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Table of Contents – Types, Formats, Examples

Table of Contents

Definition:

Table of contents (TOC) is a list of the headings or sections in a document or book, arranged in the order in which they appear. It serves as a roadmap or guide to the contents of the document, allowing readers to quickly find specific information they are looking for.

A typical table of contents includes chapter titles, section headings, subheadings, and their corresponding page numbers.

The table of contents is usually located at the beginning of the document or book, after the title page and any front matter, such as a preface or introduction.

Table of Contents in Research

In Research, A Table of Contents (TOC) is a structured list of the main sections or chapters of a research paper , Thesis and Dissertation . It provides readers with an overview of the organization and structure of the document, allowing them to quickly locate specific information and navigate through the document.

Importance of Table of Contents

Here are some reasons why a TOC is important:

• Navigation : It serves as a roadmap that helps readers navigate the document easily. By providing a clear and concise overview of the contents, readers can quickly locate the section they need to read without having to search through the entire document.
• Organization : A well-structured TOC reflects the organization of the document. It helps to organize the content logically and categorize it into easily digestible chunks, which makes it easier for readers to understand and follow.
• Clarity : It can help to clarify the document’s purpose, scope, and structure. It provides an overview of the document’s main topics and subtopics, which can help readers to understand the content’s overall message.
• Efficiency : This can save readers time and effort by allowing them to skip to the section they need to read, rather than having to go through the entire document.
• Professionalism : Including a Table of Contents in a document shows that the author has taken the time and effort to organize the content properly. It adds a level of professionalism and credibility to the document.

Types of Table of Contents

There are different types of table of contents depending on the purpose and structure of the document. Here are some examples:

Simple Table of Contents

This is a basic table of contents that lists the major sections or chapters of a document along with their corresponding page numbers.

Example: Table of Contents

I. Introduction …………………………………………. 1

II. Literature Review ………………………………… 3

III. Methodology ……………………………………… 6

IV. Results …………………………………………….. 9

V. Discussion …………………………………………. 12

VI. Conclusion ……………………………………….. 15

Expanded Table of Contents

This type of table of contents provides more detailed information about the contents of each section or chapter, including subsections and subheadings.

A. Background …………………………………….. 1

B. Problem Statement ………………………….. 2

C. Research Questions ……………………….. 3

II. Literature Review ………………………………… 5

A. Theoretical Framework …………………… 5

B. Previous Research ………………………….. 6

C. Gaps and Limitations ……………………… 8 I

II. Methodology ……………………………………… 11

A. Research Design ……………………………. 11

B. Data Collection …………………………….. 12

C. Data Analysis ……………………………….. 13

IV. Results …………………………………………….. 15

A. Descriptive Statistics ……………………… 15

B. Hypothesis Testing …………………………. 17

V. Discussion …………………………………………. 20

A. Interpretation of Findings ……………… 20

B. Implications for Practice ………………… 22

VI. Conclusion ……………………………………….. 25

A. Summary of Findings ……………………… 25

B. Contributions and Recommendations ….. 27

Graphic Table of Contents

This type of table of contents uses visual aids, such as icons or images, to represent the different sections or chapters of a document.

I. Introduction …………………………………………. [image of a light bulb]

II. Literature Review ………………………………… [image of a book]

III. Methodology ……………………………………… [image of a microscope]

IV. Results …………………………………………….. [image of a graph]

V. Discussion …………………………………………. [image of a conversation bubble]

Alphabetical Table of Contents

This type of table of contents lists the different topics or keywords in alphabetical order, along with their corresponding page numbers.

A. Abstract ……………………………………………… 1

B. Background …………………………………………. 3

C. Conclusion …………………………………………. 10

D. Data Analysis …………………………………….. 8

E. Ethics ……………………………………………….. 6

F. Findings ……………………………………………… 7

G. Introduction ……………………………………….. 1

H. Hypothesis ………………………………………….. 5

I. Literature Review ………………………………… 2

J. Methodology ……………………………………… 4

K. Limitations …………………………………………. 9

L. Results ………………………………………………… 7

M. Discussion …………………………………………. 10

Hierarchical Table of Contents

This type of table of contents displays the different levels of headings and subheadings in a hierarchical order, indicating the relative importance and relationship between the different sections.

    A. Background …………………………………….. 2

      B. Purpose of the Study ……………………….. 3

      A. Theoretical Framework …………………… 5

             1. Concept A ……………………………….. 6

                    a. Definition ………………………….. 6

                     b. Example ……………………………. 7

              2. Concept B ……………………………….. 8

       B. Previous Research ………………………….. 9

III. Methodology ……………………………………… 12

       A. Research Design ……………………………. 12

             1. Sample ……………………………………. 13

               2. Procedure ………………………………. 14

       B. Data Collection …………………………….. 15

            1. Instrumentation ……………………….. 16

            2. Validity and Reliability ………………. 17

       C. Data Analysis ……………………………….. 18

          1. Descriptive Statistics …………………… 19

           2. Inferential Statistics ………………….. 20

IV. Result s …………………………………………….. 22

    A. Overview of Findings ……………………… 22

B. Hypothesis Testing …………………………. 23

V. Discussion …………………………………………. 26

A. Interpretation of Findings ………………… 26

B. Implications for Practice ………………… 28

VI. Conclusion ……………………………………….. 31

A. Summary of Findings ……………………… 31

B. Contributions and Recommendations ….. 33

Table of Contents Format

Here’s an example format for a Table of Contents:

I. Introduction

C. Methodology

II. Background

A. Historical Context

B. Literature Review

III. Methodology

A. Research Design

B. Data Collection

C. Data Analysis

IV. Results

A. Descriptive Statistics

B. Inferential Statistics

C. Qualitative Findings

V. Discussion

A. Interpretation of Results

B. Implications for Practice

C. Limitations and Future Research

VI. Conclusion

A. Summary of Findings

B. Contributions to the Field

C. Final Remarks

VII. References

VIII. Appendices

Note : This is just an example format and can vary depending on the type of document or research paper you are writing.

When to use Table of Contents

A TOC can be particularly useful in the following cases:

• Lengthy documents : If the document is lengthy, with several sections and subsections, a Table of contents can help readers quickly navigate the document and find the relevant information.
• Complex documents: If the document is complex, with multiple topics or themes, a TOC can help readers understand the relationships between the different sections and how they are connected.
• Technical documents: If the document is technical, with a lot of jargon or specialized terminology, This can help readers understand the organization of the document and locate the information they need.
• Legal documents: If the document is a legal document, such as a contract or a legal brief, It helps readers quickly locate specific sections or provisions.

How to Make a Table of Contents

Here are the steps to create a table of contents:

• Organize your document: Before you start making a table of contents, organize your document into sections and subsections. Each section should have a clear and descriptive heading that summarizes the content.
• Add heading styles : Use the heading styles in your word processor to format the headings in your document. The heading styles are usually named Heading 1, Heading 2, Heading 3, and so on. Apply the appropriate heading style to each section heading in your document.
• Insert a table of contents: Once you’ve added headings to your document, you can insert a table of contents. In Microsoft Word, go to the References tab, click on Table of Contents, and choose a style from the list. The table of contents will be inserted into your document.
• Update the table of contents: If you make changes to your document, such as adding or deleting sections, you’ll need to update the table of contents. In Microsoft Word, right-click on the table of contents and select Update Field. Choose whether you want to update the page numbers or the entire table, and click OK.

Purpose of Table of Contents

A table of contents (TOC) serves several purposes, including:

• Marketing : It can be used as a marketing tool to entice readers to read a book or document. By highlighting the most interesting or compelling sections, a TOC can give readers a preview of what’s to come and encourage them to dive deeper into the content.
• Accessibility : A TOC can make a document or book more accessible to people with disabilities, such as those who use screen readers or other assistive technologies. By providing a clear and organized overview of the content, a TOC can help these readers navigate the material more easily.
• Collaboration : This can be used as a collaboration tool to help multiple authors or editors work together on a document or book. By providing a shared framework for organizing the content, a TOC can help ensure that everyone is on the same page and working towards the same goals.
• Reference : It can serve as a reference tool for readers who need to revisit specific sections of a document or book. By providing a clear overview of the content and organization, a TOC can help readers quickly locate the information they need, even if they don’t remember exactly where it was located.

About the author

Muhammad Hassan

Researcher, Academic Writer, Web developer

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6.4: Formal Report—Table of Contents and List of Figures

Learning objectives.

• Identify the role and format of a table of contents and list of figures

What Is a Table of Contents?

The table of contents shows readers what topics are covered in the report, how those topics are discussed (the subtopics), and on which page numbers those sections and subsections start.

In creating a  table of contents, you have a number of design decisions:

• Levels of headings to include: In longer reports, consider including only the top two levels of headings. This keeps the table of contents from becoming long and unwieldy. The table of contents should provide an at-a-glance way of finding information in the report quickly.
• Indentation, spacing, and capitalization: Notice in the illustration below that items in each of the levels of headings are aligned with each other. Although you can’t see it in the illustration, page numbers are right-aligned with each other.
• Vertical spacing: Notice that the first-level sections have extra space above and below, which increases readability.

Using the automatic table of contents creator in Word can help you produce a clean, professional document. Make sure the words in the table of contents are the same as they are in the text. As you write and revise, you might change some of the headings—don’t forget to change the table of contents accordingly.

Example: Table of Contents

What Is a List of Figures?

If your document has more than two figures or tables, create a separate list of figures. The list of figures has many of the same design considerations as the table of contents. Readers use the list of figures to quickly find the illustrations, diagrams, tables, and charts in your report.

Complications arise when you have both tables and figures. Strictly speaking, figures are illustrations, drawings, photographs, graphs, and charts. Tables are rows and columns of words and numbers; they are not considered figures.

For longer reports that contain dozens of figures and tables each, create separate lists of figures and tables. Put them together on the same page if they fit, as shown in the illustration below. You can combine the two lists under the heading, “List of Figures and Tables,” and identify the items as figure or table as is done in the illustration below.

Example: List of Figures

References & Attributions

Attributions

Content is adapted from  Technical Writing  by Allison Gross, Annemarie Hamlin, Billy Merck, Chris Rubio, Jodi Naas, Megan Savage, and Michele DeSilva, which is is licensed under a  Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License , except where otherwise noted.

Original content for this page was derived by Annemarie Hamlin, Chris Rubio, and Michele DeSilva, Central Oregon Community College from  Online Technical Writing  by David McMurrey –  CC: BY 4.0

Writing in a Technical Environment (First Edition) Copyright © 2022 by Centennial College is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License , except where otherwise noted.

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Table of Contents

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A guide to the table of contents page

Inhaltsverzeichnis

• 1 Definition: Table of Contents
• 3 Everything for Your Thesis
• 5 Create in Microsoft Word
• 6 In a Nutshell

Definition: Table of Contents

The table of contents is an organized listing of your document’s chapters, sections and, often, figures, clearly labelled by page number. Readers should be able to look at your table of contents page and understand immediately how your paper is organized, enabling them to skip to any relevant section or sub-section. The table of contents should list all front matter, main content and back matter, including the headings and page numbers of all chapters and the bibliography . A good table of contents should be easy to read, accurately formatted and completed last so that it is 100% accurate. Although you can complete a table of contents manually, many word processing tools like Microsoft Word enable you to format your table of contents automatically.

When adding the finishing touches to your dissertation, the table of contents is one of the most crucial elements. It helps the reader navigate (like a map) through your argument and topic points. Adding a table of contents is simple and it can be inserted easily after you have finished writing your paper. In this guide, we look at the do’s and don’ts of a table of contents; this will help you process and format your dissertation in a professional way.

When adding the finishing touches to your dissertation, the table of contents is one of the most crucial elements. It helps the reader navigate (like a map) through your argument and topic points. Adding a table of contents is simple and can be inserted easily after you have finished writing your paper. In this guide, we look at the do’s and don’ts of a table of contents; this will help you process and format your dissertation in a professional way.

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What is a table of contents?

A table of contents is a list, usually on a page at the beginning of a piece of academic writing , which outlines the chapters or sections names with their corresponding page numbers. In addition to chapter names, it includes bullet points of the sub-chapter headings or subsection headings. It usually comes right after the title page of a research paper.

How do you write a table of contents

To write a table of contents, you first write the title or chapter names of your research paper in chronological order. Secondly, you write the subheadings or subtitles, if you have them in your paper. After that, you write the page numbers for the corresponding headings and subheadings. You can also very easily set up a table of contents in Microsoft Word.

Where do you put a table of contents?

The table of contents is found on a page right at the beginning of an academic writing project. It comes specifically after the title page and acknowledgements, but before the introductory page of a writing project. This position at the beginning of an academic piece of writing is universal for all academic projects.

What to include in a table of contents?

A sample table of contents includes the title of the paper at the very top, followed by the chapter names and subtitles in chronological order. At the end of each line, is the page number of the corresponding headings. Examples of chapter names can be: executive summary,  introduction, project description, marketing plan, summary and conclusion. The abstract and acknowledgments are usually not included in the table of contents, however this could depend on the formatting that is required by your institution. Scroll down to see some examples.

How important is a table of contents?

A table of contents is very important at the beginning of a writing project for two important reasons. Firstly, it helps the reader easily locate contents of particular topics itemized as chapters or subtitles. Secondly, it helps the writer arrange their work and organize their thoughts so that important sections of an academic project are not left out. This has the extra effect of helping to manage the reader’s expectation of any academic essay or thesis right from the beginning.

