the effectiveness of its work. The Personnel Committee prepares the resolutions on HR issues concerning Executive Board members with the company. The Audit Committee is responsible for auditing the accounting processes, risk management issues and the auditing system itself, as well as ensuring the independence of the auditors, awarding the audit contract to the auditors and determining the focal points of the audits and agreements on fees. The Committee also discusses the annual financial statements, the consolidated financial statements and the management reports of MLP SE and the MLP Group and submits a recommendation for resolution to the Supervisory Board. In the financial year 2016, the Supervisory Board of MLP AG already intensively dealt with the new legislation of EU Directive 2014/56/EU regarding account auditing, as well as Germany's audit reform legislation (AReG). It adapted procedures to the new legal regulations, so that the stipulations of both the Directive and Germany's audit reform legislation (AReG) can be met. The Supervisory Board has also formed a Nomination Committee, composed exclusively of shareholder representatives, that proposes suitable Supervisory Board candidates for recommendation to the Annual General Meeting and addresses the requirements that these candidates must meet. The members of the Personnel Committee are Ms. Tina Müller, Dr. Peter Lütke-Bornefeld (Chairman of the Personnel Committee) and Mr. Burkhard Schlingermann. The Audit Committee comprises Dr. Claus-Michael Dill (Chairman of the Audit Committee), Dr. h.c. Manfred Lautenschläger, Dr. Peter Lütke-Bornefeld and Mr. Alexander Beer. The Nomination Committee comprises Ms. Tina Müller, Dr. Peter Lütke-Bornefeld (Chairman of the Nomination Committee), Dr. h.c. Manfred Lautenschläger and Dr. Claus-Michael Dill.
In 2017, the Executive and Supervisory Boards of MLP SE again dealt intensively with the German Corporate Governance Code and further new legislation significant to the work performed by the Supervisory Board and its committees. The Code, and its amendments passed on February 7, 2017, were the object of intensive discussions by the Supervisory Board. Stipulations were analysed and corresponding adjustments to the internal regulations and procedures followed by the Supervisory Board were either proposed or submitted for verification.
Intensive dialogue between the Executive and Supervisory Boards forms the basis of transparent and responsible company management. The Executive Board of MLP SE provides the Supervisory Board with regular, timely and comprehensive information on the Group’s position, including information on its risk situation, risk management and compliance. The Supervisory Board is informed of particularly important or urgent projects outside of the regular meetings. Furthermore, the Chairman of the Supervisory Board meets with the Executive Board on a regular basis to discuss specific issues. The Chairman of the Supervisory Board informs the other members of the Supervisory Board in detail about the content of his meetings with the Executive Board. The Supervisory Board discussed the Group's corporate planning and plans for strategic growth with the Executive Board.
Significant legal transactions by the Executive Board require the consent of the Supervisory Board. A set of rules of procedures issued by the Supervisory Board in particular governs the distribution of business, powers of approval outside the Articles of Association and co-operation with the Executive Board. Further details on the co-operation between Executive Board and Supervisory Board can be found in the report by the Supervisory Board.
Further disclosures on the Supervisory Board's activities can be found in the report by the Supervisory Board.
Pursuant to Art. 19 of the Market Abuse Regulation (MAR), persons assuming executive positions at the issuer of shares must notify the issuer and the Federal Financial Supervisory Authority (BaFin) of transactions in financial instruments. This obligation also applies to natural persons and legal entities that are closely linked to such a person assuming an executive position.
Transactions up to a total value of € 5,000 per calendar year are exempt from the notification requirement.
No transactions pursuant to Art. 19 of the Market Abuse Regulation (MAR) were reported to us in the financial year 2017. Transactions from previous years can be viewed on our website at www.mlp-se.com.
The trust of our clients, shareholders and business partners in our capacity to act professionally and responsibly forms the basis of our business operations. Compliance with all relevant legislation and capital market regulations represents the foundation of our business activities and an integral part of our corporate culture. Violations against applicable legislation and regulations not only have the potential to lead to legal disputes and financial losses, but can also have a negative effect on our Group's reputation. The Executive Board at MLP ensures Group-wide compliance with legal provisions and internal company guidelines, while also guaranteeing both a trusting and transparent system of corporate governance.
The basic principles of abidance to laws, integrity and economically successful business practices are firmly anchored in our internal compliance guidelines. Our compliance activities are based on a Group-wide compliance strategy, which is specifically designed with preventive measures to avoid risks that could arise from non-compliance with applicable legislation, internal standards and processes. The primary focus here is on compliance with the key legal provisions and internal company directives, as well as in particular preventing illegal practices, such as insider trading, money laundering, fraud or any other criminal conduct. In the interests of our clients, shareholders and employees, the Compliance department supports and advises the Executive Board in establishing uniform standards for all Group companies. Taking into account the constantly changing regulatory requirements and market conditions, Compliance steers the continuous further development of our internal behavioural standards and monitors implementation of all applicable requirements.
On the basis of a Group-wide risk analysis, the Compliance department identifies, analyses and evaluates the compliance risks relevant to MLP's business operations. Compliance also collaborates with the specialist departments responsible for developing risk-reducing measures and continuously monitors their effectiveness in our day-to-day business.
