PRACTICALS ONE & TWO
FACTORS AFFECTING THE DISTRIBUTION OF PROTOZOA AND NEMATODES IN THE INTESTINE OF THE COCKROACH PERIPLANETA AMERICANA.
Aims
This practical exercise is designed to:
1. Examine the distribution of protozoa found in the cockroach intestine.
2. Examine the distribution of nematodes found in the cockroach small intestine.
3. Determine the effect of dietary antibiotics on the burden of protozoa and nematodes.
Transferable Skills
This practical is designed to cover the following transferable skills:
1. To work in a small group to carry out a scientific experiment.
2. To analyse and interpret scientific information.
3. To present scientific data in a clear, concise manner using computer-based software packages.
4. To discuss the relevance of experimental data with reference to that published in the scientific literature.
5. To follow the instructions contained in the module handbook, especially those relating to the manner in which the practical report is presented.
6. To manage your time effectively and hand work in by the deadline date.
Specific Skills
1. To observe and identify nematode and protozoan parasites.
2. To perform a fine dissection.
Learning Outcomes
This practical is designed to cover the following module learning outcomes
LO1. Discuss the nature of host-parasite relationships and explain how parasites interact with other pathogens
INTRODUCTION
Cockroaches, like most metazoan animals, are hosts to a wide range of protozoan and nematode animals. The numbers and distribution of these parasites within their host is affected by many factors such as the age and gender of the host, the host’s diet, and intraspecific and interspecific competition between the parasites themselves. Not all of the animals found in the gastrointestinal tract are true parasites. For example, some may be commensals that feed on the bacteria present in the gut lumen.
Experimental Animals
You will be provided with adult male and female cockroaches, Periplaneta americana, that have been recently and humanely killed with ethyl acetate.
You should each dissect at least two cockroaches in each practical session. If you do not dissect a cockroach yourself, your partner must be prepared to dissect it for you.
Dissection
1) Determine the sex of the cockroach using the guide provided.
2) Cut off the cockroach’s wings and head and pin it ventral side down in the wax dissecting dish.
3) Flood the dish with water and dissect out the intestinal tract according to the instructions in the dissection guide.
4) Using a fine pair of scissors, cut the intestinal tract into the oesophagus, crop, gastric caecae, mesenteron, ileum, colon, and rectum regions.
NOTE: You must work FAST otherwise the parasites may have died before you are able to look at them.
Identification of Protozoa and Nematodes
1) Carefully dissect each region of the gut on a separate glass microscope slide so as to expel all the gut contents. Add a drop of insect saline and then a cover slip. The crop and other large regions may need to be divided, because they are too big to fit onto one slide.
2) Examine each slide using a high power microscope – x20 or x40 – and identify the various parasites from the illustrations provided.
3) Count the numbers of each parasite.
4) Enter your data onto the computer spreadsheet.
Determination of the effect of antibiotics on the distribution of protozoan and nematode parasites.
1) In the first week you were provided with cockroaches that had been maintained on a normal diet. You must now compare the data you obtained with that from cockroaches that received a diet supplemented with antibiotics.
2) Dissect the cockroaches and analyse their gut contents as described previously.
PRACTICAL WRITE UP
Write up your report individually using class data.
Write your report in the style of a scientific paper according to the instructions.
Write the introduction and materials and methods sections in your own words. Do not copy verbatim from this handbook. Use your own title and headings throughout. There are better ways of expressing things than the way it has been done in this handbook.
Submit your work via Blackboard
IMPORTANT
Do not restrict your introduction / discussion to nematode / protozoan parasites of cockroaches. You will find very few scientific papers that cover this topic.
The real questions are:
a) Does parasite abundance and diversity differ between regions of the digestive tract and if so, how and why? Are the patterns that you have observed similar to those of other animals?
b) Is there any evidence of parasite species interactions? Do you think that the parasites might be competing or co-existing? For example, if species A is abundant, is species B absent or low in numbers? Or if species A is abundant, is species B equally abundant? Do similar interactions (or lack of interactions) occur in other parasite species and has anyone suggested any reason(s) for this.
c) Is there any evidence of antibiotics affecting parasite numbers / diversity? If so, why might this be the case? Is it a direct or indirect affect? Is there any evidence of antibiotics affecting these factors in other animals?