Everything for Your Thesis

A table of contents is a crucial component of an academic thesis. Whether you’re completing a Bachelor’s or a postgraduate degree, the table of contents is a requirement for dissertation submissions. As a rule of thumb, your table of contents will usually come after your title page , abstract, acknowledgement or preface. Although it’s not necessary to include a reference to this front matter in your table of contents, different universities have different policies and guidelines.

Although the table of contents is best completed after you have finished your thesis, it’s a good idea to draw up a mock table of contents in the early stages of writing. This allows you to formulate a structure and think through your topic and how you are going to research, answer and make your argument. Think of this as a form of “reverse engineering”. Knowing how your chapters are going to be ordered and what topics or research questions are included in each will help immensely when it comes to your writing.

The table of contents is not just an academic formality, it allows your examiner to quickly get a feel for your topic and understand how your dissertation will be presented. An unclear or sloppy table of contents may even have an adverse effect on your grade because the dissertation is difficult to follow.

Examiners are readers, after all, and a dissertation is an exercise in producing an argument. A clear table of contents will give both a good impression and provide an accurate roadmap to make the examiner’s job easier and your argument more persuasive.

Your table of contents section will come after your acknowledgements and before your introduction. It includes a list of all your headers and their respective pages and will also contain a sub-section listing your tables, figures or illustrations (if you are using them). In general, your thesis can be ordered like this:

1. Title Page 2. Copyright / Statement of Originality 3. Abstract 4. Acknowledgement, Dedication and Preface (optional) 5. Table of Contents 6. List of Figures/Tables/Illustrations 7. Chapters 8. Appendices 9. Endnotes (depending on your formatting) 10. Bibliography / References

The formatting of your table of contents will depend on your academic field and thesis length. Some disciplines, like the sciences, have a methodical structure which includes recommended subheadings on methodology, data results, discussion and conclusion. Humanities subjects, on the other hand, are far more varied. Whichever discipline you are working in, you need to create an organized list of all chapters in their order of appearance, with chapter subheadings clearly labelled.

Sample table of contents for a short dissertation:

Abstract ………………………………………………………………………………………………….. ii Acknowledgements ………………………………………………………………………………………………….. iii Dedication ………………………………………………………………………………………………….. iv List of Tables ………………………………………………………………………………………………….. x List of Figures ………………………………………………………………………………………………….. xi Chapter 1: Introduction ………………………………………………………………………………………………….. 1 Chapter 2: Literature Survey ………………………………………………………………………………………………….. 13 Chapter 3: Methodology ………………………………………………………………………………………………….. 42 Chapter 4: Analysis ………………………………………………………………………………………………….. 100 Chapter 5: Conclusion ………………………………………………………………………………………………….. 129 Appendices ………………………………………………………………………………………………….. 169 References ………………………………………………………………………………………………….. 172

When producing a more significant and longer dissertation, say for a Master’s degree or even a PhD, your chapter descriptions should contain all subheadings. These are listed with the chapter number, followed by a decimal point and the subheading number.

Sample table of contents for a PhD dissertation:

Chapter 1 1.1 Introduction 1.2 Literature Review 1.3 Data 1.4 Findings 1.5 Conclusion

Chapter 2, and so on.

The key to writing a good table of contents is consistency and accuracy. You cannot list subheadings for one chapter and forget them for another. Subheadings are not always required but they can be very helpful if you are dealing with a detailed topic. The page numbers in the table of contents must match with the respective pages in your thesis or manuscript.

What’s more, chapter titles and subheading titles must match their corresponding pages. If your first chapter is called “Chapter 1: The Beginning”, it must be written as such on both the table of contents and first chapter page. So long as you remain both accurate and consistent, your table of contents will be perfect.

Create in Microsoft Word

Fortunately, the days of manually writing a contents page are over. You can still produce a contents page manually with Microsoft Word, but consider using their automatic feature to guarantee accuracy and save time.

To produce an automatically-generated table of contents, you must first work with heading styles. These can be found in the home tab under “Styles”. Select top-level headings (your chapter titles) and apply the Heading 1 style. This ensures that they will be formatted as main headings. Second-level headings (subheadings) can be applied with the Heading 2 style. This will place them underneath and within each main heading.

Once you have worked with heading styles, simply click on the “References” tab and select “Table of Contents”. This option will allow you to automatically produce a page with accurate page links to your document. To customize the format and style applied to your table of contents, select “Custom Table of Contents” at the bottom of the tab. Remember to update your table of contents by selecting the table and choosing “Update” from the drop-down menu. This will ensure that your headings, sub-headings and page numbers all add up.

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In a Nutshell

• The table of contents is a vital part of any academic thesis or extensive paper.
• It is an accurate map of your manuscript’s content – its headings, sub-headings and page numbers.
• It shows how you have divided your thesis into more manageable chunks through the use of chapters.
• By breaking apart your thesis into discrete sections, you make your argument both more persuasive and easier to follow.
• What’s more, your contents page should produce an accurate map of your thesis’ references, bibliography, illustrations and figures.
• It is an accurate map of the chapters, references, bibliography, illustrations and figures in your thesis.

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A Table of Contents in APA Format

Kendra Cherry, MS, is a psychosocial rehabilitation specialist, psychology educator, and author of the "Everything Psychology Book."

Adah Chung is a fact checker, writer, researcher, and occupational therapist. 

General Guidelines

• Table of Contents

APA style does not require a table of contents, but there are cases where you may need to include one. For example, your instructor may specify that your paper must be submitted with a table of contents. A table of contents can be particularly helpful in cases where your paper is lengthy or covers a lot of material, such as a thesis paper or dissertation. Research papers, in particular, may benefit from the addition of a table of contents.

APA style is the official publication style of the American Psychological Association. APA style is used in psychology courses as well as other social science classes including those in social science, behavioral sciences, and education.

The table of contents serves as a basic roadmap of your paper. It should list all of the major headings and subheadings within the body of your paper. For a standard psychology paper, it might include listings for the introduction, method, results, and discussion sections of your paper.

While the APA may not specify guidelines for a table of contents, you should use the basic APA format for formatting your table of contents:

• Use one-inch margins on all sides
• Use 12-point Times New Roman font
• Double-space

Since APA does not require a table of contents, you should always refer to your instructor’s guidelines when deciding whether or not to include one.

It is also important to note that the 7th edition of the Publication Manual of the American Psychological Association was published in 2020, and included updated guidelines on many topics.

For example, while the previous edition of the style manual required a running head on each page of a paper, the 7th edition has eliminated that requirement on student papers unless your instructor specifies to include it. Always ask first.

If you are using a standard APA paper format, your table of contents should include the following sections:

• Introduction

The above format may work well for a standard lab report or research paper. However, your table of contents will look much different if you are writing something such as a critique, essay, or case study.

Notice, that the table of contents does not include the abstract or acknowledgments pages. When applicable, it should list the appendices and the lists of tables and figures.

The exact order of your paper depends largely on the type of paper you are writing. In general, your paper should be presented in the following order:

• Main Body of Paper

Table of Contents Format

Because there is no standard format for a table of contents in APA style, you should always defer to the provided guidelines for your assignment.

If your instructor does not have a preferred format, consider using the following:

• Title the page “Table of Contents” and center the title at the top of the page.
• Most papers should include at least two levels of headings, up to five levels.
• Level one headings will be for main topics, such as chapter titles like "Chapter One; Name of Chapter," or research sections like "Method," "Results," and "Discussion."
• All level-one headings should be flush-left and sub-headings should be indented five spaces deeper than the last. 
• All heading levels should be in title case, capitalizing the first letter of each word. The font type, style, and size stay the same for each level.
• The page number for each heading is formatted flush-right. Include dot leaders between the headings and the page number to improve readability.

While you might not think that following APA format is important, it is one of those areas where students can lose points for making small errors. It pays to spend a little extra time and attention making sure that your paper is formatted in proper APA style.

• If you need help, you can get assistance from your school's writing lab.
• Getting your own copy of the latest edition of the APA publication manual can be very helpful.
• Always refer to any instructions or guidelines that were provided by your course instructor.
• There is a helpful feature in most word processors that you can use to pre-format your paper in APA style. It takes a little effort to set it up, but well worth it in the end, especially for longer documents. You can save the style to apply to your future papers saving you the effort next time.

For those writing a paper to submit for publication, check with the publisher for any specific formatting requirements that they may have.

American Psychological Association. Publication Manual of the American Psychological Association (7th ed.) ; 2020.

By Kendra Cherry, MSEd Kendra Cherry, MS, is a psychosocial rehabilitation specialist, psychology educator, and author of the "Everything Psychology Book."

10.4 Table of contents

You are familiar with tables of contents (TOC) but may never have stopped to look at their design. The TOC shows readers what topics are covered in the report, how those topics are discussed (the subtopics), and on which page numbers those sections and subsections start.

In creating a TOC, you have a number of design decisions:

• Levels of headings to include. In longer reports, consider not including only the top two levels of headings. This keeps the TOC from becoming long and unwieldy. The TOC should provide an at-a-glance way of finding information in the report quickly.
• Indentation, spacing, and capitalization. Notice in the illustration below that items in each of the three levels of headings are aligned with each other. Although you can’t see it in the illustration, page numbers are right-aligned with each other. Notice also the capitalization: Main chapters or sections are all caps; first-level headings use initial caps on each main word; lower-level sections use initial caps on the first word only.
• Vertical spacing. Notice that the first-level sections have extra space above and below, which increases readability.

Using the automatic TOC creator in your word processor can help you produce a clean, professional document. If you prefer to make your own, learn to use dot leader tabs in order to line up the page numbers correctly.

One final note: Make sure the words in the TOC are the same as they are in the text. As you write and revise, you might change some of the headings—don’t forget to change the TOC accordingly. See the example of a table of contents:

Chapter Attribution Information

This chapter was derived by Annemarie Hamlin, Chris Rubio, and Michele DeSilva, Central Oregon Community College, from  Online Technical Writing by David McMurrey – CC: BY 4.0

Technical Writing Copyright © 2017 by Allison Gross, Annemarie Hamlin, Billy Merck, Chris Rubio, Jodi Naas, Megan Savage, and Michele DeSilva is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License , except where otherwise noted.

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• Research Paper Appendix | Example & Templates

Research Paper Appendix | Example & Templates

Published on August 4, 2022 by Tegan George and Kirsten Dingemanse. Revised on July 18, 2023.

An appendix is a supplementary document that facilitates your reader’s understanding of your research but is not essential to your core argument. Appendices are a useful tool for providing additional information or clarification in a research paper , dissertation , or thesis without making your final product too long.

Appendices help you provide more background information and nuance about your thesis or dissertation topic without disrupting your text with too many tables and figures or other distracting elements.

We’ve prepared some examples and templates for you, for inclusions such as research protocols, survey questions, and interview transcripts. All are worthy additions to an appendix. You can download these in the format of your choice below.

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Table of contents

What is an appendix in a research paper, what to include in an appendix, how to format an appendix, how to refer to an appendix, where to put your appendices, other components to consider, appendix checklist, other interesting articles, frequently asked questions about appendices.

In the main body of your research paper, it’s important to provide clear and concise information that supports your argument and conclusions . However, after doing all that research, you’ll often find that you have a lot of other interesting information that you want to share with your reader.

While including it all in the body would make your paper too long and unwieldy, this is exactly what an appendix is for.

As a rule of thumb, any detailed information that is not immediately needed to make your point can go in an appendix. This helps to keep your main text focused but still allows you to include the information you want to include somewhere in your paper.

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An appendix can be used for different types of information, such as:

• Supplementary results : Research findings  are often presented in different ways, but they don’t all need to go in your paper. The results most relevant to your research question should always appear in the main text, while less significant results (such as detailed descriptions of your sample or supplemental analyses that do not help answer your main question), can be put in an appendix.
• Statistical analyses : If you conducted statistical tests using software like Stata or R, you may also want to include the outputs of your analysis in an appendix.
• Further information on surveys or interviews : Written materials or transcripts related to things such as surveys and interviews can also be placed in an appendix.

You can opt to have one long appendix, but separating components (like interview transcripts, supplementary results, or surveys ) into different appendices makes the information simpler to navigate.

Here are a few tips to keep in mind:

• Always start each appendix on a new page.
• Assign it both a number (or letter) and a clear title, such as “Appendix A. Interview transcripts.” This makes it easier for your reader to find the appendix, as well as for you to refer back to it in your main text.
• Number and title the individual elements within each appendix (e.g., “Transcripts”) to make it clear what you are referring to. Restart the numbering in each appendix at 1.

It is important that you refer to each of your appendices at least once in the main body of your paper. This can be done by mentioning the appendix and its number or letter, either in parentheses or within the main part of a sentence. It’s also possible to refer to a particular component of an appendix.

Appendix B presents the correspondence exchanged with the fitness boutique. Example 2. Referring to an appendix component These results (see Appendix 2, Table 1) show that …

It is common to capitalize “Appendix” when referring to a specific appendix, but it is not mandatory. The key is just to make sure that you are consistent throughout your entire paper, similarly to consistency in  capitalizing headings and titles in academic writing .

However, note that lowercase should always be used if you are referring to appendices in general. For instance, “The appendices to this paper include additional information about both the survey and the interviews .”

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The simplest option is to add your appendices after the main body of your text, after you finish citing your sources in the citation style of your choice. If this is what you choose to do, simply continue with the next page number. Another option is to put the appendices in a separate document that is delivered with your dissertation.

Remember that any appendices should be listed in your paper’s table of contents .

There are a few other supplementary components related to appendices that you may want to consider. These include:

• List of abbreviations : If you use a lot of abbreviations or field-specific symbols in your dissertation, it can be helpful to create a list of abbreviations .
• Glossary : If you utilize many specialized or technical terms, it can also be helpful to create a glossary .
• Tables, figures and other graphics : You may find you have too many tables, figures, and other graphics (such as charts and illustrations) to include in the main body of your dissertation. If this is the case, consider adding a figure and table list .