Regular training sessions, during which all employees of the MLP Group familiarise themselves with the key regulations as a way of preventing any accidental infractions while also providing support in applying our compliance guidelines, represent an important element of our risk prevention measures. These in particular include web-based training events on market abuse legislation, securities compliance, data and consumer protection as well as the prevention of money laundering, financing of terrorist activities and criminal conduct. Compliance is also available to all employees as a point of contact for reporting anything suspicious with regard to criminal activities or violations against our compliance regulations. Any violations determined are investigated immediately, comprehensively clarified and then used to eliminate any weaknesses identified. The Executive Board and Supervisory Board are regularly informed of all relevant actions and measures taken by the Compliance department.
The Compliance Policy in the MLP Group also sets out the measures for insider trading prevention and describes the internal guidelines for execution of employee transactions. The compliance guidelines also ensure that confidential information is handled responsibly at MLP and define standards for advising and supporting our clients, as well as the policy on giving and accepting invitations and gifts. To prevent any impairment of client interests, we have defined policies regarding the avoidance and monitoring of conflicts of interest and the acceptance and granting of benefits. These policies are regularly reviewed and adapted to changing requirements.
MLP defined its core values; a process in which a large number of employees and consultants were involved. "Performance" and "Trust" were identified as values which the corporate mission relies and builds on. You can find details on our corporate mission on our homepage at www.mlp-se.com. In a further step, the following management principles were then derived from this for MLP.
ensure systematic development of managers and staff.
In accordance with the recommendation of the Corporate Governance Code in Section 4.1.5., the Executive Board has further reinforced its efforts to secure diversity when filling management positions. It will also continue to test the effectiveness of the adopted measures in the financial year 2018 and either make any changes deemed necessary or implement additional measures to ensure that women are given appropriate consideration at the management levels of the company, taking into account the company's specific situation.
An explanation of the business and risk strategy as well as the risk management system can be found in the chapter "Risk and opportunity report" of the Annual Report.
The legislation on equal participation of women and men in management positions came into force on May 1, 2015. It stipulates an obligation to specify targets for members of supervisory and executive boards, as well as the two upper management levels, although it does not define an actual minimum target. Insofar as the percentage of women is below 30%, the targets must then not fall below the percentage reached. The deadline for achievement of the first targets was fixed for June 30, 2017. After that deadlines can be up to five years. The targets and deadlines are to be specified in concrete terms. In addition to this, it must be stated whether the specified targets have been achieved and, if not, information as to the reasons must be specified.
MLP places great emphasis on promoting women and helping employees combine a career with a family. The company has therefore already made this topic a focus over the last few years and implemented various measures to promote women into management positions. The concept for example includes measures such as family-friendly meeting arrangements or flexible workplace designs. As a sign of its public commitment to diversity, MLP signed the Diversity Charter in 2014. To help staff more effectively combine their career with a family, MLP opened a parent & child office in February 2015 to provide parents that are struggling to find childcare with the option of taking their children to work with them.
Based on the experience gained when implementing the individual measures, the MLP Group will stick to the targets already achieved for management levels and thereby comply with the minimum legal requirements. In addition to this, MLP has set internal rules for the composition of its Executive Board and Supervisory Board. These stipulate a 25% proportion of women, assuming equal personal and professional aptitude.
MLP SE has met this quota on the shareholder side of the Supervisory Board. This leads to a quota of 16.66% for the entire Supervisory Board. Alongside the three-member Executive Board and one first level manager, as a holding company MLP SE does not employ any other managers and currently no woman holds any of these positions. The objectives for the Supervisory Board and Executive Board at MLP Finanzdienstleistungen AG are the same as those at MLP SE. As a result of the demerger to create MLP Finanzberatung SE and MLP Banking AG, there are now two separate legal entities, each with a dedicated Supervisory Board and the following quotas on the shareholder side: MLP Banking AG 25% and MLP Finanzberatung SE 0%. In the 2017 financial year, the quotas for the first management level below the Executive Board were 50% at MLP Banking AG and 5.26% at MLP Finanzberatung SE. For the second management level below the Executive Board, the quotas were 33.33% and 8.11% respectively. With measures such as the Top Talents Programme for targeted development of junior staff, as well as strengthening the role of the equal opportunities officer, MLP will continue to consistently pursue the goal of increasing the number of women in management positions.
The Transparency of Remuneration Act (EntgTranspG) came into force on July 6, 2017 to counteract wage differences between women and men doing the same or equivalent work. The legislation promotes disclosure of company remuneration systems and prescribes an individual right to information regarding in-house remuneration structures for staff at enterprises with more than 200 employees. The right to information can be asserted for the first time by employees at MLP Finanzberatung SE from January 6, 2018.
A "remuneration system" works agreement, which applies to MLP SE, MLP Banking AG and MLP Finanzberatung SE, was concluded in December 2015 as the basis for establishing improved remuneration transparency at MLP. This agreement was then used to assess and subsequently assign each job type to a salary range. The awarding of salaries for new recruitments is based on this assignment, which also forms the basis for salary adjustments together with the concepts of professional experience and performance. Private employers that generally have more than 500 employees are called upon to use operational audit procedures to regularly review the application of their remuneration systems and the various remuneration components paid for compliance with the equal pay requirement in the meaning of this legislation. MLP has decided to perform these audits for MLP SE, MLP Banking AG and MLP Finanzberatung SE.
For the first time in 2018 a report on the current status and the provisions determined to establish equal pay and equality will be included with the management report as an appendix and also published in the Federal Gazette (Bundesanzeiger).