PREPARATION OF A SCIENTIFIC PAPER
Scientific results are of little importance unless they can be conveyed in a format easily interpreted by other scientists. In the course of this module you will be asked to prepare your work in two of the formats routinely used by the scientific community for the dissemination of information: the verbal presentation, which is the focus of the problem-based learning and seminar sessions, and, most usefully, the written presentation of a scientific paper.
To ensure that you understand the principles governing the layout and presentation of these papers we are asking you to prepare your report on this practical in paper format.
By this stage in your career you should be familiar with finding and assimilating information from scientific journals. The vast majority of journals are published at monthly or quarterly intervals and contain the best of the current research within their subject area e.g. vector biology, molecular parasitology, germanic philosophy etc. For a scientist to have his/her work considered for publication, he/she must first prepare a submission copy of the work in strict accordance with that journal’s ‘Instructions to Authors’. On submission to the journal, the editor will then send copies of the manuscript to two anonymous referees who are experts in the field. It is on advice from the referees that the work will be accepted for publication or rejected by the journal. Often, the referees will ask the authors to alter the script to answer specific questions or perform further studies.
Each journal will decree its own format and if this is not strictly followed the paper will be rejected before even being sent to the referees. To give you practice in the production of a submission-ready manuscript, we are asking you to follow the ‘Instructions to Authors’.
1) Use published papers as models of the formats that can be used to present data.
2) You should treat all the class data as replicates, just as if you had obtained all the data yourself.
3) No raw data or basic calculations should be presented.
4) Only data that has undergone the appropriate statistical analysis should be considered.
5) Present your worked data in the clearest appropriate format – either tabular or graphic.
6) You will need to make extensive use of the literature to introduce your topic and discuss your results.
LJMU Journal of Parasitology
Instructions to Authors
Preparation of Manuscripts
Manuscripts should be typed 1.5x-spaced throughout (excluding references and figure legends). Spelling should conform to the Oxford English Dictionary.
Pages should be numbered and manuscripts should be arranged in the following order: Title page, Abstract, Introduction, Materials and Methods, Results, Discussion, Acknowledgements, References.
Title Page
The title and author’s name and address (cite the School and University) should appear on a separate title page. The title should NOT be the same as that used in the module handbook.
Abstract
The Abstract, of approximately three quarters of a page, should state the objectives of the study and the methods used and should summarise the results and conclusions. Abbreviations and references are not allowed.
Prepare your abstract using the following sub-headings: Each subheading should consist of one or two short paragraphs.
Background: Provide a bit of background information and state why the work was undertaken.
Methodology: Briefly explain how the work was undertaken. You do not have to provide fine detail.
Principal Findings: Summarise the most important findings. You do not have to list all of the results.
Conclusions: What can one conclude from the findings?
Significance: State what the conclusions mean.
As an example of how this looks in practice see Galvan-Moroyoqui, J.M. et al., (2008)
The interplay between Entamoeba and enteropathogenic bacteria modulates epithelial cell damage. PLoS Neglected Tropical Diseases 2(7): e266. doi:10.1371/journal.pntd.0000266
The easiest way to find this paper is to go to Google Scholar and enter the author’s name and Entamoeba
Introduction
The Introduction should set the study in context by briefly reviewing relevant knowledge on the subject. This should be followed by a concise statement of the objectives of the study.
Materials and Methods
The full binomial Latin name should be given for all animals.
Sufficient information should be provided so other workers can repeat the study. Do not copy out material from the book verbatim.
Statistical Analysis
Authors should give sufficient details of the experimental design and analysis so that readers can assess their adequacy and validity for testing the hypothesis of interest. In particular, the number of experimental units and the way in which they have been allocated to treatments should be described. If observations have been omitted from the analysis this must be justified. Methods of analysis should be described precisely and any necessary assumptions clearly stated because these may affect the conclusions that can be drawn from the experiment.
Results
Results should be presented in text, tables and figures as appropriate. Data given in tables and figures should not be repeated in the text.
Tables
Tables should be concise and informative. Tables should be self-contained, that is they should not require further explanation. They should also be numbered (Arabic numerals) and cited in the text. All abbreviations used in the table should be explained in the footnotes. Each column should have a separate heading. Each table should be accompanied by a fully descriptive legend.