Checklist: Appendix

All appendices contain information that is relevant, but not essential, to the main text.

Each appendix starts on a new page.

I have given each appendix a number and clear title.

I have assigned any specific sub-components (e.g., tables and figures) their own numbers and titles.

My appendices are easy to follow and clearly formatted.

I have referred to each appendix at least once in the main text.

Your appendices look great! Use the other checklists to further improve your thesis.

If you want to know more about AI for academic writing, AI tools, or research bias, make sure to check out some of our other articles with explanations and examples or go directly to our tools!

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Yes, if relevant you can and should include APA in-text citations in your appendices . Use author-date citations as you do in the main text.

Any sources cited in your appendices should appear in your reference list . Do not create a separate reference list for your appendices.

An appendix contains information that supplements the reader’s understanding of your research but is not essential to it. For example:

• Interview transcripts
• Questionnaires
• Detailed descriptions of equipment

Something is only worth including as an appendix if you refer to information from it at some point in the text (e.g. quoting from an interview transcript). If you don’t, it should probably be removed.

When you include more than one appendix in an APA Style paper , they should be labeled “Appendix A,” “Appendix B,” and so on.

When you only include a single appendix, it is simply called “Appendix” and referred to as such in the main text.

Appendices in an APA Style paper appear right at the end, after the reference list and after your tables and figures if you’ve also included these at the end.

You may have seen both “appendices” or “appendixes” as pluralizations of “ appendix .” Either spelling can be used, but “appendices” is more common (including in APA Style ). Consistency is key here: make sure you use the same spelling throughout your paper.

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Table of Contents Format

For Academic Papers

This table of contents is an essential part of writing a long academic paper, especially theoretical papers.

This article is a part of the guide:

• Outline Examples
• Example of a Paper
• Write a Hypothesis
• Introduction

Browse Full Outline

• 1 Write a Research Paper
• 2 Writing a Paper
• 3.1 Write an Outline
• 3.2 Outline Examples
• 4.1 Thesis Statement
• 4.2 Write a Hypothesis
• 5.2 Abstract
• 5.3 Introduction
• 5.4 Methods
• 5.5 Results
• 5.6 Discussion
• 5.7 Conclusion
• 5.8 Bibliography
• 6.1 Table of Contents
• 6.2 Acknowledgements
• 6.3 Appendix
• 7.1 In Text Citations
• 7.2 Footnotes
• 7.3.1 Floating Blocks
• 7.4 Example of a Paper
• 7.5 Example of a Paper 2
• 7.6.1 Citations
• 7.7.1 Writing Style
• 7.7.2 Citations
• 8.1.1 Sham Peer Review
• 8.1.2 Advantages
• 8.1.3 Disadvantages
• 8.2 Publication Bias
• 8.3.1 Journal Rejection
• 9.1 Article Writing
• 9.2 Ideas for Topics

It is usually not present in shorter research articles, since most empirical papers have similar structure .

A well laid out table of contents allows readers to easily navigate your paper and find the information that they need. Making a table of contents used to be a very long and complicated process, but the vast majority of word-processing programs, such as Microsoft WordTM and Open Office , do all of the hard work for you.

This saves hours of painstaking labor looking through your paper and makes sure that you have picked up on every subsection. If you have been using an outline as a basis for the paper, then you have a head start and the work on the table of contents formatting is already half done.

Whilst going into the exact details of how to make a table of contents in the program lies outside the scope of this article, the Help section included with the word-processing programs gives a useful series of tutorials and trouble-shooting guides.

That said, there are a few easy tips that you can adopt to make the whole process a little easier.

The Importance of Headings

In the word processing programs, there is the option of automatically creating headings and subheadings, using heading 1, heading 2, heading 3 etc on the formatting bar. You should make sure that you get into the habit of doing this as you write the paper, instead of manually changing the font size or using the bold format.

Once you have done this, you can click a button, and the program will do everything for you, laying out the table of contents formatting automatically, based upon all of the headings and subheadings.

In Word, to insert a table of contents, first ensure that the cursor is where you want the table of contents to appear. Once you are happy with this, click 'Insert' on the drop down menu, scroll down to 'Reference,' and then across to 'Index and Tables'.

Click on the 'Table of Contents' tab and you are ready to click OK and go. OpenOffice is a very similar process but, after clicking 'Insert,' you follow 'Indexes and Tables' and 'Indexes and Tables' again.

The table of contents should appear after the title page and after the abstract and keywords, if you use them. As with all academic papers, there may be slight variations from department to department and even from supervisor to supervisor.

Check the preferred table of contents format before you start writing the paper , because changing things retrospectively can be a little more time consuming.

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• Dissertation Table of Contents in Word | Instructions & Examples

Dissertation Table of Contents in Word | Instructions & Examples

Published on 15 May 2022 by Tegan George .

The table of contents is where you list the chapters and major sections of your thesis, dissertation, or research paper, alongside their page numbers. A clear and well-formatted table of contents is essential, as it demonstrates to your reader that a quality paper will follow.

The table of contents (TOC) should be placed between the abstract and the introduction. The maximum length should be two pages. Depending on the nature of your thesis, dissertation, or paper, there are a few formatting options you can choose from.

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Table of contents

What to include in your table of contents, what not to include in your table of contents, creating a table of contents in microsoft word, table of contents examples, updating a table of contents in microsoft word, other lists in your thesis, dissertation, or research paper, frequently asked questions about the table of contents.

Depending on the length of your document, you can choose between a single-level, subdivided, or multi-level table of contents.

• A single-level table of contents only includes ‘level 1’ headings, or chapters. This is the simplest option, but it may be too broad for a long document like a dissertation.
• A subdivided table of contents includes chapters as well as ‘level 2’ headings, or sections. These show your reader what each chapter contains.
• A multi-level table of contents also further divides sections into ‘level 3’ headings. This option can get messy quickly, so proceed with caution. Remember your table of contents should not be longer than 2 pages. A multi-level table is often a good choice for a shorter document like a research paper.

Examples of level 1 headings are Introduction, Literature Review, Methodology, and Bibliography. Subsections of each of these would be level 2 headings, further describing the contents of each chapter or large section. Any further subsections would be level 3.

In these introductory sections, less is often more. As you decide which sections to include, narrow it down to only the most essential.

Including appendices and tables

You should include all appendices in your table of contents. Whether or not you include tables and figures depends largely on how many there are in your document.

If there are more than three figures and tables, you might consider listing them on a separate page. Otherwise, you can include each one in the table of contents.

• Theses and dissertations often have a separate list of figures and tables.
• Research papers generally don’t have a separate list of figures and tables.

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All level 1 and level 2 headings should be included in your table of contents, with level 3 headings used very sparingly.

The following things should never be included in a table of contents:

• Your acknowledgements page
• Your abstract
• The table of contents itself

The acknowledgements and abstract always precede the table of contents, so there’s no need to include them. This goes for any sections that precede the table of contents.

To automatically insert a table of contents in Microsoft Word, be sure to first apply the correct heading styles throughout the document, as shown below.

• Choose which headings are heading 1 and which are heading 2 (or 3!
• For example, if all level 1 headings should be Times New Roman, 12-point font, and bold, add this formatting to the first level 1 heading.
• Highlight the level 1 heading.
• Right-click the style that says ‘Heading 1’.
• Select ‘Update Heading 1 to Match Selection’.
• Allocate the formatting for each heading throughout your document by highlighting the heading in question and clicking the style you wish to apply.

Once that’s all set, follow these steps:

• Add a title to your table of contents. Be sure to check if your citation style or university has guidelines for this.
• Place your cursor where you would like your table of contents to go.
• In the ‘References’ section at the top, locate the Table of Contents group.
• Here, you can select which levels of headings you would like to include. You can also make manual adjustments to each level by clicking the Modify button.
• When you are ready to insert the table of contents, click ‘OK’ and it will be automatically generated, as shown below.

The key features of a table of contents are:

• Clear headings and subheadings
• Corresponding page numbers

Check with your educational institution to see if they have any specific formatting or design requirements.

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Write yourself a reminder to update your table of contents as one of your final tasks before submitting your dissertation or paper. It’s normal for your text to shift a bit as you input your final edits, and it’s crucial that your page numbers correspond correctly.

It’s easy to update your page numbers automatically in Microsoft Word. Simply right-click the table of contents and select ‘Update Field’. You can choose either to update page numbers only or to update all information in your table of contents.

In addition to a table of contents, you might also want to include a list of figures and tables, a list of abbreviations and a glossary in your thesis or dissertation. You can use the following guides to do so:

• List of figures and tables
• List of abbreviations

It is less common to include these lists in a research paper.

All level 1 and 2 headings should be included in your table of contents . That means the titles of your chapters and the main sections within them.

The contents should also include all appendices and the lists of tables and figures, if applicable, as well as your reference list .

Do not include the acknowledgements or abstract   in the table of contents.

To automatically insert a table of contents in Microsoft Word, follow these steps:

• Apply heading styles throughout the document.
• In the references section in the ribbon, locate the Table of Contents group.
• Click the arrow next to the Table of Contents icon and select Custom Table of Contents.
• Select which levels of headings you would like to include in the table of contents.

Make sure to update your table of contents if you move text or change headings. To update, simply right click and select Update Field.

The table of contents in a thesis or dissertation always goes between your abstract and your introduction.

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George, T. (2022, May 15). Dissertation Table of Contents in Word | Instructions & Examples. Scribbr. Retrieved 15 April 2024, from https://www.scribbr.co.uk/thesis-dissertation/contents-page/

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How do I format a table of contents in MLA style?

Note: This post relates to content in the eighth edition of the MLA Handbook . For up-to-date guidance, see the ninth edition of the MLA Handbook .

Tables of contents may be formatted in a number of ways. In our publications, we sometimes list chapter numbers before chapter titles and sometimes list the chapter titles alone. We also sometimes list section heads beneath the chapter titles. After each chapter or heading title, the page number on which the chapter or section begins is provided. The following show examples from three of the MLA’s books.

From Elizabeth Brookbank and H. Faye Christenberry’s  MLA Guide to Undergraduate Research in Literature  (Modern Language Association of America, 2019):

From  Approaches to Teaching Bechdel’s  Fun Home, edited by Judith Kegan Gardiner (Modern Language Association of America, 2018):

From the  MLA Handbook , 8th ed. (Modern Language Association of America, 2016):

Need more information? Read about where to place a table of contents in your paper .

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How to Write a Table of Contents

Last Updated: February 16, 2024 Fact Checked

This article was co-authored by Stephanie Wong Ken, MFA . Stephanie Wong Ken is a writer based in Canada. Stephanie's writing has appeared in Joyland, Catapult, Pithead Chapel, Cosmonaut's Avenue, and other publications. She holds an MFA in Fiction and Creative Writing from Portland State University. This article has been fact-checked, ensuring the accuracy of any cited facts and confirming the authority of its sources. This article has been viewed 1,043,520 times.

The Table of Contents in a document acts as a map for the reader, making it easier for them to find information in the document based on title and page number. A good Table of Contents should be organized, easy to read and simple to use. You can write a Table of Contents manually on your computer or have a word processing tool create it for you. Make sure the Table of Contents is formatted properly in your final document so it is as accurate and accessible as possible.

Sample Tables of Contents

Creating the Table of Contents on a Word Processor

• The Table of Contents should be on its own page. Do not include the introduction or a dedication on the same page as the Table of Contents.

• For example, you may write down main headings like, “Introduction,” “Case Study 1,” or “Conclusion.”

• For example, under the main heading “Introduction” you may write the subheading, “Themes and Concepts.” Or under the main heading “Conclusion” you may write, “Final Analysis.”
• You can also include sub-subheadings underneath the subheadings, if applicable. For example, under the subheading “Themes and Concepts” you may have the sub-subheading, “Identity.”
• Some papers do not have subheadings at all, only main headings. If this is the case, skip this step.

• For example, if the “Introduction” section begins on page 1, you will attach “page 1” to the Introduction heading. If the “Conclusion” section begins on page 45, attach “page 45” to the Conclusion heading.

• Check that the subheadings are located underneath the correct headings, indented to the right.
• Make sure there are page numbers for the subheadings listed as well.
• You can center the content in the table using the table options if you want the content to appear a few spaces away from the lines of the table. You can also leave the content indented to the left if you'd prefer.

• You can put the title above the table or in a separate row on the top of the rest of the content.

Using a Word Processing Tool

• You should also confirm the page numbers are correct in the document. Each page should be numbered in order. Having the correct page numbers will ensure the Table of Contents is created correctly when you use the word processing tool.

• If there are subheadings in your document, label them “Heading 2.” Highlight each subheading and click on “Heading 2” in the Styles tab.
• If there are sub-subheadings in your document, label them “Heading 3.” Highlight each subheading and click on “Heading 3” in the Styles tab.
• The text and font for each main heading may change based on the settings for “Heading 1,” “Heading 2,” and “Heading 3.” You can choose your preferred text and font for each main heading so they appear as you like in the Table of Contents.

• You can choose the built-in Table of Content options, where the tool will automatically choose a font size and style for you.
• You can also go for from a list of custom Table of Contents, where you choose the font color and size based on your preferences.

Polishing the Table of Contents

• You should also check the subheadings or sub-subheadings in the Table of Contents, if applicable, to ensure they match those in the document.

• If you created the Table of Contents manually, do this by going in and adjusting the headings and/or the page numbers when they change.
• If you created the Table of Contents with a word processing tool, update it by clicking the Update option by the Table of Contents option on the Reference tab. You can side clicking on the Table of Contents and choosing “update” that way.