By law, the shareholders are involved in all fundamentally important decisions at MLP SE, such as decisions on amendments to the Articles of Association and the issue of new shares. In order to help shareholders assert their rights, MLP offers them the option of having their voting rights exercised in writing by non-discretionary proxies appointed by the company or by postal vote. We report on the main content of the Annual General Meeting on our website at www.mlp-se.com, where the Chairman’s speech can also be accessed online.
We also use the internet in order to provide comprehensive and timely information on the company’s position and significant changes in a way that ensures all stakeholders are treated equally. We provide access to both German and English versions of annual and quarterly reports, press releases, conference calls and presentations on our homepage at www.mlp-se.com. Our financial calendar includes important events and dates for investors. Analysts’ and media conferences are held at least once a year. In accordance with legal provisions, ad-hoc notices are published on our website, where we also provide comprehensive information on corporate governance at MLP. We provide access to our Declaration of Compliance on our homepage for at least five years.
Group accounting is performed in line with International Financial Reporting Standards (IFRS). KPMG AG Wirtschaftsprüfungsgesellschaft, Berlin, was appointed as auditor by the Annual General Meeting and has audited the 2017 consolidated financial statements. The Supervisory Board gave its assurance that the relations existing between the auditors and MLP or its governing bodies give no cause for doubting the independence of the auditor. The Supervisory Board at MLP SE also discusses the annual and consolidated financial statements.
Within the scope of our 2017 Sustainability Report, we report on the non-financial aspects of our business activities. The focuses of our sustainability reporting in terms of content result from the materiality analysis performed in 2017, on the basis of which we identified the key aspects for our company.
To ensure our sustainability activities have a comparable and transparent framework at all times, we have aligned our reporting with the reporting standard of the German Sustainability Code (DNK). Please refer to the Declaration of Compliance with the German Sustainability Code for further information and details on our sustainability management. We publish this Declaration of Compliance, as well as our sustainability report on our website at https://mlp-se.com/company-profile/sustainability. You can also find the sustainability report as part of this Annual Report.


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Art. 28


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The prophenoloxidase-activating (PO activating) system plays an important role in the crustacean innate immunity, particularly in wound healing and pathogen defense. A key member of this system is prophenoloxidase-activating enzyme (PPAE), which is the direct activator of prophenoloxidase (proPO). Despite their importance in crustacean PO activating system, the studies on them remain limited.
Here we report on a PPAE of white shrimp, Litopenaeus vannamei (lvPPAE1), which showed 94% similarity to PPAE1 of Penaeus monodon. We found that lvPPAE1 in fluid hemocytes was down regulated after challenge by Vibrio harveyi but was enhanced when shrimps were exposed to a bacteria-rich environment for long-term. In vivo gene silence of lvPPAE1 by RNAi can significantly reduce the phenoloxidase activity (PO) and increase the susceptibility of shrimps to V. harveyi. Although lvPPAE1 was down-regulated in fluid hemocytes by Vibrio challenge, its expression increased significantly in gill after bacteria injection, which is the primary bacteria-clearance tissue.
Suppressed expression in fluid hemocytes and enhanced expression in gill indicates selectively enhanced expression at the bacterial clearance site. This is a novel feature for PPAE expression. The results will contribute to our understanding of the PO activating system in crustaceans.
Innate immunity is of great importance to insects and crustaceans because they lack antibodies . Innate immunity involves phagocytosis, encapsulation, hemocyte coagulation and activation of the prophenoloxidase (proPO) or melanization cascade [2, 3]. Activation of proPO generates phenoloxidase (PO), which catalyzes the oxygenation of monophenols to o-diphenols and the oxidation of o- diphenols to the corresponding o-quinones . These are reactive intermediates for melanin synthesis and other physiological processes such as cuticle sclerotization, wound healing and pathogen sequestration .
In most cases, the proPO cascade is triggered by a small amount of microbe-derived molecules such as lipopolysaccharides (LPSs), β-1, 3-glucans, or peptidoglycan. Pattern-recognition proteins bind these molecules and initiate the proPO system through a quick proteolytic cascade, and many proteins involved in the proteolytic cascade are serine proteinases . The final serine proteinase that converts the inactive proPO into its active form is called prophenoloxidase-activating enzyme (PPAE) .
The proPO genes of insects and crustaceans have been intensively studied [7–14], and models for the serine proteinase cascades regulating proPO activation have been studied in detail in some insects such as Manduca sexta and Tenebrio molitor[15–23]. Studies on proPO-activating cascades are limited in crustaceans. Hitherto, few PPAEs in crustaceans have been reported, except for in crayfish, Pacifastacus leniusculus and shrimp, Penaeus monodon[24–26]. In this study, the PPAE1 of white shrimp Litopenaeus vannamei was identified and its expression feature was reported. The new findings of the study will contribute to our understanding of PO activating system of crustaceans.
The full-length lvPPAE1 cDNA was 1557 bp, with an ORF of 1389, a 5'-UTR of 57 bp, and a 3'-UTP of 111 bp. The theoretical pI and Mw were 6.96 and 50.4 kDa. The proteolytic activation site was found between Arg228 and Ile229. The pI and Mw of activated lvPPAE1 were 4.5 and 25.4 kDa. The lvPPAE1 protein showed 94% identity to pmPPAE1 and 61% to plPPAE. Three amino acid residues (H270, D319, and S412) corresponded to the catalytic triad of the SP domain and another three amino acid residues (D406, S433, and G435) to the substrate binding sites.
