The Plant and the Insects

The text in this submission was adapted from my project manuscript which was submitted as part of my Zoology Honours course.

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Water hyacinth (Eichornia crassipes)

Water hyacinth (Eichhornia crassipes) (Mart.) Solms-Laub, is an invasive South American water weed that was originally imported into South Africa more than 100 years ago because of its beauty as an ornamental plant in ponds and aquariums (Guillarmod 1979). Much like the other major invasive water weeds in South Africa, water hyacinth has spread to many water sources throughout the country and has grown to become the most invasive water weed in South Africa (Cilliers 1991). Water hyacinth forms dense mats of vegetation that interfere with aquatic ecosystems, subsequently degrading the habitat of indigenous fauna and flora resulting in environmental, recreational and agricultural losses (Hill 2003).

Water hyacinth was originally controlled using herbicides and mechanical removal but because these forms of management damage indigenous biota, are expensive and offer only short term relief, biological control has become the preferred method of controlling water hyacinth as it is more efficient and sustainable than other means of control (Cilliers 1991, Hill 2003, Coetzee et al. 2011).

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Feeding damage dealt to water hyacinth by biocontrol agents

My study investigated the two species of biological control agents currently reared for water hyacinth at Waainek Mass Rearing Facility (WMRC), Rhodes University, Grahamstown. These include the water hyacinth mirid (Eccritotarsus catarinensis) and the delphacid plant hopper (Megamelus scutellaris). Both E. catarinensis and M. scutellaris are host-specific South American sap feeders that feed on the chlorophyll in water hyacinth. Continued feeding leads to a reduction in plant vigor and in some cases, death (Coetzee et al. 2005, 2011, Hernandez et al. 2011).

Eccritotarsus catarinensis is a 3mm long mirid that is found on leaves of water hyacinth, where it gathers nutrients through the uptake of chlorophyll from the leaf. The feeding process facilitates the leaf’s colour change from green to yellow and even brown, making the mirid important, because it weakens the water hyacinth and affords indigenous flora the opportunity to outcompete the invasive water weed (Coetzee et al. 2005).

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Water hyacinth mirid (Eccitotarsus catarinensis)

Megamelus scutellaris is a 3.8-4.3mm long planthopper from Argentina which has found much success in South American studies because of its ability to combat the spread of water hyacinth (Hernandez et al. 2011). The adults exhibit wing dimorphism. There is a long winged form (macropterous), which is capable of flight and a short-winged form (brachypterous), which is not capable of flight.

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Delphacid planthopper (Megamelus scutellaris)

Both insects are being reared at the WMRC for release in the field and are thus of great import to scientists because they offer the best method of controlling the spread of water hyacinth in South Africa.

References

Cilliers CJ. 1991. Biological control of water hyacinth, Eichhornia crassipes (Pontederiaceae), in South Africa. Agriculture, Ecosystems and Environment 37: 207-217.

Coetzee JA, Center TD, Byrne MJ, Hill MP. 2005, Impact of the biocontrol agent Eccritotarsus catarinensis, a sap feeding mirid, on the competitive performance of water hyacinth, Eichornia crassipes. Biological Control 32: 90-96.

Coetzee JA, Hill MP, Byrne MJ, Bownes A. 2011. A review of the biological control programmes on Eichhornia crassipes (C.Mart.) Solms (Pontederiaceae), Salvinia molesta D.S.Mitch. (Salvibiaceae), Pistia stratiotes L. (Araceae), Myriophyllum aquaticum (Vell.) Verdc. (Haloragaceae) and Azolla filiculoides Lam. (Azollaceae) in South Africa. African Entomology 19: 451-468.

Guillarmod AJ. 1979. Water weeds in Southern Africa. Aquatic Botany 6: 377-391.

Hill MP. 2003. The impact and control of alien aquatic vegetation in South African aquatic ecosystems. African Journal of Aquatic Science 28: 19-24.

Hernandez MC, Brentassi MJ, Sosa AJ, Sacco J, Elsesser G. 2011. Feeding behaviour and spatial distribution of two planthoppers, Megamelus scutellaris (Delphacidae) and Taosa longula (Dictyopharidae), on water hyacinth. Biocontrol Science and Technology 21: 941-952.

 

The methods behind my project

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I am doing a genetics project, and whilst I have come to understand the ‘fundamental’ processes which underlie molecular biology, there are many people out there that don’t. This post thus serves the purpose of teaching those of you who interested, just a little bit about what I do and how I do it. I don’t claim to be an expert nor do I claim to be good at it, but bear with me, and maybe you will come to appreciate just how simple this stuff can be.