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• ↑ https://edu.gcfglobal.org/en/word-tips/how-to-create-a-table-of-contents-in-word/1/#
• ↑ https://examples.yourdictionary.com/reference/examples/table-of-content-examples.html
• ↑ http://bitesizebio.com/21549/using-word-to-write-your-thesis-making-a-table-of-contents-inserting-captions-and-cross-referencing/
• ↑ https://guides.lib.umich.edu/c.php?g=283073&p=1886010
• ↑ https://nsufl.libguides.com/c.php?g=413851&p=2820026

About This Article

To write a table of contents, open a new document and list the major headings, titles, or chapters of the project in chronological order. Next, insert subheadings or subtopics if your project has those. Fill in the page number where each heading starts, then format the content in a table with 2 columns. Place the headings and subheadings in order in the first column, then put the page numbers in the second column. Don't forget to add a "Table of Contents" title at the top of the document! To learn more about polishing your Table of Contents, read on! Did this summary help you? Yes No

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• Writing Tips

How to Structure a Business Report

5-minute read

• 14th March 2019

The content of a business report will depend on what you are writing about. Even the writing style may depend on who you are writing for (although clear, concise and formal is usually best). However, there is a general structure that most business reports follow. In this post, then, we’ll look at how to structure a business report for maximum clarity and professionalism.

1. Title Page

Every business report should feature a title page . The title itself should clearly set out what the report is about. Typically, you should also include your name and the date of the report.

Most business reports begin with a summary of its key points. Try to include:

• A brief description of what the report is about
• How the report was completed (e.g., data collection methods)
• The main findings from the research
• Key conclusions and recommendations

A paragraph or two should suffice for this in shorter business reports. However, for longer or more complex reports, you may want to include a full executive summary .

3. Table of Contents

Short business reports may not need a table of contents, especially if they include a summary. But longer reports should set out the title of each section and the structure of the report. Make sure the headings here match those used in the main text. You may also want to number the sections.

4. Introduction

The introduction is the first part of the report proper. Use it to set out the brief you received when you were asked to compile the report. This will frame the rest of the report by providing:

• Background information (e.g., business history or market information)
• The purpose of the report (i.e., what you set out to achieve)
• Its scope (i.e., what the report will cover and what it will ignore)

These are known as the “terms of reference” for the business report.

5. Methods and Findings

If you are conducting original research, include a section about your methods. This may be as simple as setting out the sources you are using and why you chose them. But it could also include how you have collected and analyzed the data used to draw your conclusions.

After this, you will need to explain your findings. This section will present the results of your research clearly and concisely, making sure to cover all the main points set out in the brief.

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One tip here is to break the findings down into subsections, using headings to guide the reader through your data. Using charts and illustrations , meanwhile, can help get information across visually, but make sure to label them clearly so the reader knows how they relate to the text.

6. Conclusions and Recommendations

The last main section of your report will cover conclusions and recommendations. The conclusion section should summarize what you have learned from the report. If you have been asked to do so, you should also recommend potential courses of action based on your conclusions.

If you are not sure what to suggest here, think back to the objectives set out in your brief.

7. References

If you have used any third-party sources while writing your report, list them in a bibliography after the main report. This could include other business documents, academic articles, or even news reports. The key is to show what you have based your findings and conclusions upon.

8. Appendices (If Applicable)

Finally, you may have gathered extra documentation during your research, such as interview transcripts, marketing material, or financial data. Including this in the main report would make it too long and unfocused, but you can add it to an appendix (or multiple appendices) at the end of the document. It will then be available should your reader need it.

Summary: How to Structure a Business Report

If you are writing a business report, aim to structure it as follows:

• Title Page – Include a clear, informative title, your name, and the date.
• Summary – A brief summary of what the report is about, the data collection methods used, the findings of the report, and any recommendations you want to make.
• Table of Contents – For longer reports, include a table of contents.
• Introduction –Set out the brief you were given for the report.
• Methods and Findings – A description of any methods of data collection and analysis used while composing the report, as well as your findings.
• Conclusions and Recommendations – Any conclusions reached while writing the report, plus recommendations for what to do next (if required).
• References – Sources used in your report listed in a bibliography.
• Appendices – If you have supporting material (e.g., interview transcripts, raw data), add it to an appendix at the end of the document.

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Home » Research Methodology » Contents and Layout of Research Report

Contents and Layout of Research Report

Contents of  research  report.

The researcher must keep in mind that his research report must contain following aspects:

• Purpose of study
• Significance of his study or statement of the problem
• Review of literature
• Methodology
• Interpretation of data
• Conclusions and suggestions
• Bibliography

These can be discussed in detail as under:

(1) Purpose of study:

Research is one direction oriented study. He should discuss the problem of his study. He must give background of the problem. He must lay down his hypothesis of the study. Hypothesis is the statement indicating the nature of the problem. He should be able to collect data, analyze it and prove the hypothesis . The importance of the problem for the advancement of knowledge or removed of some evil may also be explained. He must use review of literature or the data from secondary source for explaining the statement of the problems.

(2) Significance of study:

Research is re-search and hence the researcher may highlight the earlier research in new manner or establish new theory. He must refer earlier research work and distinguish his own research from earlier work. He must explain how his research is different and how his research topic is different and how his research topic is important. In a statement of his problem, he must be able to explain in brief the historical account of the topic and way in which he can make and attempt. In his study to conduct the research on his topic.

(3) Review of Literature :

Research is a continuous process. He cannot avoid earlier research work. He must start with earlier work. He should note down all such research work, published in books, journals or unpublished thesis. He will get guidelines for his research from taking a review of literature . He should collect information in respect of earlier research work. He should enlist them in the given below:

• Author/researcher
• Title of research /Name of book
• Year of publication
• Objectives of his study
• Conclusion/suggestions

Then he can compare this information with his study to show separate identity of his study. He must be honest to point out similarities and differences of his study from earlier research work.

(4) Methodology:

It is related to collection of data. There are two sources for collecting data; primary and secondary. Primary data is original and collected in field work, either through questionnaire interviews. The secondary data relied on library work. Such primary data are collected by sampling method . The procedure for selecting the sample must be mentioned. The methodology must give various aspects of the problem that are studied for valid generalization about the phenomena. The scales of measurement must be explained along with different concepts used in the study.

While conducting a research based on field work, the procedural things like definition of universe, preparation of source list must be given. We use case study method , historical research etc. He must make it clear as to which method is used in his research work. When questionnaire is prepared, a copy of it must be given in appendix.

(5) Interpretation of data :

Mainly the data collected from primary source need to be interpreted in systematic manner. The tabulation must be completed to draw conclusions. All the questions are not useful for report writing . One has to select them or club them according to hypothesis or objectives of study .

(6) Conclusions/suggestions:

Data analysis forms the crux of the research problem . The information collected in field work is useful to draw conclusions of study. In relation with the objectives of study the analysis of data may lead the researcher to pin point his suggestions. This is the most important part of study. The conclusions must be based on logical and statistical reasoning. The report should contain not only the generalization of inference but also the basis on which the inferences are drawn. All sorts of proofs, numerical and logical, must be given in support of any theory that has been advanced. He should point out the limitations of his study.

(7) Bibliography:

The list of references must be arranged in alphabetical order and be presented in appendix. The books should be given in first section and articles are in second section and research projects in the third. The pattern of bibliography is considered convenient and satisfactory from the point of view of reader.

(8) Appendices:

The general information in tabular form which is not directly used in the analysis of data but which is useful to understand the background of study can be given in appendix.

Layout of the Research Report

There is scientific method for the layout of research report . The layout of research report means as to what the research report should contain. The contents of the research report are noted below:

• Preliminary Page

(1) Preliminary Pages:

These must be title of the research topic and data. There must be preface of foreword to the research work. It should be followed by table of contents. The list of tables, maps should be given.

(2) Main Text:

It provides the complete outline of research report along with all details. The title page is reported in the main text. Details of text are given continuously as divided in different chapters.

• (a)       Introduction
• (b)     Statement of the problem
• (c)   The analysis of data
• (d)     The implications drawn from the results
• (e)   The summary

(a)       Introduction :

Its purpose is to introduce the research topic to readers. It must cover statement of the research problem , hypotheses, objectives of study, review of literature, and the methodology to cover primary and secondary data, limitations of study and chapter scheme. Some may give in brief in the first chapter the introduction of the research project highlighting the importance of study. This is followed by research methodology in separate chapter.

The methodology should point out the method of study, the research design and method of data collection.

(b)     Statement of the problem :

This is crux of his research. It highlights main theme of his study. It must be in nontechnical language. It should be in simple manner so ordinary reader may follow it. The social research must be made available to common man. The research in agricultural problems must be easy for farmers to read it.

(c)       Analysis of data :

Data so collected should be presented in systematic manner and with its help, conclusions can be drawn. This helps to test the hypothesis . Data analysis must be made to confirm the objectives of the study.

(d)     Implications of Data :

The results based on the analysis of data must be valid. This is the main body of research. It contains statistical summaries and analysis of data. There should be logical sequence in the analysis of data. The primary data may lead to establish the results. He must have separate chapter on conclusions and recommendations. The conclusions must be based on data analysis. The conclusions must be such which may lead to generalization and its applicability in similar circumstances. The conditions of research work limiting its scope for generalization must be made clear by the researcher.

(e)       Summary :

This is conclusive part of study. It makes the reader to understand by reading summary the knowledge of the research work. This is also a synopsis of study.

(3) End Matter:

It covers relevant appendices covering general information, the concepts and bibliography. The index may also be added to the report.

Related Posts:

• Sources of Hypothesis in Research
• Referencing a Research Report
• Primary stages of research process
• Interpretation of Research Data
• Pre-Testing Research Data Collection Instruments
• Exploratory research and it's methods
• Significance and Problems of Social Research
• Descriptive research and it's methods
• The Role of Business Research
• Secondary Data Sources for Research

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Enhancement of the in-plane shear properties of carbon fiber composites containing carbon nanotube mats (English)

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Abstract The in-plane shear property of carbon fiber laminates is one of the most important structural features of aerospace and marine structures. Fiber-matrix debonding caused by in-plane shear loading is the major failure mode of carbon fiber composites because of the stress concentration at the interfaces. In this study, carbon nanotube mats (CNT mat) were incorporated in two different types of carbon fiber composites. For the case of woven fabric composites, mechanical interlocking between the CNTs and the carbon fibers increased resistance to shear failure. However, not much improvement was observed for the prepreg composites as a result of incorporation of the CNT mats. The reinforcement mechanism of the CNT mat layer was investigated by a fractographic study using scanning electron microscopy. In addition, the CNT mat was functionalized by three different methods and the effectiveness of the functionalization methods was determined and the most appropriate functionalization method for the CNT mat was air oxidation.

More details on this result

• Title: Enhancement of the in-plane shear properties of carbon fiber composites containing carbon nanotube mats
• Contributors: Kim, Hansang ( author )
• Published in: Metals and Materials International ; 21, 1 ; 185-193
• Publisher: The Korean Institute of Metals and Materials
• New search for: The Korean Institute of Metals and Materials
• Place of publication: Seoul
• Publication date: 2015-01-01
• Size: 9 pages
• DOI: https://doi.org/10.1007/s12540-015-1023-7
• Type of media: Article (Journal)
• Type of material: Electronic Resource
• Language: English
• Keywords: composites , carbon and graphite , nanostructured materials , mechanical properties , strength Material Science , Metallic Materials , Operating Procedures, Materials Treatment , Magnetism, Magnetic Materials , Engineering Thermodynamics, Heat and Mass Transfer , Characterization and Evaluation of Materials , Continuum Mechanics and Mechanics of Materials
• Source: Springer Verlag

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Volume 628 Issue 8007, 11 April 2024

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LETTER TO THE EDITOR

Successful control of morbihan disease with dapsone: a case report and literature review.

• 1 Department of Dermatology, Komaki City Hospital, Komaki, Aichi, Japan
• 2 Department of Dermatology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan

Dear Editors,

Morbihan disease is a persistent inflammatory condition characterized by chronic erythema, non-pitting edema, and swelling, primarily manifesting in the upper two-thirds of the face [ 1 ]. While there is mounting evidence suggesting links between Morbihan disease and conditions like rosacea and acne vulgaris, it is noteworthy that this condition can also manifest in individuals without any current or previous medical history. One particular point of contention revolves around the nomenclature of this condition, as Morbihan disease is an eponymous term and is often used interchangeably with solid facial edema. This discrepancy in naming adds to the challenges surrounding the understanding and classifying of this disease. Despite ongoing research and clinical observations, the available information remains limited. Although there are reports of treatment of Morbihan disease with isotretinoin, doxycycline, and minocycline, the treatment is sometimes insufficient and limited. Here, we report a 48-year-old Japanese woman with Morbihan disease that was successfully controlled with dapsone. As far as we searched, there were no other reports administrating dapsone for Morbihan disease other than us.