Multiple alignments between the sequences of lvPPAE1, plPPAE, and pmPPAE1 showed that this gene is relatively well conserved in crustaceans (Figure 1). lvPPAE1 exhibited a typical clip-domain structure, composed of six cysteines at the N-terminus. Similar to other crustacean PPAEs, a glycine-rich domain was found between residues 40 and 130, with 17.6% glycines (16/91, pI = 12.2), and a proline-rich domain was found between residues 194 and 226, with 39.4% prolines (13/33, pI = 12.5).
Alignment of crustacean prophenoloxidase-activating enzymes. The signal peptides are bold and double-underlined. The six clip-domain cysteines are indicated with the letter "C," and the black triangle indicates the active cleavage site. The amino acid residues corresponding to the serine proteinase catalytic triad are marked by black stars. Glycine-rich and proline-rich domains are marked separately by solid lines. Gene Bank accession numbers are as follows: pmPPAE1 (Penaeus monodon, Genebank ID: ACP19558), plPPAE (Pacifastacus leniusculus, Genebank ID: CAB63112), ptSP (Portunus trituberculatus, Genebank ID: ACI46638).
A phylogenetic tree was constructed using the neighbor-joining method (Figure 2) by comparing the deduced amino acid sequence of the conserved SP domain to the 82 clip-domain serine proteinases showing the most similarity or any reported PPAEs. The 83 proteinases were separated into two main branches: one mainly composed of insect prophenoloxidase-activating proteins and the other composed of insect pro-clotting enzymes. The PPAEs of crustaceans were classified into the second main branch and they shared the same and a unique sub-branch. Except for seven pro-clotting enzymes and five trypsins, the biological roles of the clip-domain serine proteinases from the second main branch are still unclear.
Neighbor-joining phylogenetic tree constructed with the prophenoloxidase-activating enzyme (lvPPAE1) SP domain and the other 82 clip-domain serine proteinases showing the most similarity in prophenoloxidase-activating proteins in insects. Bootstrap sampling was reiterated 1000 times. The scale bar refers to a phylogenetic distance of 0.1 amino acid substitutions per site. Prophenoloxidase-activating proteins are highlighted in red, and easter-type serine proteases are in green, hemolymph proteases are in blue, and pro-clotting enzymes are in orange. PPAEs in crustaceans are underlined.
lvPPAE1 gene expression was analyzed by qRT-PCR. lvPPAE1 in SPF shrimps was mainly expressed in hemocytes and gill, whilst low level was detected in the other tissues. The relative expression in FHs was about 3.5-fold in gill. Forty eight hours after Vibrio challenge, the relative expression level of lvPPAE1 in gill was as same as that in FHs, which indicated an increase in the gill (Figure 3). Gill samples from the challenged shrimps were checked using microscope with phase contrast mode. Intensive hemocyte aggregation was found in gill canals (Figure 5A, B).
Tissue distribution of the prophenoloxidase-activating enzyme mRNA from white shrimp Litopenaeus vannamei (lvPPAE1) before and after Vibrio harveyi challenge. The transcription level was detected by Taqman qRT-PCR. Gene expression level was normalized to 18 s rRNA. Five samples were taken as replicates.
Hemocytes and gill were sampled at each time point after the challenge. qRT-PCR showed that the lvPPAE1 mRNA expression level began to decline in FHs in a short time post challenge. This suppression became more obvious after 4 hpc (hour post challenge); the expression level decreased by 60% compared to the control (0 h). After 16 hpc, the expression recovered slightly but then declined again and remained suppressed after 48 hpc. Multiple comparison between challenged and saline injected groups at each time point showed the suppression of lvPPAE1 were significant at 2, 4, 8 and 24 hpc. Temporal expression in saline injected shrimp was not changed significantly. The two results, normalized to 18 s and β-actin separately, were well matched (Figure 4A, B). However, the relative change in expression in gills was different. At 4 hpc, lvPPAE1 expression in gill was significantly enhanced to about 2.5-fold than that of the control (0 h). After a relative decline at 8 hpc, the expression level increased to 3-fold compared to the control and the high expression was sustained for 48 hpc (Figure 5C).
Time-course mRNA expression of the prophenoloxidase-activating enzyme from white shrimp Litopenaeus vannamei (lvPPAE1) in fluid hemocytes after the shrimp was injected with saline or Vibrio harveyi. Sampling time points were 0, 1, 2, 4, 8, 16, 24 and 48 h post-injection. Transcript levels were detected by Taqman qRT-PCR. Two sets of data were obtained by normalized to different inferences separately. Five individuals were sampled as replicates. Data were analyzed with Dunnett's t-test and Tukey's range test. Significant differences from the control (0 h, P < 0.05) are indicated by asterisks. Significant differences between challenged and control at each time point are noted by the P value above. (A) Normalized to 18 S rRNA. (B) Normalized to β-actin.
Hemocyte aggregation and analysis in gills after Vibrio harveyi challenge. (A) Intensive hemocyte aggregation in gill 4 h after bacterial challenge. White arrows show aggregated hemocytes in gill canal. (B) Hemocytes released from gill by dissecting. Bar = 20 μm. (C) Time-course expression of the prophenoloxidase-activating enzyme from white shrimp Litopenaeus vannamei (lvPPAE1) in gill after challenge. Sampling times are 0, 4, 8, 16, and 48 h post-infection. Five samples were tested as replicates at each time point. Gene expression level was normalized to β-actin. Data were analyzed with Dunnett's t-test. Significant differences from the control (0 h, P < 0.05) are indicated with asterisks.