Figures
Figures should be bar charts showing mean values for parasite burden, with error bars showing standard deviations or standard errors of the mean. Line graphs are not appropriate for data which has discrete categories. Graphs should be prepared using a computer software package, such as Excel or SPSS. Figures should be accompanied by a fully descriptive legend. For example, ‘The relationship between increasing numbers of Varroa destructor (1-11 mites) infesting developing worker honeybees (Apis mellifera) and (a) the live weight at emergence of deformed and non-deformed bees and (b) the body water content at emergence of deformed and non-deformed bees.
Statistical analysis
Guidance on statistical analysis will be provided in the statistics workshop in week 11. During the workshop, a new analysis technique available in SPSS, the generalised linear model (GLZM), will be introduced. As the practical involves counting parasites, we can take advantage of the fact that count data usually has a Poisson distribution, rather than using the more simplistic categories of “normal” and “not normal” for data distribution like you learned at level 5. However, if you would prefer to use a more familiar data analysis technique such as a 2-way ANOVA, this is also perfectly acceptable, as long as the analysis is performed and interpreted correctly.
The first thing to do is to decide which data sets you will be analysing- if even in the control animals there are very few parasites in very few animals for a given gut region, then statistical analysis won’t be possible- you can’t do stats with a lot of zeros! So reporting these findings descriptively will be adequate. Copy the datasets you want to analyse into SPSS, very carefully- most errors occur because of copying and pasting the wrong number of cells over. Then make your graphs- a separate graph for each parasite, and for males and females, makes sense- so 6 graphs in total. Your graphs should be bar charts, showing means and standard errors.
To test whether antibiotic treatment affects parasite count in the different gut regions, it is simpler to look at the male and female datasets separately. There are then still 2 independent variables or factors- gut region, and treatment group. So you need to include both of these in your generalised linear model or ANOVA analysis. Make sure you perform post-hoc tests to find out which treatment group is different to which other, and when doing this make sure you compare only biologically relevant things. You will perform a separate analysis for each parasite- and for males and females- so like the 6 graphs, you will have 6 sets of results.
To investigate whether parasite species are interacting or competing, you should use correlation analysis (presented with a scatter plot graph) to see whether there is competition between parasites- if the parasites compete, we should see a negative correlation, with numbers of one increasing as another decreases. Or we may see a positive correlation, where animals with a higher burden of one parasite are more susceptible to other parasitic infections.
When reporting your results, NEVER paste SPSS tables into your report- always write biologically meaningful sentences, putting the relevant statistics in brackets at the end, eg. “In male cockroaches, penicillin treatment reduced the mean Nyctotherus count in the ileum from 24 parasites (+/-9) to less than 1 parasite (p< 0.001)”.
Discussion
The interpretation of results should be discussed, observations should be related to other relevant studies and the implications of the results for future research should be outlined. Results should not be repeated in this section.
Acknowledgement
Acknowledgement of technical help and of financial and material support should be mentioned in this section.
References
Ensure that all references cited in the text are included in the reference list and vice versa. The reference list should contain only articles that are accessible to most scientists. Unpublished work, including personal communications should be referred to in the text as (A. Stone, unpublished).
In Text, references should be cited in chronological order and et al should be used where there are more than two authors e.g. (Frost et al., 1992).
In Reference List, references should be cited in alphabetical order. Papers by the same author should be given in the order, (1) Single author (2) Two authors alphabetically according to the name of the second author (3) three or more authors chronologically with a), b), c) etc for papers published in the same year. References to Journal articles should contain the author’s names with initial, year of publication, full title of article, Journal's full title, volume of publication and first and last page numbers.
Reference to books should include, authors’ names, year of publication, chapter title, book title, names of editors, name of publisher and city of publication - in that order.
Examples of reference
a) A journal reference
Bowen-Walker, P.L., Martin, S.J. & Gunn, A. (1999) The transmission of deformed wing virus between honeybees (Apis mellifera L.) by the ectoparasitic mite Varroa jacobsoni Oud. Journal of Invertebrate Pathology 73, 101-106.
b) A book reference
Bailey, L. & Ball, B.V. (1991) Honey Bee Pathology 2nd ed. Academic Press, London, UK.
c) A chapter in a book
Martin, S.J. (1997) Life and death of Varroa. In Fight the Mite (P. Munn, Ed.), pp. 3-10. International Bee Research Association. Cardiff, UK.
Practical Report Marking Scheme
Title and Abstract/ Introduction/ Materials & Methods 30
Results 30
Discussion (includes references) 40