A 48-year-old Japanese woman presented to our department with complaints of swelling in her right upper eyelid that had persisted for 6 months, despite antihistamine drugs and topical therapy with hydrocortisone butyrate, tacrolimus, and delgocitinib, respectively. On examination, there was moderate edema with erythema confined to the right upper eyelid ( Figure 1A ). A skin biopsy showed the infiltration of lymphocytes and histiocytes around the perifollicular area in the middle dermis ( Figure 1B ). There was no neutrophil and giant cell infiltration commonly seen in rosacea. Immunohistochemical examination revealed the proliferation of mast cells and mucin deposition around the hair follicles by Alcian blue staining ( Figures 1C, D ). Blood tests showed no abnormalities. These findings presented a diagnostic dilemma due to the clinical similarity among Morbihan disease, cutaneous mucinosis, and rosacea as the principal differential diagnoses. However, the absence of the infiltration of neutrophil or giant cells, the localized perifollicular mucin deposition observed in Alcian blue staining and the restriction of lesions to the upper two-thirds of the face led to the conclusive diagnosis of Morbihan disease. Since antihistamines and topical therapy proved insufficient in improving the symptoms, we started doxycycline alone. However, the erythema of the right upper eyelid worsened after doxycycline therapy for 4 months ( Figure 1E ). Even after the additional administration of prednisolone (PSL) 10 mg daily, the erythema remained unchanged after 1 month. Therefore, we discontinued PSL. Since histopathology showed numerous infiltrations of mast cells, daily 75 mg of dapsone was administered to inhibit the mast cell activities such as the production of reactive oxygen species. Following this, the erythema and swelling of the right eyelid decreased after 4 months ( Figure 1F ). No adverse effects emerged with oral dapsone. In addition, the patient remained free of recurrence after discontinuing dapsone.

Figure 1 . Eyelids were edematous and erythematous at the initial visit (A) . Histopathology shows infiltration of lymphocytes and histiocytes around the perifollicular area in the middle dermis (HE) (B) . Alcian blue staining shows the mucin deposition around the hair follicles (red arrows) (C) . Alcian blue staining reveals the proliferation of mast cells in the dermis (arrowheads) (D) . The swelling of the right upper eyelid increased after four month-treatment with doxycycline (E) . The swelling and erythema of the eyelids decreased after 4 months of treatment with dapsone (F) .

Morbihan disease is a relatively rare disorder initially reported in 1957 by Robert Degos [ 1 ]. Seventy-four cases, including our present case, have been reported to date. These cases are comprehensively summarized in Supplementary Table S1 , providing a detailed overview of their respective characteristics ( Supplementary References S1–S54 ). There was a male predominance, with a male-to-female ratio of 41:27, although gender information for six cases was unavailable. The median age at the initial presentation was 54.5 years, emphasizing that Morbihan disease predominantly affects middle-aged individuals. Furthermore, the median duration between symptom onset and seeking medical attention was 6 months, underscoring potential delays in diagnosis or patient awareness of the condition. The facial distribution of symptoms was elucidated in 69 cases, with the eyelids including periorbital area being the most commonly affected site in 51 cases (73.9%), followed by the cheeks (11 cases, 15.9%), forehead (4 cases, 5.8%), and nose (3 cases, 4.3%). These findings are consistent with the characteristic localization of Morbihan disease. Treatment modalities such as isotretinoin and tetracycline exhibited efficacy in 20 and 18 cases, respectively, indicating their potential usefulness in managing Morbihan disease. Supplementary Table S2 summarizes the duration of treatment in previous case reports for isotretinoin, the most commonly used treatment for Morbihan disease, and for doxycycline. No reports mentioned using dapsone, a traditionally used agent in dermatology due to its anti-inflammatory and antioxidant properties [ 2 ]. Dapsone exhibits antimicrobial effects due to its sulfonamide-like capacity to inhibit the synthesis of dihydrofolic acid. Additionally, it possesses anti-inflammatory properties, including the inhibition of reactive oxygen species production, reduction of eosinophil peroxidase’s impact on mast cells, and downregulation of neutrophil-mediated inflammatory responses. These characteristics enable its application in the treatment of a diverse range of inflammatory and infectious skin conditions [ 3 ]. The consideration of dapsone introduces a potential new therapeutic avenue; however, caution is warranted due to its association with adverse effects such as dapsone hypersensitivity syndrome, and hematologic complications like blood cell reduction.

Although our case offers a new treatment option for Morbihan disease, certain limitations were encountered. Morbihan disease, being toponymic, may share similarities with conditions like solid facial edema, yet instances not explicitly reported as Morbihan disease might not be captured in our search methodologies. Moreover, Morbihan disease presents a diagnostic challenge due to its diverse differential diseases, with some cases lacking clear differentiation as in the present case. The primary differential diagnoses include rosacea and cutaneous mucinosis. Distinguishing between these conditions is particularly challenging. Morbihan disease is considered to present localized, often pronounced swelling than rosacea. Pathological examination of Morbihan disease may reveal mast cell infiltration, a feature that has also been reported in some cases of rosacea [ 4 ]. In addition, it may be useful to observe mucin deposition by alcian blue staining to distinguish cutaneous mucinosis.

Despite advancements, the pathophysiology of Morbihan disease remains elusive. Nevertheless, our findings suggest that dapsone could emerge as a viable therapeutic option, although further research is imperative to validate its efficacy and safety profile in treating this condition. This scarcity of conclusive data underscores its complexity, and further study is needed to better clarify the diagnostic criteria and recommend management strategies for this condition.

Data availability statement

The original contributions presented in the study are included in the article/ Supplementary Material , further inquiries can be directed to the corresponding author.

Ethics statement

Because this is a single case report, ethics approval was not required for this study. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the participant/patient(s) for the publication of this case report.

Author contributions

YA and KS collected the data, all participated in analyzing the results of the data and reviewing the manuscript, and YM, RF, and HK wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that no financial support was received for the research, authorship, and/or publication of this article.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontierspartnerships.org/articles/10.3389/jcia.2024.12849/full#supplementary-material

1. Degos, R, Civatte, J, and Beuve-Mery, M. Nouveau cas d’oedeme erythemateux facial chronique. Bull Soc Fr Dermatol Syph (1973) 80:257.

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3. Ghaoui, N, Hanna, E, Abbas, O, Kibbi, AG, and Kurban, M. Update on the use of dapsone in dermatology. Int J Dermatol (2020) 59:787–95. doi:10.1111/ijd.14761

4. Ramirez-Bellver, JL, Perez-Gonzales, YC, Chen, KR, Diaz-Recuero, JL, Requena, L, Carlson, JA, et al. Clinicopathological and immunohistochemical study of 14 cases of morbihan disease: an insight into its pathogenesis. Am J Dermatopathol (2019) 41:701–10. doi:10.1097/DAD.0000000000001378

Keywords: eyelids, diphenyl sulfone, mast cell, solid facial edema, rosacea

Citation: Morita Y, Koizumi H, Arisawa Y, Fukaura R and Sugawara K (2024) Successful control of Morbihan disease with dapsone: a case report and literature review. J. Cutan. Immunol. Allergy 7:12849. doi: 10.3389/jcia.2024.12849

Received: 15 February 2024; Accepted: 04 April 2024; Published: 16 April 2024.

Copyright © 2024 Morita, Koizumi, Arisawa, Fukaura and Sugawara. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Haruka Koizumi, [email protected]

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Published on 11.4.2024 in Vol 8 (2024)

Correction: Implementation Documentation and Process Assessment of the PharmNet Intervention: Observational Report

Authors of this article:

Corrigenda and Addenda

• Lori Ann Eldridge 1 , PhD   ; 
• Beth E Meyerson 2, 3 , PhD   ; 
• Jon Agley 4 , MPH, PhD  

1 Department of Health Education and Promotion, College of Health and Human Performance, East Carolina University, Greenville, NC, United States

2 Harm Reduction Research Lab, Family and Community Medicine, College of Medicine-Tucson, University of Arizona, Tucson, AZ, United States

3 Comprehensive Center for Pain and Addiction, University of Arizona Health Sciences, University of Arizona, Tucson, AZ, United States

4 Prevention Insights, Department of Applied Health Science, School of Public Health Bloomington, Indiana University Bloomington, Bloomington, IN, United States

Corresponding Author:

Jon Agley, MPH, PhD

Prevention Insights, Department of Applied Health Science

School of Public Health Bloomington

Indiana University Bloomington

809 E 9th Street

Bloomington, IN, 47405

United States

Phone: 1 812 855 3123

Email: [email protected]

Related Article Correction of: https://formative.jmir.org/2024/1/e54077 JMIR Form Res 2024;8:e59427 doi:10.2196/59427

In “Implementation Documentation and Process Assessment of the PharmNet Intervention: Observational Report” (JMIR Form Res 2024;8:e54077) two corrections were made.

The peer reviewer information has been updated. The first line in the publication information box has been changed from:

...peer-reviewed by D Carpenter...

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As one peer reviewer aptly noted, “the optimal means of naloxone community distribution remains to be determined.”

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As one peer reviewer (Steven H Linder MD) aptly noted, “the optimal means of naloxone community distribution remains to be determined.”

The correction will appear in the online version of the paper on the JMIR Publications website on April 11, 2024, together with the publication of this correction notice. Because this was made after submission to PubMed, PubMed Central, and other full-text repositories, the corrected article has also been resubmitted to those repositories.

This is a non–peer-reviewed article. submitted 11.04.24; accepted 11.04.24; published 11.04.24.

©Lori Ann Eldridge, Beth E Meyerson, Jon Agley. Originally published in JMIR Formative Research (https://formative.jmir.org), 11.04.2024.

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Original research article, short-term and long-term exposure to combined elevated temperature and co 2 leads to differential growth, toxicity, and fatty acid profiles in the harmful dinoflagellate karlodinium veneficum.

• 1 College of Earth, Ocean and Environment, School of Marine Science and Policy, University of Delaware, Lewes, DE, United States
• 2 Horn Point Laboratory, University of Maryland Center for Environmental Science, Cambridge, MD, United States
• 3 Department of Microbiology, The University of Tennessee, Knoxville, TN, United States
• 4 Department of Chemistry, Henson School of Science and Technology, Salisbury University, Salisbury, MD, United States

Ocean warming and acidification may significantly alter the distribution and intensity of harmful algal blooms as well as their effects on marine food webs. Estimating such effects rely, in part, on understanding the physiological response of individual algal species to controlled laboratory simulations of climate change conditions. Here we report the physiological response of the harmful dinoflagellate Karlodinium veneficum to the combined effects of elevated temperature and CO 2 (29°C/1000 ppm CO 2 ). We first examined these effects by comparing ambient control (25°C/441 ppm CO 2 ) and elevated conditions under short-term (~20 generations) growth. Next, we compared the short-term elevated condition to a longer-term (~200 generations) growth scenario under the same elevated temperature and CO 2 . Under the short-term elevated conditions, K. veneficum growth declined, cell toxicity increased, and saturated and mono-unsaturated fatty acid (FA) composition varied significantly from ambient conditions. Meanwhile, after ~ 200 generations of growth under elevated temperature and CO 2 , K. veneficum carbon assimilation, growth, and cell toxicity were significantly higher than the short-term elevated treatment. Further, while total saturated FA declined, essential fatty acids increased and likely represented an adaptive temporal response to long-term exposure to high temperature and CO 2 . Such shifts in FA profiles and cell toxicity may possibly alter K. veneficum nutritional quality as prey and its mixotrophic behavior, thereby affecting the energy and mass transfer through the marine food webs as well as bloom dynamics.

1 Introduction

With the continued release of greenhouse gases, the upper ocean has warmed by 0.11°C per decade from 1971–2010, and the surface ocean pH has decreased by 0.1 since the beginning of the industrial era ( IPCC, 2014 , 2021 ). It is virtually certain that ocean warming and acidification will continue beyond 2100 ( IPCC, 2014 , 2021 ). These changes directly and indirectly affect phytoplankton physiology and their role in several marine ecosystems. How climate change could affect phytoplankton that cause harmful algal blooms (HABs) is particularly concerning because of their negative effects on aquatic ecosystems, and their impacts to socio-economics, as well as human health and wellbeing.

Over the past several decades, there is mounting evidence that climate change has increased HAB intensity, frequency, and range ( Hallegraeff, 2010 ; Wells et al., 2015 ; Gobler et al., 2017 ; Trainer et al., 2019 ; Glibert, 2020 ; Gobler, 2020 ), and toxic dinoflagellate blooms are often projected to increase under future climate change scenarios ( Tatters et al., 2013a ; Glibert et al., 2014 ; Hattenrath-Lehmann et al., 2015 ; Gobler et al., 2017 ; Brandenburg et al., 2019 ; Glibert, 2020 ). However, dinoflagellate growth and cell toxicity do not always increase in response to ocean warming and/or acidification. For example, elevated CO 2 and temperature (750-1000 ppm and +4-5°C), led to increased growth and saxitoxin content in Alexandrium catenella ( Tatters et al., 2013a ), but did not affect the growth and/or the amount of brevetoxin in Karenia brevis ( Errera et al., 2014 ). In addition to species-specific differences, there may be considerable variation to warming and acidification among individual genotypes within a single species. For example, elevated CO 2 and temperature exposure led to strain-specific differences in growth in eight isolates of the dinoflagellate Alexandrium ostenfeldii , while total toxin production remained unchanged in most strains ( Kremp et al., 2012 ). Furthermore, toxicity responses to elevated CO 2 and temperature across dinoflagellate taxa may vary due to underlying metabolic pathways ( Brandenburg et al., 2019 ).

Ocean warming and acidification may significantly alter phytoplankton cell biochemistry, and in particular lipid composition. Algal fatty acids, especially omega-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA) such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) are germane since they are exclusively synthesized by phytoplankton and are predicted to decline in response to ocean warming ( Hixson and Arts, 2016 ) and acidification ( Meyers et al., 2019 ). Lipid profile adjustments are also taxon-specific ( Hixson and Arts, 2016 ) and do not always change under elevated CO 2 ( Wang et al., 2017 ). Mesozooplankton (e.g., copepod) growth and life history, such as fecundity, egg hatching, and naupliar survival, are critically dependent on algal-derived fatty acids, and n-3 fatty acids in particular. In addition, shifts in phytoplankton fatty acid content strongly affect not only organismal fitness, but also trophic energy transfer efficiency among phytoplankton and zooplankton ( Jónasdóttir et al., 2009 ; Cripps et al., 2016 ; McLaskey et al., 2019 ; Meyers et al., 2019 ).