Vibrio in the biofloc and filtered seawater was calculated following colony forming unit method after a 24-h culture. The Vibrio number in biofloc seawater was 3.5 × 104 CFU/ml, whereas 45 CFU/ml were found in filtered seawater (Figure 6A). The relative lvPPAE1 mRNA expression level in FHs of shrimp reared in different bacterial environments was analyzed by qRT-PCR. Five samples were taken as replicates for expression analysis. The relative lvPPAE1 mRNA expression level in shrimps exposed to a higher bacteria environment was 2.5-fold than that of those exposed to a lower level of bacteria (Figure 6B).
Vibrio amounts in different water conditions and corresponding lvPPAE1 mRNA expression levels. Five samples were taken as replicates. (A) Log value of the Vibrio colony amount per milliliter biofloc water and filtered seawater. (B) qRT-PCR analysis of lvPPAE1 mRNA expression level in FHs of shrimp reared in biofloc or filtered seawater. Data were analyzed with the unpaired t-test. Different letters indicate significantly different means (P < 0.05).
qRT-PCR was used to detect the dsRNA interference effect 12 and 24 h after the first dsRNA injection and 24 h after the second injection. Total RNA extracted from the hemocytes of gene-silenced shrimp was analyzed. RNAi strongly inhibited lvPPAE1 expression. The amount of mRNA decreased to less than 5% of the control 24 h after the second lvPPAE1 dsRNA injection (0 h, Figure 7A).
Gene silencing of the prophenoloxidase-activating enzyme from white shrimp Litopenaeus vannamei (lvPPAE1) and related bioassays. (A) RNAi of lvPPAE1 using gene-specific dsRNA analyzed by qRT-PCR and normalized to β-actin. Five samples were mixed and analyzed 12 and 24 h after the first dsRNA injection and another 24 h after the second injection. (B) Phenoloxidase activity assay after gene silencing, analyzed by analysis of variance (ANOVA) followed by Duncan's multiple range test. Five individuals were taken as replicates. Bars with different letters are significantly different (P < 0.05). (C) Mortality of shrimps due to Vibrio harveyi after gene silencing. The experiment was conducted three times.*Occasional mortality occurred after the first lvPPAE1 dsRNA infection.
Susceptibility of shrimps to V. harveyi was tested after gene silence. About 1 h after the challenge, shrimps in the gene-silenced group began to show symptoms of slowed mobility and imbalance, as well as mortality after 2 h. Saline-injected shrimp showed symptoms after at least 4 h, and mortality began after 8 h. Mortality in the saline pre-injected group reached about 50% after 48 h, and all gene-silenced shrimp died within 24 h. No mortality was found in the saline-only injected group and occasional mortality was found in the lvPPAE1 dsRNA-only injected group (Figure 7B).
PO activity was analyzed in shrimp injected with gene-specific dsRNA, GFP dsRNA, or saline solution. PO-induced activity in the gene-silenced shrimp decreased by approximately 50% compared to the saline-injected control group. No such decrease was found in the GFP dsRNA-injected shrimp (Figure 7C).
We identified and characterized a clip-domain serine proteinase from the white shrimp, L. vannamei. The deduced amino acid of this gene showed high similarity to that of plPPAE (61%) and particularly high similarity to pmPPAE1 (94%), which is the terminal enzyme in the prophenoloxidase-activating cascade [25, 26]. Thus, we named this gene lvPPAE1. Multiple alignment results indicated that this gene is rather well conserved in crustaceans. Interestingly, phylogenetic analyses revealed that all PPAEs in crustaceans branched off into the cluster of insect pro-clotting proteinases rather than insect PPAEs. Besides with molecular structure deviation, PPAEs in crustaceans can proteolyses proPO dependently , unlike most of insect PPAEs that need cofactors to active proPOs . Another major difference between PPAEs in crustaceans and insects is the different regulation type to pathogen invasion. Three prophenoloxidase activating proteinase of the horn worm Manduca sexta were reported to be stimulated or up-regulated by injection of Micrococcus lysodeikticus or Escherichia coli[17, 28, 29]. Five putative PPAEs of the fly Drosophila were induced to higher levels after E. coli and Micrococcus luteus infection . Two potential PPAEs of the mosquito Anopheles gambiae were up-regulated by bacteria E. coli or M. luteus and parasite Plasmodium berghei. A prophenoloxidase activation proteinase of the silk worm Samia cynthia ricini was inducible by cell wall components of gram-positive bacteria . Contrary to those insect PPAEs, The crayfish PPAE transcript level was reported to be unaffected upon Aeromonas hydrophila challenge . The pmPPAE1 transcript level in P. monodon was down regulated 3-24 h after Vibrio injection . In the present study, the lvPPAE1 transcript abundance was reduced within 48 h after Vibrio challenge. All reported crustacean PPAEs as well as our study showed their transcript levels were either not affected or down-regulated within 48 h, contrary to PPAEs in insects that were up-regulated or stimulated within 24 h after challenge. Dissimilarity in proteinase structures and functional features imply certain degree of difference exists between crustaceans and insects for the prophenoloxidase-activating mechanism, particularly in the down stream.