My project is using the DNA from spotted skaapsteker (Psammophylax rhombeatus rhombeatus)  tail clippings to determine whether the snakes on the east coast, and the snakes on the west coast of South Africa, are different species. I will admit, Genetics is not exactly the most accessible science, but when you peel away the prestige created by ‘full-of-them-self’ scientists and shed your own self-doubt, you will begin to find the methods can be rather simple, and better yet, fun.

Firstly the tissue has to be acquired and this can be done in one of two ways. Either you sub-sample a specimen in a museum (ie: cut a piece of tissue off a preserved animal) or you capture the animal yourself and cut off a piece of tissue. Due to time constraints, I did not capture my own specimens but rather opted to sub-sample museum specimens from the Bayworld museum in the Eastern Cape and SANBI in the Western Cape. Once you have your sub-samples, you have to log the numbers and further sub-sample your original sub-sample. This is done to ensure that there is back-up tissue, in the event that you lose, or contaminate the sub-sample that you are working with. (FYI: I will use the word ‘sub-sample’ more sparingly from here on out)

Figure 1: To left-Test tubes, containing DNA samples from spotted skaapstekers across the southern Cape. To Right- A skaapsteker tail being sub-sampled.

Following this, the original sample is returned to the freezer, to preserve the integrity of the DNA for future use, and the sub-sample is extracted. Extraction is the process whereby the the DNA is purified and isolated from all other compounds that may be present in the piece of tissue. This is an integral step, because the more pure the DNA, the more likely it will be translated into a usable DNA sequence at the end of the entire process.Firstly the DNA sample is broken up to expose the DNA, then the fats and proteins inherent in the tissue are broken-up, using salt and detergents and finally, the DNA is separated from the solution using alcohol and centrifugation. Basically, The DNA is isolated and purified.

Figure 2: To left- process of adding and removing alcohol in the seperation step of extraction. To Right- Centrifuge used for separating DNA from alcohol

Once extracted, a micro-liter of the DNA solution is removed from the test tube and analysed using a Nano-drop to determine the concentration of the DNA in each test tube. This is done to ensure that the DNA was properly extracted and in addition, enables you to modify the ratio of water and DNA going into the PCR step to optimize the reaction. Once all samples have been nano dropped, the DNA from each test tube is transferred into new tubes and primer, mastermix and water are added to the test tube.

The purpose of a Polymerase Chain Reaction is to amplify the DNA already present in the test tubes and create higher quantities, and longer strands of DNA, that can be analysed in the last step of the process.  The primer which is specific, amplifies a particular gene and the mastermix supplies the building blacks, facilitates the binding process and ensures the right conditions are met for DNA amplification.

In my project, I am analyzing the 16s RNA gene so naturally I used a 16s primer to amplify the gene I was looking for. The entire process takes place inside a PCR machine which cycles the temperatures for specific periods of time in order to facilitate the amplification process inside the test tubes.

Figure 3: To left- Nano-drop, used to test the DNA concentration of samples after extraction step. To right- PCR machine, cycles temperature thereby facilitating the amplification of DNA.

Once the PCR machine has run its course, the test tubes are removed, hopefully containing many copies of the gene you want to sample. To test whether the entire process has worked, the amplified DNA samples from the PCR step are inserted into wells in an electrophoresis gel. When an electric current is run through the gel, the negatively charged DNA molecules are pulled through the gel by the positive charge created on the other end of the gel. The gel is porous so the smaller fragments move further through the gel than the bigger fragments, and thus a ladder of different-sized DNA molecules is created. Once 30 minutes have elapsed, the gel is removed from the gel tank and is placed in a machine that takes a picture of the gel.

If you have have been accurate and everything has worked as it was supposed to, you should get a picture with a row of dark black DNA bands across your gel. A dark band is created if the desired gene has been amplified correctly in a particular sample and a row of bands is created if multiple samples have worked together.

PCR 1

 

Figure 4: Row 1- Ladder of differently sized DNA fragments, against which you can compare your samples. Row 2-6- Bands created by the presence of large quantities of the 16s gene, which bunch together because they share the same DNA fragment size. Row 7- Blank, contains no DNA and is inserted as control to ensure that no contamination is present. This sample had a little contamination as you can see above

The above picture shows the result of 5 successful extractions, with each dark band representing the amplified DNA of a individual spotted skaapsteker. Seen as though this picture was taken a few months ago, the DNA has already been sent to Macrogen, along with 10 other samples, for sequencing.  I am happy to say that from this batch, 9 out of the 15 DNA samples produced viable sequences, and following the successful acquisition of more sequences from more localities, these samples will be used to determine whether there is a need for taxonomic reevaluation in the spotted skaapstekers.

 

picture of sequence

Figure 5: Example of a sequence showing the bases along a  section of an DNA strand in a spotted skaapsteker.