Many experiments designed to test the effects of climate change to phytoplankton are short-term, typically lasting less than 15 algal generations (e.g. Fu et al., 2010 ; Errera et al., 2014 ; Hennon et al., 2017 ; Bercel and Kranz, 2019 ; Seto et al., 2019 ). However, some longer experiments have provided nuanced and unique insights into phytoplankton physiological acclimation and/or adaptation to future climate change that deviate from shorter duration experiments (e.g. Collins and Bell, 2004 ; Flores-Moya et al., 2012 ; Tatters et al., 2013b ). For example, after growing diatoms for 2 years under elevated temperature (30°C), essential fatty acids that significantly declined when shifted from 26°C to 30°C for only seven days partially recovered after the longer growth period ( Jin et al., 2020 ).

Karlodinium veneficum is a common coastal estuarine toxic dinoflagellate with a near-global distribution that produces a suite of toxic compounds called karlotoxins (KmTx) that have cytotoxic, ichthyotoxic, hemolytic, and grazer deterrent properties ( Deeds et al., 2002 ; Adolf et al., 2007 ; Waggett et al., 2008 ; Fu et al., 2010 ; Place et al., 2012 ). Karlodinium veneficum blooms often cause massive fish kills and are also toxic to oyster embryos, larvae, and juveniles ( Adolf et al., 2007 ; Glibert et al., 2007 ; Brownlee et al., 2008 ; Stoecker et al., 2008 ; Waggett et al., 2008 ). As a mixotroph, K. veneficum bloom initiation is affected not only by the availability of nutrients but also by prey ( Adolf et al., 2008 ; Li et al., 2015 ; Lin et al., 2018 ). Temperature, light intensity, and dissolved inorganic carbon availability also affect growth ( Adolf et al., 2007 , 2009 ; Fu et al., 2010 ). Karlodinium veneficum growth is often greater at an intermediate temperature (e.g., 25°C) than at slightly warmer temperatures (e.g., 28–30°C; Lin et al., 2018 ; Vidyarathna et al., 2020 , 2023 ), and is also resilient to low pH (7.8–6.0; Nielsen et al., 2010 ), and increases at high p CO 2 (e.g., 745 ppm) ( Fu et al., 2010 ). In addition, high temperature (i.e. ≥ 28°C) or high p CO 2 can increase K. veneficum cell toxicity ( Fu et al., 2010 ; Vidyarathna et al., 2020 ), but these factors were treated in isolation and under short-term exposure. In this regard, it is unclear how the combined effects of elevated temperature and CO 2 may affect K. veneficum physiology and long-term acclimation or adaptation.

Here, we tested the physiological response of a mid-Atlantic strain of Karlodinium veneficum to short- and long-term growth under combined warming and acidification. Our central objective was to compare growth, photosynthesis, cellular biochemistry, and toxicity after short-term (~20 generations, 40 days) and long-term exposure (~200 generations, 380 days) to projected future high temperature and CO 2 conditions. Long-term treatments resulted in greater growth, toxicity and essential fatty acids than in short-term treatments thereby providing potential implications for both Karlodinium prey and its predators.

2 Materials and methods

2.1 general experimental design.

Short-term experiments were conducted at two temperatures and CO 2 concentrations: ‘ambient’ conditions were set as 25°C and 400 ppm p CO 2 , while the ‘treatment’ conditions were designed to simulate warming and acidification conditions projected for the end of the 21st century (baseline RCP scenarios, IPCC, 2014 ) and set to 29°C and 1000 ppm p CO 2 . Cultures were acclimated to short-term ambient and treatment conditions for at least 20 generations in steady state before examining the short-term response (abbreviated as ST hereafter) to elevated temperature and CO 2 . The treatment cultures were then further acclimated for ~200 generations to measure if the long-term (abbreviated as LT hereafter) response to elevated temperature and CO 2 differed from the ST response to these same conditions.

2.2 Phytoplankton cultures and acclimation to experimental conditions

Karlodinium veneficum , originally collected from the Delaware Inland Bays, Delaware, USA (CCMP2936; National Center for Marine Algae and Microbiota, Bigelow, USA), was initially grown in replicate (n=4) non-axenic batch cultures in modified f/2 media (320 µM NO 3 - and 20 µM PO 4 3- ; Guillard and Ryther, 1962 ) at 25°C under a light intensity of 100 µmol photons m -2 s -1 provided by cool white fluorescent bulbs set to a 12:12 hour light:dark cycle. Media seawater was collected from the Indian River Inlet, DE, diluted with laboratory-grade fresh water to a salinity of 20, and the total alkalinity was adjusted to ~2200 µmol kg -1 by adding NaHCO 3 . This TA adjustment was done to avoid disrupting the carbonate chemistry of the stock cultures and to bring the carbonate chemistry parameters back to the baseline level since the dilution reduced the DIC and TA significantly (~1000 and ~800 umol kg- 1 for TA and DIC respectively). Water was then filtered (0.2µm, Whatman Polycap 75 TC) and autoclaved. Algal cultures were slowly acclimated to aeration by bubbling filtered (0.2 µm) air through fine glass frits (Prism Research Glass Inc., USA), and were kept in log-phase growth by weekly inoculations into fresh f/2 media.

Cultures were then transferred to 2L Pyrex bottles (Corning) and maintained in temperature- and pH-controlled cyclostats, containing filter-sterilized media (n=4, 25°C, 400 ppm p CO 2 ) under the same light conditions as described above (see MacIntyre and Cullen (2005) for detailed description of continuous algal culturing). Cultures were sampled every two days to monitor cell growth by cell number and in vivo chlorophyll a (chl a ) fluorescence. The media inflow rate was adjusted to match the intrinsic growth rate of acclimated cultures (based on cell number) until cultures reached steady state (~3 weeks) and was held constant through the rest of the experimental period. Steady-state conditions were confirmed when cell density and in vivo chl a fluorescence no longer changed during the acclimation phase and for several days after inflow rate adjustments stopped. Once cultures were fully acclimated to the ambient conditions, they were grown for a further ~18 generations (40 days) in pH-controlled continuous culture.

Treatment batch cultures were initially acclimated to 29°C by ramping the temperature at a rate of 0.5°C day -1 in temperature-controlled incubators (Percival Scientific), and were held for ~6 generations prior to transferring to pH-controlled cyclostats. For elevated temperature conditions, cyclostat bottles were kept in a clear acrylic water jacket connected to a recirculating water bath set to 29°C. The p CO 2 in each bottle was then slowly raised to ~1000 ppm at a rate of 100 ppm day -1 . Cell growth was regularly monitored after the p CO 2 ramping period, and the media inflow rate was adjusted to match the intrinsic growth rate as explained above for ambient cultures. To limit biofouling, cultures were transferred to new sterile bottles every three weeks. Short-term (ST) treatment conditions lasted for ~22 generations (58 days) after which cultures were sampled for physiological analyses. The remaining cultures were further acclimated to treatment conditions for an additional ~180 generations. After ~200 generations at steady state (381 days total), treatment cultures were considered acclimated to long-term (LT) conditions and were sampled again for physiological analyses. All routine samples (i.e., prior to terminating an experiment) for biomass estimation and carbonate chemistry parameter analyses were withdrawn directly from the cultures using a sample tube attached to the culture vessel.

2.3 Carbonate chemistry control and measurement

CO 2 levels (~400 and 1000 ppm) were controlled with a pH-stat system (Qubit Systems Inc, Canada), with pH levels set to 8.2 and 7.9 for ambient and treatment conditions respectively. The pH in each culture bottle was continually recorded and logged in computer-controlled software (Logger Pro) that controlled a 3-way solenoid delivering either CO 2 (certified 2%, Tech Gas Inc, USA) or CO 2 -free air passed through a soda lime column. pH electrodes were cleaned and re-calibrated with NBS buffer standards (pH 4.01 and 9.18, Orion™) every 7-14 days. The pH stability and accuracy of the system was confirmed by independent measurements of culture outflow with a laboratory grade pH system (Fisher Scientific A815 Plus pH meter equipped with Fisherbrand™ accumet™ pH sensor).

The total dissolved inorganic carbon (DIC) of each container was analyzed using an in-house custom DIC analyzer ( Friedrich et al., 2002 ). Water samples were acidified, followed by gas stripping and delivery to a non-dispersive infrared gas analyzer (LI-Cor) for total CO 2 measurement. Media and sample total alkalinity (TA) were measured using a bromocresol purple colorimetric assay ( Yao and Byrne, 1998 ) with a spectrometer (Ocean Optics, USB4000-ES) and a titrator (Metrohm 876 Dosimat plus, Switzerland). All DIC and TA measurements were compared to certified reference materials (Dickson laboratory, San Diego, USA), and seawater carbonate chemistry was calculated with the CO2SYS software ( Pierrot et al., 2006 ) based on DIC and pH (NBS) ( Table 1 ).

Table 1 Seawater carbonate chemistry parameters for ambient and treatment conditions.

2.4 Cell growth and chlorophyll a

Cell growth was monitored every two days by measuring in vivo chl a fluorescence (10 AU; Turner Designs, USA) and active chl a fluorescence (fast repetition rate fluorometry, Chelsea Instruments, UK), and periodic cell counts confirmed estimates by in vivo fluorescence. Cell density was calculated by counting glutaraldehyde-preserved (final concentration 1% v/v) sub samples (1 mL) with a Neubauer hemocytometer and a light microscope (100x magnification). Chl a concentration was determined according to Welschmeyer (1994) , with acetone extracts measured on a fluorometer (10 AU; Turner Designs, USA). Once cultures reached steady-state, cell biomass was monitored several times each week by in vivo chl a fluorescence. Cell specific growth rate (µ, day -1 ) was calculated according to Brading et al. (2011) , using the following formula:

where t = time (days), Δ t = time interval between sampling, C = in vivo chl a fluorescence, V o l = culture volume in the vessel at the time of sampling, V o l w a s t e = outflow volume (mL) generated during the time interval between sampling and V o l v e s s e l = the maximum volume of medium (mL) in the vessel.

2.5 Cell photochemistry

Cells were dark acclimated for 15 min and algal photochemistry was monitored by active chl a fluorescence by fast repetition rate fluorometry (FRRf) with a FASTtracka II fluorometer with a FASTact light and temperature control assembly (Chelsea Instruments, UK) connected to a constant temperature water bath set to the growth temperature. Fluorescence induction was recorded by applying a single-turnover fluorescence protocol that consisted of 100 1µs flashlets at ∼1-μs intervals provided by a bank of blue LEDs (peak excitation 450 nm, 200 µs total induction time), followed by 50 flashlets spaced at increasing intervals to allow for PSII reaction center re-oxidation. Measurements were recorded from the average of 15–30 acquisitions at 100 ms intervals. Photochemical activity in the light activated state was then assessed by the same fluorescence induction protocol after exposing cells to white light for 3 min that was provided by a bank of LEDs set to 104 µmol photons m -2 s -1 . Fluorescence data were fit in FASTpro software (v3.0, Chelsea Instruments) and used to calculate the maximum quantum yield of photosystem II ( F v /F m ) and the quantum yield of photosystem II in the light activated state ( F q ’/F m ’ ) ( Cosgrove and Borowitzka, 2010 ).

2.6 Photosynthetic carbon assimilation

Subsamples were removed from each replicate culture (5 mL) and combined with 12.5 µL of 14 C sodium bicarbonate (specific activity 0.1 µCi mL -1 MP Biomedicals) and incubated in a temperature-controlled aluminum block set to 25°C or 29°C for 30 min. Light in each well was set to 200 µmol photons m -2 s -1 and was provided by a cool white LED under each sample well. Preliminary measurements confirmed that photosynthesis in all samples was saturated at this light intensity (average E k ~ 160 µmol photons m -2 s -1 ). Dark controls were run in duplicate. Incubations were stopped by adding 1% glutaraldehyde (final concentration) to each vial, and residual inorganic C was removed by adding hydrochloric acid (1.2 M final concentration), followed by shaking samples overnight. After adding 5 mL scintillation cocktail (Ultima Gold, Perkin Elmer), radioactivity was measured in a liquid scintillation counter (Beckman LS-6500). Carbon assimilation in the light was corrected for any dark incorporation and expressed as the assimilation rate normalized to cell number (cell h -1 ). As samples were not filtered after 14 C assimilation, presented values include both particulate and dissolved production.

2.7 Particulate organic carbon, nitrogen and phosphorus

Samples (25-50 mL) for particulate organic carbon (POC), nitrogen (PON) and phosphorus (POP) were filtered onto 25 mm pre-combusted glass-fiber filters (2h at 450°C, GF/C, Whatman), dried at 60°C for 24 h, and then stored in a desiccator prior to analyses. Cellular C and N were quantified with a CHN elemental analyzer (ECS 4010 Elemental combustion system; Costech Instruments, USA), with phenylalanine and EDTA used as standards. POP was measured following the method of Solórzano and Sharp (1980) using Phosphate standard (RICCA, 1000 ppm PO 4 3- ).