It has been reported that the PPAE1 mRNA of crustaceans were found specially in hemocyte and free from other tissues . Our tissue distribution analysis by qRT-PCR also revealed that lvPPAE1 transcript level was very low in muscle, antenna gland, nerve cord and digesting system, and this minimum expression also could be caused by a few amount of hemocytes penetrated into the tissues . The most convincing proof was that the lvPPAE1 transcript level in those tissues didn't change after bacteria challenge, suggesting that these tissues were in fact free of lvPPAE1 transcripts. This conclusion is in accordance with the previous report . However, it is notable that during bacteria clearance process in shrimps, bacteria or foreign materials would be encapsulated by hemocytes to form hemocyte-foreign material aggregation and this kind of aggregation happens mainly in gill, which is the main bacteria or alien materials clearance site . This phenomenon was also confirmed by our microscope observation (Figure 5A, B). In another word, gill is closely related with hemocytic immunity in host defense of shrimps. By sensitive qRT-PCR method, we successfully found out the inevitable repercussion of this close relation: The lvPPAE1 mRNA transcripts represented a considerable level and even higher after Vibrio challenge.
Our RNAi experiment showed silence of lvPPAE1 significantly reduced the PO activity and increased the susceptibility to V. harveyi. qRT-PCR revealed shrimps exposed in a bacteria rich environment for a long-term presented a higher lvPPAE1 transcript level. Those results suggested the importance of lvPPAE1 in the host defense of L. vannamei. It is thus odd to find the expression of lvPPAE1 was depressed when shrimps tried to fight against the injected bacteria. Same phenomenon was found in another shrimp species P. monodon, yet the author didn't give an explanation. Our findings in gill gave a clue to answer the doubtful question. Although the transcript expression of lvPPAE1 in gill was in fact due to the fixed hemocytes (GH), the transcript level of GHs showed different regulation type than in FHs. Contrary to the continuous dropping trend in FHs, the transcript level of lvPPAE1 in gill was stimulated quickly and arrived to a peak at 4 h when the depression in FHs was at the upmost degree. Higher lvPPAE1 mRNA transcript level was maintained in gill within 48 h when the lvPPAE1 was continuously depressed in FHs. By now, a new conclusion was drawn that the lvPPAE1 was not simply down-regulated by Vibrio challenge but also up-regulated in bacteria clearance site. By the current data, it can not be asserted that the overall lvPPAE1 transcript level was depressed or enhanced shortly after the Vibrio challenge; we thus call the expression feature as the "selective enhanced expression". Because the selective enhanced expression in gill happens simultaneously with the suppression in fluid hemocytes, the endogenous regulation mechanism for the short time enhanced expression in bacteria clearance site is probably different from the enhanced expression in FHs under a long-term, bacteria-rich environment stress. Our current study on lvPPAE1 indicated that the endogenous regulation mechanism for shrimp immunity is far from clarified.
A prophenoloxidase activating enzyme (lvPPAE1) of the white shrimp L. vannamei was cloned and sequenced. In vivo gene silence of lvPPAE1 significantly deduced the PO activity and increased the susceptibility of shrimps to gram negative bacteria V. harveyi. The overall tissue distribution analysis showed the lvPPAE1 was mainly expressed in fluid hemocytes (FH) and gill due to the aggregated hemocytes in canal (GH). The lvPPAE1 expression was significantly enhanced in gill within 2 h after V. harveyi challenge. In the case of FH, lvPPAE1 expression was suppressed within 48 h after a single challenge, whereas higher expression was found when shrimp was exposed to a high bacterial environment over the long term. The different gene expression responses to bacterial challenge in FH and gill suggest enhanced lvPPAE1 expression at the bacteria-clearance site.
In conclusion, the lvPPAE1 is involved in melanization cascade and plays an important role in shrimp host defense against bacteria. The expression in FH was suppressed in a short term after V. harveyi challenge but was up regulated under bacterial press for a long term. The lvPPAE1 represents a feature of selective enhanced expression in bacteria clearance site after challenge. This is a novel characteristic for crustacean PPAEs expression.
Specific pathogen-free (SPF) L. vannamei (10 g ± 1.2 g mean ± SD) were obtained from a biofloc water culture system at the NFRDI (Seoul, Korea) and were maintained in filtered running seawater for two weeks, if needed. All tissue preparations were performed as bellow: Tissues such as muscle, stomach, antenna gland, nerve cord, intestine, hepatopancreas, or gill were obtained by dissection. Hemolymph was collected from the ventral hemolymph sinus of L. vannamei with a 3 ml RNase-free syringe containing on ice pre-cooled anticoagulant (113 mM glucose, 27.2 mM sodium citrate, 2.8 mM citric acid, and 71.9 mM NaCl). The hemocyte cell pellet was collected by centrifugation at 700 × g and 4°C for 10 min followed by two rinses with anticoagulant. All tissues were placed immediately in 200 μl RNA later reagents after preparation (Ambion, Austin, TX, USA). Total RNA was extracted with the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and further purified with DNase I (Qiagen), according to the manufacturer's protocol.