 

 

 

The frog, the plant and the insect

Unlike last year which saw me producing one project in pursuit of my zoological degree, this year is quite different. This year I am expected to produce two projects. Both fall within the field of herpetology but that’s about all they have in common. My other project which ventures to unravel the secrets of spotted skaapsteker distribution patterns’ is a genetics project. The project that I will explain now is a biological control project.

Unlike last year however, this project doesn’t detail the effect of water-stressed aquatic weeds on insects but rather the effect of painted reed frogs on water hyacinth biocontrol agents. Biological invasions are common place in the world, with many invasive species being introduced into new ecosystems both intentionally and unintentionally. Some are very bad for the local ecosystem and its animal and plant inhabitants, but some invasions confer no negative effect on the environments.

Water hyacinth and it’s spread through South African aquatic ecosystems is neither good nor neutral, one could venture to call it a parasite on the nation’s natural water resources. Water hyacinth is a menace because unlike indigenous water weeds, it has no natural enemies and thus it grows out of control, thereby chocking species of indigenous plant life, which struggle to keep up.

Water hyacinth is a very real threat to South Africa’s water bodies, and although there are many ways of combating it’s spread, very few are successful. Herbicidal control kills the water hyacinth but kills indigenous plants and animals too. Mechanical removal is painstaking and ineffective because the smallest piece of plant can re-establish somewhere else, if it is not killed completely.

These failures led scientists to look at water hyacinths’ natural enemies for answers. Biological control involves bringing in enemies from the plants natural environment and introducing the foreign enemies in our ecosystems, granted they do not pose any threat to local fauna or flora. In doing this the plant is exposed to an organism that can recognise, eat and thereby deplete its density.

In water hyacinths case, this natural enemy is not one but many, but the ones we are going to focus on is a plant hopper (M. scutellaris) and and a mirid (E. catarinensis) species from Brazil. Both biocontrol agents have been relatively successful in controlling water hyacinth. Both biocontrol agents are currently being reared at the Waainek Mass Rearing Centre at Rhodes University. The problem is, recently, painted Reed Frogs (Hyperolius marmoratus verrucosus) have been found in the mass rearing pools, the same pools that rear the insects.

The project thus aims to determine whether the frog predates upon the two insects. If the frog does in fact incorporate either of the two insects into its diet, it could affect the Waainek Mass Rearing Centres’ ability to rear and distribute the insect, and if they cannot rear the insect in high enough quantities, then water hyacinth will continue to take over South African waterways without restriction from the biocontrol agents.

 

The beginning of my project

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I have loved snakes for as long I can remember. They fascinate and inspire me to read more and more. They very well may be the thing I choose to base my life’s work on one day, and it’s this love that has steered me towards my latest Zoological endeavour: the study of spotted skaapstekers and their distribution In South Africa

Unlike my first project which saw me growing buckets upon buckets of invasive water weeds, this project has me in an air-conditioned lab with finely-tuned scientific instruments – a hop, a skip and a mile away from my previous greenhouse and spade. It may not be as glamorous as birthing a T-rex but it has its perks. Firstly, it doesn’t pay. Secondly, it makes me feel stupid and thirdly, it makes me feel more stupid than the laboratory’s pet fish that eats its own semen. Granted, these are not perks but the semen eating Siamese fighting fish is weirdly frightening and fascinating all at the same time. (P.S. I don’t know if the fish’s diet is completely factual but I have it from reliable sources that it is).

Regardless of the fish and its diet, I believe this project is both challenging and rewarding at the same time because it promises new knowledge and new skills that are tantamount to the arsenal of any aspiring herpetologist.

Thus far I have completed the DNA preparation for most of the DNA samples from the Bayworld museum in Port Elizabeth. The preparation which involves clipping body tissue, extracting, amplifying and running DNA is a rather painstaking and unpredictable process which leaves even the most robust knees shaking from side to side. The end product, a small picture with an array of black and white lines can make or break your day and luckily for me, my day was made the first time I delved into the world of genetics.

My DNA samples were good and for this reason they were packaged and sent to South Korea only weeks ago for further preparation. The product of the preparation carried out in South Korea will be inputted into a rather sophisticated programme to determine whether the samples ascertained from the museum prove or disprove the existence of multiple subspecies of spotted skaapsteker in South Africa.

The project is still in its early stages because I have only done approximately a third of the necessary DNA preparations for the project. The next step which was begun only days ago will involve me preparing spotted skaapsteker DNA samples from SANBI (South African National Biodiversity Institute) in Cape Town. Once prepared, the large batch of samples will be sent to South Korea and following that will be analysed in conjunction with the samples from the Bayworld to determine whether spotted skaapstekers are perhaps more than just one species.

Although I am only at the beginning of this sure-to-be stressful endeavour, I am excited to see what the future holds and whether the spotted skaapstekers warrant taxonomic reevaluation.