2.8 Carbohydrates and soluble proteins

Twenty-five to fifty milliliter samples were centrifuged (4,800 x g) and cell pellets were stored at -20°C until further analyses. Carbohydrate content was determined by the phenol-sulphuric acid method using D-(+)-glucose as a standard ( DuBois et al., 1951 ) after acid hydrolyzing the cell pellets in 1M H 2 SO 4 at 100°C for 1 h. Proteins were extracted by bead beating (BioSpec) cell pellets with 0.5 mm glass beads for 60 s in 500 µL of filtered sea water (FSW, nominal pore 0.2 µm) followed by centrifugation (3,380 x g, 5 min). Soluble protein was determined with a linearized Bradford assay ( Ernst and Zor, 2010 ). All absorbance measurements were recorded using a plate reader (FLUOstar Omega, BMG Labtech).

2.9 Fatty acids

Fifty milliliter samples were centrifuged (4,800 x g) and the pellets were freeze-dried (Virtis, 12SL) and stored in a desiccator. Prior to lipid extraction, samples were spiked with 20 µL of C13:0ME internal standard (10 mg mL -1 , Sigma Aldrich, St. Louis, MO). Acid-catalyzed transesterification of lipids to fatty acid methyl esters (FAMEs) and the extraction of FAMEs into hexane was performed as in Van Wychen et al. (2013) . FAME extracts were stored at -20°C until analysis. Supelco 37 Component FAME mix (Sigma Aldrich, St. Louis, MO) was spiked with the 5 µL of the same C13:0ME internal standard as above and were used as standards (total fatty acid concentration ranging from 0.01 to 0.5 mg mL -1 ). FAMEs were analyzed by gas chromatography (GC) on a Hewlett Packard HP 7890B Series (Palo Alto, CA) equipped with a flame ionization detection and a Zebron ZB-1 wax column (30m x 250µm x 25µm, Phenomenex, Torrance, CA). FAMEs were separated using splitless injection (injection volume =3 µL, inlet temperature = 250°C). An initial column heating to 100°C was maintained for 1 min followed by ramping at 25°C min -1 up to 170°C, then 2°C min -1 up to 200°C, which was held for 1 min prior to ramping at 18°C min -1 up to 250°C, which was held for 8 min. FAMEs were identified based on their retention times, and peaks were compared to the standard curve in Open LAB CDS GC software (Agilent Technologies). Fatty acid recovery was corrected for the recovery of the internal standard, and FAME concentration normalized to cell number was then converted to the corresponding fatty acid concentration using individual Sheppard factors ( Sheppard, 1992 ).

2.10 Cellular toxicity

Fifty milliliter samples were centrifuged (4,800 x g) at 4°C (Sorval RC-5B, USA), and cell pellets were extracted in 1–2 mL of methanol for 24 h in the dark at -20°C. Cell toxicity was assessed by the hemolytic assay according to Eschbach et al. (2001) and with a Rainbow Trout (RT) fish gill cell (FGC) assay according to Dayeh et al. (2005) with modifications ( Vidyarathna et al., 2020 ) and was normalized to total cellular carbon.

2.11 Data analyses

Statistical analyses were performed using R (v 4.1.1; R Core Team 2021 ). The data were checked for normality and homogeneity of the variance by Shapiro-Wilk and Bartlett tests respectively. Treatment effects (increased temperature and CO 2 ) were tested between ambient and ST cultures and the effect of acclimation time to increased temperature and CO 2 (short term and long term) between ST and LT cultures were analyzed separately using one-way analysis of variance (ANOVA). Results were considered significant at α = 0.05.

3.1 Cell growth, carbon assimilation, photophysiology and chlorophyll a

The ambient cell growth rate was 0.32 ± 0.02 day -1 , while the treatment growth significantly decreased in the short-term (ST) cultures to 0.27 ± 0.01 (ANOVA, p=0.006, on day 50); however, after ~200 generations, growth in the long-term (LT) treatment increased to 0.36 ± 0.01 day -1 (ANOVA, p=0.0004 compared to ST cultures, Figure 1 ). Carbon assimilation on the other hand increased in response to both treatment effect (ANOVA, p= 0.03 between ambient and ST cultures) and to acclimation time (ANOVA, p= 0.03 between ST and LT cultures) ( Figure 2 ). In contrast, ambient K. veneficum cultures had a higher PSII maximum quantum yield ( Fv/Fm ), than the ST cultures (ANOVA, p= 0.0004, Figure 3A ). Of the two treatments, Fv/Fm was significantly higher in the LT cultures than in the ST cultures (ANOVA, p=0.01). There was less variability in the effective quantum yield of PSII ( F q ’/F m ’ ) in the light activated state ( Figure 3B ), as F q ’/F m ’ was significantly lower in the ST cultures than in the ambient and LT cultures (ANOVA, p=0.02 and 0.005, respectively). Overall, Fv/Fm and Fq’/Fm’ ranged between 0.44-0.48 and 0.39-0.41 respectively, and no warming or acidification induced stress on K. veneficum photochemistry was evident. Cellular chl a remained unchanged between ambient and ST cultures (ANOVA, p=0.16) and increased significantly in LT cultures compared to ST cultures (ANOVA, p<0.001, Figure 3C ).

Figure 1 Steady-state growth rates (µ, day -1 ) of K. veneficum acclimated to Ambient (Amb.) and treatment conditions (short term: ST and long term: LT). Error bars denote the standard deviation of 40, 76 and 84 replicates for Amb., ST and LT cultures, respectively. Letters above the bars indicate significant differences between ambient and ST cultures (treatment effects) while asterisks indicate significant differences between ST and LT cultures (acclimation time effect) (ANOVA, p < 0.05).

Figure 2 Carbon assimilation rates of K. veneficum (µg C Cell -1 , h -1 ) acclimated to ambient (Amb.) and treatment conditions (short term: ST and long term: LT). Error bars denote the standard deviation of 4 replicates (n=4) for Amb and ST cultures and 3 replicates (n=3) for LT cultures. Letters above the bars indicate significant differences between ambient and ST cultures (treatment effects) while asterisks indicate significant differences between ST and LT cultures (acclimation time effect) (ANOVA, p < 0.05). Carbon assimilation rates per chlorophyll a (µg C Chl a -1 , h -1 ) and cellular carbon (µg C C -1 , h -1 ) yielded the same results (data not shown).

Figure 3 Maximum photochemical efficiency of photosystem II ( Fv/Fm ) (A) , effective light acclimated photochemical efficiency of PSII ( F q ′/F m ′ ) (B) and cellular chlorophyll- a content (pg Chl- a cell -1 ), (C) of K . veneficum acclimated to ambient (Amb) and treatment conditions (short term: ST and long term: LT). Error bars denote the standard deviations: Amb, n=4; ST n=36 and LT, n=21 panels (A, B) and Amb, n=4; ST, n=4; LT, n=3 (panel (C) . Letters above the bars indicate significant differences between ambient and ST cultures (treatment effects) while asterisks indicate significant differences between ST and LT cultures (acclimation time effect) (ANOVA, p < 0.05).

3.2 Elemental composition and cellular carbohydrates and proteins

Cell quotas for C and P (POC and POP) were not affected by treatment (ANOVA, p= 0.08 and 0.73 for POC and POP respectively) or acclimation time (ANOVA, p= 0.40 and 0.81 for POC and POP respectively) and remained unchanged across ambient, ST, and LT cultures ( Table 2 ). In contrast, N cell quota (PON), was significantly higher in ST cultures than in ambient cultures (ANOVA, p= 0.01), but remained unchanged between ST and LT cultures (ANOVA, p= 0.49). Meanwhile, the atomic N:P and C:P ratios remained similar across ambient and ST cultures (ANOVA, p=0.57 and 0.81 for N:P and C:P ratios respectively) and ST and LT cultures (ANOVA, p=0.94 and 0.15 for N:P and C:P ratios respectively, Table 2 ), while the C:N ratio was significantly lower in ST cultures than the ambient and LT cultures (ANOVA, p=0.004 and p=0.002, respectively, Table 2 ).

Table 2 Cell densities, cellular carbon, nitrogen, phosphorus, elemental ratios, and cellular carbohydrate and soluble protein of K. veneficum acclimated to ambient, treatment-ST and treatment-LT conditions.

Cellular carbohydrates were significantly higher in ST cultures than in ambient cultures (ANOVA, p=0.01, Table 2 ), but decreased slightly in the LT cultures in comparison to the ST cultures (ANOVA, p=0.2). ST culture cellular protein was also significantly higher than both ambient (ANOVA, p=0.0007) and LT cultures (ANOVA, p=0.002, Table 2 ).

3.3 Cellular fatty acid composition

Total fatty acid content remained similar across each culture condition and ranged between 21-33 pg cell -1 (ANOVA, p=0.09 and 0.99 for ambient vs. ST and ST vs. LT comparisons respectively, Figure 4A ). In contrast, fatty acid composition changed considerably in response to (short-term treatment and time ( Figures 4B, C ). The percentage of saturated fatty acids (SFA) was higher in the ST cultures than the ambient cultures (ANOVA, p=0.0007), but this increase was not sustained through time under the treatment conditions, as SFAs decreased significantly in the LT cultures (ANOVA, p<0.0001, Figure 4B ). In contrast, the percentage of monounsaturated fatty acids (MUFA) in the ST cultures was significantly lower than the ambient cultures (ANOVA, p<0.0001), while the LT culture MUFAs were significantly higher than the ST cultures (ANOVA, p<0.0001, Figure 4B ).

Figure 4 Total fatty acid contents (pg cell -1 ) (A) , and percent composition of saturated fatty acids (SFA), mono-unsaturated fatty acids (MUFA) (B) , and polyunsaturated fatty acids (PUFA) (C) of K . veneficum acclimated to ambient (Amb) and treatment conditions (short term: ST and long term: LT). Error bars denote the standard deviation of 4 replicates (n=4) for Amb and ST cultures and 3 replicates (n=3) for LT cultures. Letters above the bars indicate significant differences between ambient and ST cultures (treatment effects) while asterisks indicate significant differences between ST and LT cultures (acclimation time effect) (ANOVA, p < 0.05). n3 FA, omega-3 fatty acids; EPA, Eicosapentaenoic acid; DHA, Docosahexaenoic acid.

Polyunsaturated fatty acids (PUFA) accounted for only 2.4–8.6% of total FA content and were similar between ambient and ST (ANOVA, p=0.61), and ST and LT culture conditions (ANOVA, p=0.89, Figure 4C ). However, the PUFA composition changed substantially in the LT cultures. In particular, the percentage of n3 fatty acids (n3 FA), a subgroup of PUFA, was significantly greater than those recorded in the ST cultures (ANOVA, p=0.0003, Figure 4C ). Notably, both major essential fatty acids EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) were significantly greater in LT cultures than in the ST cultures (ANOVA, p<0.0001, Figure 4C ). We also observed an increase in the percentage of very long chain fatty acids over time (VLCFA, i.e. FA with carbon chains >20) from ~6% in ST cultures to approximately 15% in the LT cultures ( Supplementary Table 1 ).

Twenty-seven fatty acids were identified ( Supplementary Table 1 ), and the two most abundant SFA in K. veneficum were palmitic acid (16:0) and myristic acid (14:0), which together represented 66%, 76% and 45% of the total FA content at ambient, ST and LT cultures, respectively. Oleic and elaidic acids (18:1 cis+trans) together represented the major MUFA in ambient cultures, while eicosenoic acid (20:1) was the dominant MUFA in the treatment cultures. Karlodinium veneficum n3 PUFA included alpha-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), and docosahexaenoic acid (DHA, 22:6n-3). In addition, eicosatrienoic acid (ETE, 20:3n-3) was also detected in the LT cultures.

3.4 Cell toxicity

Hemolytic activity normalized to either cell number or carbon remained unchanged between the ambient and ST treatment and when comparing ST vs LT conditions (ANOVA, p>0.5, Figure 5 ); however, toxicity against the fish gill cell (FGC) line increased (i.e., EC 50 decreased) significantly in both ST and LT cultures ( Figure 5 ). Specifically, the FGC toxicity in the ST cultures was 2.5 times greater than the ambient cultures (ANOVA, p=0.001), while FGC toxicity was 2.6 times greater in the LT cultures than in the ST cultures (ANOVA, p=0.005). Likewise, when normalized to cellular carbon (EC50: µg C mL -1 ), FGC toxicity followed a similar pattern and increased significantly from ambient to ST (ANOVA, p=0.0001) and ST to LT (ANOVA, p= 0.01) culture conditions.

Figure 5 Hemolytic activity (pg pg C -1 - Left Y axes) and the rainbow trout fish gill cell toxicity (FGC; EC 50 : µg Carbon mL -1 - Right Y axes) of K. veneficum acclimated to ambient (Amb) and treatment (short term: ST and long term: LT) conditions. Error bars denote the standard deviation of four replicates (n=4) for Amb and ST cultures and three replicates (n=3) for LT cultures. Letters above the bars indicate significant differences between ambient and ST cultures (treatment effects) while asterisks indicate significant differences between ST and LT cultures (acclimation time effect) (ANOVA, p < 0.05).

4 Discussion

Acclimating K. veneficum to long term (LT, ~200 generations) combined elevated temperature and CO 2 increased carbon assimilation, growth, and FGC toxicity. The long-term treatment also resulted in major changes in key fatty acid classes, as the percent of monounsaturated (MUFA) and essential fatty acids (n3 FA) increased, while saturated fatty acids (SFA) decreased. However, some of these differences were not evident or changed in opposite ways when K. veneficum was acclimated to short-term (ST, ~20 generations) elevated temperature and CO 2 . For example, ST growth and % MUFA were lower and % SFA were higher than the ambient cultures, while carbon fixation remained similar in both conditions. In contrast, other cellular parameters, such as FGC toxicity, were consistently higher in ST cultures than in ambient conditions. These different results between the ST and LT experiments suggest that complete acclimation to elevated temperature and p CO 2 required >20 generations.