The first-strand cDNA for 5' rapid amplification of cDNA ends (RACE) and 3' RACE was synthesized separately with the SMARTer™ RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA), according to the user's manual. The first-strand cDNA was diluted 20 times to serve as the RACE template. A degenerate of the gene-specific primer lvPPAERACE3' was designed based on other crustacean PPAE sequences [25, 26]. PCR amplification was performed with the specific primer and two universal primer provided by the kit. The touchdown PCR conditions were as follows: five cycles of 94°C for 30 s and 72°C for 3 min; five cycles of 94°C for 30 s, 70°C for 30 s, and 72°C for 3 min; 25 cycles of 94°C for 30 s, 68°C for 30 s, and 72°C for 3 min. PCR products were then subcloned into the TOPO vector (Invitrogen, Carlsbad, CA, USA) and sequenced. The fragment sequence was confirmed by the BLAST program on NCBI by comparison with other shrimp serine proteases. A new primer, lvPPAERACE5', was designed based on the partial sequence of lvPPAE1. 5' RACE was performed with the new primer and universal primers mentioned above with the same PCR conditions as 3' RACE. Then the 5' RACE product was subcloned into the TOPO vector and sequenced. After the 5' RACE sequence was confirmed by BLAST, 3' RACE fragment and 5' RACE fragment sequences were joined using the Mega 4 program .
The open reading frame (ORF) of the gene was analyzed by the ORF finder program on NCBI. The nucleotide sequence and deduced amino acid sequences were analyzed by the BLAST program on NCBI. Multiple alignments were performed with the Clustal W2 program http://www.ebi.ac.uk/Tools/clustalw2/, and the result was viewed with Genedoc 2.7.0 . A neighbor-joining phylogenetic tree was constructed based on the SP domain of the deduced amino acid sequences of lvPPAE1 and the 82 clip-domain serine proteinases showing highest similarity or the prophenoloxidase-activating proteins of insects using the Mega 4 program . The lvPPAE1 signal peptide was predicted with the SignalP 3.0 server http://www.cbs.dtu.dk/services/SignalP/. The theoretical isoelectric point (pI) and molecular weight (Mw) of the proteins were estimated online http://isoelectric.ovh.org/.
A Taqman probe-based quantitative reverse transcription PCR (qRT-PCR) technique was taken to test the lvPPAE1 transcript level. Tissue distribution or mRNA expression under different circumstance stresses can thus be evaluated by this method. A pair of gene-specific primers qRTPPAEf, qRTPPAEr and a Taqman qRTPPAEp probe were designed from the full-length lvPPAE1 cDNA sequence using PrimerExpress software (Applied Biosystems Pty Ltd., Melbourne, Australia). One-step qRT-PCR was accomplished with the One Step PrimeScript™ RT-PCR perfect real time Kit (Takara Bio). The reaction mixture consisted of 10 μl 2× One-Step RT-PCR Buffer III, 2 units of Takara Ex Hot Start Taq enzyme, 0.4 μl reverse transcript enzyme Mix II, and 0.4 μM each of forward primer, reverse primer, and Taqman probe in a final reaction volume of 20 μl. The PCR conditions were as follows: 42°C for 5 min, 95°C for 10 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Fluorescent signal detection was started from the first cycle of the annealing stage. The 18 s ribosomal RNA (18 s) and β-actin was taken as references with primers and probe shown in Table 1.
V. harveyi KCCM 40866 obtained from the Korean Culture Center of Microorganism (KCCM) was used in a preliminary challenge trial to determine its pathogenicity. Then it was further selectively enriched from the hepatopancreas of freshly killed shrimp in the preliminary challenge on tryptic soy agar (Difco, Franklin Lakes, NJ, USA) plate. Harvested bacteria were diluted with physiological saline (154 mM) to an OD540 of 0.09 (106 CFU). The diluted bacteria (100 μl) were injected into the third abdominal segment. SPF shrimps (about 10 gram) from biofloc culture system were raised in filtered sea water for 2 weeks before challenge. One hundred and twenty individuals were challenged and a control group of 50 individuals were injected with the same volume of saline. Gill and hemocyte were sampled at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h and 48 h after bacteria or saline injection. Five shrimps for each group were sampled as replicates at each time point separately. Temporal expression was detected by qRT-PCR and the relative expression data were analyzed by Dunnett's t- test and Tukey's range test.
Water samples (200 μl) from biofloc water or filtered sea water were spread on 0.2% sodium-chloride thiosulfate citrate bile salt agar (Difco) and incubated at 28°C for 24 h before the colonies were counted. Vibrio colony numbers were counted three times everyday continuously for 4 days. Fluid hemocytes were extracted from SPF shrimps (about 10 gram) raised in biofloc water culture system or filtered running seawater and the lvPPAE1 mRNA level were measured and compared with qRT-PCR. Five shrimps were sampled as replicates from the two culture systems separately. The deference of PO activity was analyzed by unpaired t-test for unequal variance.
First-strand cDNA was synthesized with the PrimeScript™ First-Strand cDNA Synthesis Kit (Takara Bio) with the oligo dT Primer, according to the manufacturer's protocol. A pair of forward and reverse primers with a T7 promoter oligo anchor (dslvPPAEf, dslvPPAEr) amplifying a fragment of 542 lvPPAE1 nuclei fragments was designed. The PCR amplification was conducted with a reaction mixture of 2 μl 10× PCR buffer, 1.6 μl dNTP mixture, and 0.1 μl ExTaq enzyme with a final primer concentration of 0.4 μM. One μl first-strand cDNA was added to make a final volume of 20 μl with PCR water. The PCR conditions were as follows: 35 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 1 min 30 s. PCR fragments were purified by agarose gel electrophoresis and subcloned into the TOPO vector (Invitrogen). Sequences were confirmed by sequence alignment and then used as the template for a secondary PCR with T7 promoter primers only. The secondary PCR product was then used as a template to produce dsRNA. dsRNA was produced and purified using the RiboMAX™ T7 Express System (Promega, Madison, WI, USA), according to the user's manual. dsRNA concentration was calculated by measuring the absorbance at 260 nm with a spectrophotometer (Cary 100 scan spectrophotometer, Varian). An alien GFP protein sequence was amplified with the PEGFP-1 (Clontech) reconstructed vector, using the dsGFPf forward primer and the dsGFPr reverse primer (encoding 303 nuclei), and the produced dsRNA fragment was used as a control.
lvPPAE1 and GFP protein dsRNA were diluted in 0.9% saline separately. Diluted dsRNA was injected into the third abdominal segment using a 1 ml sterile hypodermic syringe with a 26 gauge needle. Approximately 1 μg lvPPAE1 or GFP dsRNA was used per 1 g shrimp. Another injection with the same amount of dsRNA was performed 24 h later. The gene silencing effect was tested by qRT-PCR.
Susceptibility of lvPPAE1 gene silenced or control shrimp to V. harveyi was evaluated. SPF shrimps from biofloc system were pre-raised in 26°C filtered sea water for two weeks and used for experiment. Shrimps were challenged simultaneously with the second dsRNA injection for gene silencing. Mortality of gene silenced or control shrimp was monitored within 48 h. Two additional groups injected with only saline or only lvPPAE1 dsRNA were set as controls to determine mortality from the procedure. Thirty individuals for each group were used to calculate the mortality rate and the experiment were repeated for three times.
Hemolymph of shrimps from the gene silenced or control group were extracted with anticoagulant buffer, centrifuged at 700 × g and 4°C for 15 min. The cell pellet was rinsed twice with anticoagulant buffer, resuspended, and centrifuged at 16000 × g and 4°C for 5 min. An aliquot (500 μl) of ice-cold homogenizer buffer containing 10 mM sodium cacodylate, 5 mM CaCl2, pH 7.0 (Sigma, Munich, Germany) was added, and the cell pellet was homogenized for 2 min at full speed to release the proteins. The cell homogenate was then centrifuged at 10000 × g and 4°C for 10 min, and the supernatant (LPS) was used for the PO activity test. LPS (100 μl) and 50 μl laminarin (0.1 mg/ml, Sigma) was mixed in a 96-well ELISA plate and incubated at 27°C for 10 min. L-3, 4-dihydroxyphenylalanine (50 μl, 3 mg/ml) was added, and PO activity was measured with an ELISA reader, using dynamic read style, for 20 min at room temperature. Absorbance at 490 nm was measured from 10 to 20 min. Protein concentration was measured with a BCA™ Protein Assay Kit (Pierce, Rockford, IL, USA). PO activity was calculated as OD490/mg LPS protein/min. Data was analyzed by analysis of variance followed with Duncan's multiple range test. Five individuals were sampled as replicates.
This work was supported by the Project "Environmentally-friendly BFT shrimp culture technology" (No. RP2011-AQ-122), National Fisheries Research & Development Institute (NFRDI), Republic of Korea.
IKJ designed the study and gave important comments to the MS. ZGP performed the statistical analysis and wrote the MS. ZGP and JPY carried out sequencing, sequence alignment, qRT-PCR analysis, RNAi and challenge experiment and PO assay. SKK participated in PO assay and Vibrio counting. HCS and YRC prepared materials and the field facility. They also donated animals for the study.

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In 1996, the Supreme Court heard a landmark case on software copyright. Lotus Development Corporation had sued Borland International, Inc. for copying the names and hierarchical structure of the menu commands in Lotus’s blockbuster spreadsheet program, 1-2-3, and offering them in Borland’s competing Quattro Pro software. However, the Supreme Court did not make binding precedent in Lotus v. Borland. One justice’s recusal left the remaining justices in a 4-4 tie, and so the Court let stand, without opinion, the First Circuit’s judgment that the names and hierarchical structure of the menu commands in Lotus 1-2-3 were uncopyrightable.
Nearly twenty years later, similar questions of software copyright remain unresolved. A high-profile case involving copyright in computer programs, Oracle v. Google, was denied review by the Supreme Court in late June of 2015. Google’s petition for certiorari to the Supreme Court maintained that Oracle v. Google “directly implicates the unanswered question in Lotus...” and its outcome will exert “enormous” influence over innovation in the computer business. Lotus v. Borland is an essential piece of context in the contemporary debate over digital intellectual property—a debate that has persisted for decades without definitive resolution by the Supreme Court. Studying the case can offer a practical immersion in legal doctrine, litigation procedure and tactics, policymaking, and business strategy.
This case study contextualizes Lotus v. Borland in order to analyze the issues of jurisprudence, policy, commerce, and legal practice that the case implicated. Students study internal government documents—made public during Justice Elena Kagan’s confirmation hearings—that give insight into the rarely seen debates that shape the government’s official position on pending litigation. By providing important legal and historical context, this case study challenges students to examine Lotus v. Borland’s influence on the contemporary software ecosystem.
This case study is based on the syllabus and pedagogy of “Anatomy of a Copyright Case,” a course taught at HLS in Spring 2015 by Henry Gutman, who argued Lotus’s side before the Supreme Court.
Discuss software copyright from legal, commercial, and policy perspectives.
Understand legal and business strategies in copyright litigation.
Practice argumentation and brief-writing in a variety of legal, governmental, and commercial roles.
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