4.1 The effects of combined elevated temperature and CO 2 on K. veneficum growth and toxicity

While our experimental design did not allow us to test the independent effects of elevated temperature and CO 2 , K. veneficum and some other dinoflagellates often grow faster under elevated CO 2 ( Fu et al., 2010 ; Errera et al., 2014 ) but grow slower under elevated temperature ( Seto et al., 2019 ; Vidyarathna et al., 2020 , 2023 ). However, pre-acclimation to heating prior to changing CO 2 can lead to different outcomes, as Coyne et al. (2021) noted higher growth after acclimating K. veneficum to 30°C for over one year prior to applying short-term high CO 2 (24 days/750 ppm CO 2 ). Excess CO 2 may provide this K veneficum isolate some relief from heating, whereby elevated CO 2 reduces the energetic demand for acquiring and fixing carbon at higher temperatures. We hypothesize that lower growth rates in the ST cultures were likely caused by a stronger negative effect of temperature than high CO 2 . This may be common in many short-term experiments (typically < ~20 generations) and explains similar growth responses in this and other dinoflagellates when comparing ‘combined’ and ‘warming only’ treatments in similar experimental designs ( Feng et al., 2008 ; Fu et al., 2008 ; Seto et al., 2019 ). Likewise, our previous work has noted that the thermal optima for growth in this strain of K. veneficum is closer to 25°C ( Vidyarathna et al., 2020 ). While carbon fixation remained similar between the ambient and ST treatment, the lower photochemical performance in the ST treatment ( Figures 3A, B ) that was not apparent in the LT treatment suggests that some aspect of photosynthetic electron transport in the light reactions may have been the rate limiting step in the ST acclimation. Although not evaluated in our study, the possible shifts in bacterial consortia [e.g. Alteromonas ( Deng et al., 2022 )] associated with K. veneficum cultures may also have contributed to promoting algal growth under elevated temperature and CO 2 as has been reported for other photosynthetic organisms such as Prochlorococcus ( Hennon et al., 2018 ).

While growth was higher in the LT treatment, carbon assimilation still exceeded the growth rate while elemental composition remained unchanged. Such decoupling between photosynthesis and growth may signal a mechanism to remove excess photosynthate as dissolved organic carbon, perhaps as transparent exopolymeric particles ( Engel et al., 2014 ; Passow and Laws, 2015 ), when electron transport and carbon fixation are high ( Claquin et al., 2008 ). In contrast, when exposed to long term high CO 2 (~900 generations), the diatom Phaeodactylum tricornutum reduced photosynthesis and respiration, thereby allocating a similar amount of carbon for growth as the low CO 2 selected population with no change in growth rates ( Jin et al., 2022 ). Similar to our LT treatments, phytoplankton elemental composition, including in other dinoflagellates, does not necessarily change in response to elevated CO 2 ( Burkhardt et al., 1999 ; Clark et al., 2014 ; Wynn-Edwards et al., 2014 ; Sugie and Yoshimura, 2016 ; Bercel and Kranz, 2019 ; Xu et al., 2023 ), but intracellular distributions can still change, as Chl a content approximately doubled in our LT treatments. In contrast, nitrogen, protein, and carbohydrate quotas increased in the ST cultures and this excess stored carbon may reflect a more immediate physiological response to elevated temperature and CO 2 .

The greater K. veneficum FGC toxicity under elevated temperature and CO 2 noted between ambient and ST cultures as well as ST and LT cultures is similar (~ 52%) to increased FGC toxicity in this same isolate in response to elevated temperature alone, which peaked at 25°C–28°C ( Vidyarathna et al., 2020 ). Hence, the increased FGC toxicity in our ST treatment was possibly due more to elevated temperature than CO 2 . Meanwhile, the even greater increase in FGC toxicity in the LT cultures (57% higher than the ST cultures) perhaps represented further cellular adjustments to carbon allocation in response to the combined effects of elevated temperature and CO 2 .

In contrast to FGC toxicity, hemolytic toxicity remained similar across ambient and the ST treatment cultures and also did not change in the LT treatment. Notably, elevated temperature alone led to increased hemolytic toxicity in late-exponential phase batch cultures of K. veneficum , and batch grown K. veneficum hemolytic toxicity cell -1 at 25°C was 4x higher ( Vidyarathna et al., 2020 ) than in the continuous cultures reported here. Further, although K. veneficum hemolytic activity increased with elevated CO 2 and P-limitation ( Fu et al., 2010 ), the absolute toxicity cell -1 was lower than data from batch cultures [> ~50 ng cell -1 vs. 95 ng cell -1 in Vidyarathna et al. (2020) ]. While we cannot discount possible synergestic effects of elevated temperature and CO 2 , K. veneficum hemolytic activity may be affected more by nutrient limitation and starvation.

4.2 Temporal changes in K veneficum lipids under elevated temperature and CO 2 and the possible implications for trophic transfer and mixotrophy

Phytoplankton often change their fatty acid (FA) composition to maintain membrane homeoviscocity in response to changing temperature ( Sinensky, 1974 ). Increasing saturated fatty acid content at higher temperature helps to maintain membrane rigidity ( Fuschino et al., 2011 ; Baker et al., 2018 ; Hixson and Arts, 2016 ), which we also noted in our ST treatment. However, not all shifts in FA content in our ST treatment were similar to previous studies where the % PUFA often declines at higher temperature ( Fuschino et al., 2011 ; Hixson and Arts, 2016 ; Torstensson et al., 2013 ; Hyun et al., 2016 ) but remained unchanged here. Further, while FA composition (% SFA and MUFA) did not change in phytoplankton within mesocoms exposed to elevated temperature and CO 2 for 20 days ( Meyers et al., 2022 ), we found a significant decrease in the % MUFA in our ST treatment. Hence, K. veneficum FA reaction norms may change rapidly in response to both elevated temperature and CO 2 but are likely species- and strain- dependent ( Jin et al., 2020 ) and not necessarily reflective of the larger phytoplankton community.

In contrast to the ST cultures, the significant increase in growth and decline in total saturated FA in the LT cultures may have been an adaptive response; however, our experimental design did not have a long-term ambient control to directly compare against the LT treatment. Furthermore, this also prevented us from explicitly testing for trait adaptation by performing reciprocal shifts between ambient and treatment conditions (i.e., comparing high temperature and CO 2 treated algae against the ambient control samples after shifting them into the high temperature and CO 2 and vice versa) ( Schlüter et al., 2014 ). Nevertheless, the decline in saturated FA is similar to that noted in the diatom Thalassiosira pseudonana , where saturated FA were lower after 500 generations at 31°C, providing evidence for some thermal adaptation ( O’Donnell et al., 2019 ). Importantly, the measured response traits in long-term experiments can return to pre-exposure levels or fall below those of the original non-adapted population ( Schlüter et al., 2016 ; Collins et al., 2020 ). For example, initial FA and lipids declined in three diatom species and then fully recovered after two years of long-term growth at 30°C ( Jin et al., 2020 ). However, the FA profile shift in our LT cultures was a significant re-arrangement in lipid composition that included a larger proportion of essential FA and very long chain FA (VLCFA) not seen in the ST cultures. Furthermore, n-3 PUFA and n-6 PUFA increased in LT cultures ( Figure 4C ; Supplementary Table 1 ) and contrasts with the suggestion that global heating alone could favor phytoplankton n-6 PUFA over n-3 PUFA ( Hixson and Arts, 2016 ). Hence, the increased FA elongation and desaturation in K. veneficum in response to long-term exposure to elevated temperature and CO 2 may represent a unique interactive effect of these combined climate stressors.

Altered algal fatty acid (FA) profiles due to climate change has important ecological implications for marine food webs, as primary consumers acquire most FA through phytoplankton grazing. While total FA content remained unchanged, elevated temperature and CO 2 led to major changes in key K. veneficum FA classes. Studies with other algal species have reported a broad range of FA variability from little to no change (e.g. Fitzer et al., 2019 ; O’Donnell et al., 2019 ; Meyers et al., 2022 ) to a significant decline in response to elevated temperature and/or CO 2 ( Rossoll et al., 2012 ; Hyun et al., 2016 ; Jacob et al., 2017 ). Increased essential fatty acids, including EPA, DHA, in the LT treatment holds particular ecological relevance as they are exclusively synthesized by phytoplankton and affect both consumer growth and fitness. Declining phytoplankton n3 FA in response to elevated temperature potentially weakens energy and mass transfer through the marine food web ( Fuschino et al., 2011 ; Rossoll et al., 2012 ; Fitzer et al., 2019 ; Meyers et al., 2019 ) where a temperature increase of 2.5°C may reduce phytoplankton EPA by 8.2% and DHA by 27.8% ( Hixson and Arts, 2016 ). In contrast, we found a consistent increase in the percentage of n3-PUFA when moving from ambient to ST and ST to LT treatments. Additionally, since the total FA cell -1 did not change, the increased n3-PUFA may have resulted from desaturating pre-existing fatty acids–a process that requires reducing power from NADPH or NADH ( Sato et al., 2003 ). Here, n3-PUFA increased proportionally with photosynthetic C fixation across ambient and treatment cultures ( Figure 4 ), hence excess energy not used for growth may have been used to support desaturation of SFA to n3-FA.

Altered biochemical composition of algal prey can affect the growth, reproduction, survival, and biochemical stoichiometry of grazers such as copepods and bivalves ( Rossoll et al., 2012 ; Cripps et al., 2016 ; Fields et al., 2022 ; Pan et al., 2023 ), and leads to cascading effects to fitness at higher trophic levels. For example, Cripps et al. (2016) estimated that at elevated CO 2 , carbon trophic transfer efficiency from phytoplankton to copepods declined to <50% of the control population. However, other studies have reported greater fitness in the copepod, Calanus finmarchicus when raised at elevated CO 2 ( Fields et al., 2022 ) and copepod biomass (particularly in smaller size classes) can increase at elevated CO 2 , likely due to indirect effects through increased food availability (e.g. Taucher et al., 2017 ). In contrast, others have reported that ocean acidification alone or, in combination with elevated temperature, did not affect prey or zooplankton FA composition ( Meyers et al., 2022 ). While these responses are results of a complex interplay of trophic interactions, short-term acclimation vs long-term adaptation as well as differences in sensitivities to elevated temperature and/or CO 2 among different taxa may explain some of this variability ( Lohbeck et al., 2012 ; Tatters et al., 2013b ; Bermúdez et al., 2015 ; Jin et al., 2020 ; Xu et al., 2023 ). Among the order Gymnodiniales, Karlodinium species are globally prevalent ( Le Bescot et al., 2016 ), and K. veneficum is a common constituent in coastal mid-Atlantic waters such as the Chesapeake Bay ( Zhang et al., 2008 ). Further, given that K. veneficum is consumed by micrograzers, including other heterotrophic dinoflagellates in this region ( Johnson et al., 2003 ) as well as the copepod Acartia tonsa ( Hong et al., 2012 ), possible shifts in algal biochemical composition under future warming and acidification as shown here could affect such trophic interactions.

In addition to autotrophy, mixotrophic feeding by K. veneficum can significantly increase bloom formation ( Li A. et al., 2001 ; Place et al., 2012 ; Li et al., 2015 ). While not included in our experimental design, mixotrophy could play an important role in some climate change scenarios, especially in regard to elevated temperature, which can significantly increase prey ingestion and digestion ( Li A. et al., 2001 ; Wilken et al., 2013 ; Li et al, 2022 ). Other factors related to bloom progression, such as nutrient starvation, can negatively affect algal response to pH-induced stress, and are important to consider in future studies, as mixotrophic phytoplankton may be favored under some ocean acidification scenarios ( Flynn et al., 2015 ). Taken together our results suggest that elevated CO 2 and temperature can enhance carbon assimilation and increase K. veneficum autotrophic growth and toxicity, which could further influence bloom formation and severity. However, long-term exposure to these conditions also resulted in notable shifts in greater proportions of essential fatty acids that might counteract the negative effects of elevated cell toxicity to grazers. Such interactions, as well as possible shifts in mixotrophic behavior and subsequent changes to algal toxicity warrant further investigation in more complex future climate change simulations.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement

Ethical approval was not required for the studies on animals in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.

Author contributions

NV: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing. LS: Investigation, Writing – review & editing. KM: Investigation, Resources, Writing – review & editing. KC: Conceptualization, Funding acquisition, Resources, Writing – review & editing. JC: Conceptualization, Funding acquisition, Resources, Supervision, Writing – review & editing. MW: Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing.

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was supported by the National Oceanic and Atmospheric Administration (NOAA) through grant NA15NOS4780182.

Acknowledgments

We thank the reviewers for their valuable comments. We thank Dr. Patricia Glibert (Horn point laboratory-UMCES) for valuable comments on the manuscript. This is contribution number ECO1091 from the NOAA ECOHAB program.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmars.2024.1305495/full#supplementary-material

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Keywords: ocean acidification, climate change, harmful algae, Karlodinium veneficum , algal toxicity, fatty acids, trophic transfer

Citation: Vidyarathna NK, Smith LE, Miller KR, Coyne KJ, Cohen JH and Warner ME (2024) Short-term and long-term exposure to combined elevated temperature and CO 2 leads to differential growth, toxicity, and fatty acid profiles in the harmful dinoflagellate Karlodinium veneficum . Front. Mar. Sci. 11:1305495. doi: 10.3389/fmars.2024.1305495

Received: 01 October 2023; Accepted: 26 March 2024; Published: 09 April 2024.

Reviewed by:

Copyright © 2024 Vidyarathna, Smith, Miller, Coyne, Cohen and Warner. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Mark E. Warner, [email protected]

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