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BSc Countryside Management

April 2014

Submitted in part candidature for the degree of B.Sc., Institute of Biological,

Environmental and Rural Sciences, Aberystwyth University.

Does one of the common plants (Shepherds Purse

Capsella bursa-pastoris) in the United Kingdom

express carnivorous behaviour?

John Gibson

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I certify that, except where indicated, all material in this thesis is the result of my own

work, presented in my own words, that any direct quotations are contained within

quotation marks with appropriate citation and that all sources of information used

(textual, graphic, etc) have been correctly cited in both the text and reference list in

accordance with the IBERS referencing style. The work has not previously been

submitted as part of any other assessed module, or submitted for any other degree

or diploma. I have read and abided by the University and Departmental statements

on plagiarism. I confirm that I have read, understood and abided by this declaration.

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Does one of the common plants (Shepherds Purse

Capsella bursa-pastoris) in the United Kingdom

express carnivorous behaviour?

Abstract

Global biodiversity has come under increasing focus in recent years due to demands on land and

altering of habitats causing declines in animal and plants species. With renewed emphasis upon

ecosystem services and efficient habitat management, it would seem important to understand the

ecological interactions that take place in the habitats we aim to protect. This study focuses upon

Shepherds Purse (Capsella bursa-pastoris) and follows on from studies in the 1970’s. It was

suggested that this common UK plant may actually exhibit carnivorous traits when in its early stages

of growth. Understanding such common plants and their interactions is key if we are to manage

biodiversity effectively in an ever changing landscape. The study implemented three experiments to

directly investigate whether Capsella bursa-pastoris (plus other Brassicales) expressed carnivorous

traits starting from the germination of the seeds. Experiments set out to answer the three criteria for

being described as a carnivorous plant: Does the plant attract prey? Can the plant capture that prey?

Does the plant benefit from this interaction? The findings reported here agreed with previous studies

that the plant could attract and capture insects. However, it could not conclude that the plant

benefited from this interaction. Evidence did highlight however that all seed groups tested did have

an effect on killing the insects. The study places an emphasis to expand investigations into if

Capsella bursa-pastoris benefits in other stages of growth and opens the question “Is carnivorous

behaviour more widespread than we ever knew in our habitats”.

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Table of Contents

1. Introduction .............................................................................................................................. 5

1.1 Global biodiversity and its importance ...................................................................................... 5

1.2 Carnivorous Plants ................................................................................................................... 6

1.3 The possibility of Capsella bursa-pastoris seeds being carnivorous ......................................... 8

1.4 The changing countryside of the United Kingdom ................................................................... 10

1.5 The importance of understanding the biology of Capsella bursa-pastoris seeds ................... 10

2. Research questions ............................................................................................................. 11

3. Methods and materials ......................................................................................................... 11

3.1 Materials .................................................................................................................................. 11

3.2 Experimental insect - Fruit fly Drosophila melanogaster ........................................................ 11

3.3 The range of test plant seeds ................................................................................................ 12

3.4 Breaking seed dormancy. ....................................................................................................... 13

3.5 Germination and growth conditions . ...................................................................................... 13

4. The experimental designs .................................................................................................... 14

4.1 Experimental design one

-To test: Are Capsella bursa-pastoris seeds able to positively attract insect larvae? ........... 14

4.2 Experimental design two

-To test: Are Capsella bursa-pastoris seeds capable of increasing the mortality of insect

larvae within close proximity? ................................................................................................ 15

4.3 Data analysis for experiment one and two .............................................................................. 15

4.4 Experimental design three

-To test: Do Capsella bursa-pastoris, benefit in the early stages of growth from the presence

of deceased larvae? ................................................................................................................ 16

4.5 Data analysis for experiment three .......................................................................................... 17

5. Results .................................................................................................................................... 18

5.1 Experimental one .................................................................................................................... 18

5.2 Experimental two .................................................................................................................... 19

5.3 Experimental three ................................................................................................................. 26

6. Discussion.............................................................................................................................. 29

6.1 The attraction ......................................................................................................................... 29

6.2 Mortality rates ......................................................................................................................... 30

6.3 Have benefits been seen? ...................................................................................................... 32

7. Conclusion ............................................................................................................................ 33

8. Acknowledgements ............................................................................................................... 34

9. References ............................................................................................................................. 35

10. Appendix ................................................................................................................................ 41

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1. Introduction

This review explores the evidence that certain plants are carnivorous during germination and in the

early stages of growth. Several researchers, including Darwin (1896), have looked at plants that

exhibit insectivorous mechanisms. These include the Venus Fly Trap Dionaea muscipula, which

traps small insects (Kitchen, 1954). However studies have looked into the possibility that plants can

display such carnivorous traits at early stages of growth without visible adapted mechanisms. A brief

report by Barber (1978) published in the Carnivorous Plant Newsletter, suggested that during the

germination of Shepherds Purse Capsella bursa-pastoris seeds, they are able to attract mosquito

larvae. Killing them in order to benefit from their nutrients and therefore displaying carnivorous

characteristics. The purpose of this study was to investigate these claims that Shepherd’s Purse

seeds are carnivorous.

1.1 Global biodiversity and its importance

Recent analysis of global biodiversity has estimated that the number of flowering plant species is

352,000, but many have still not been described which means this number could rise to a more

accurate figure that is above 400,000 species (RBG, 2014). The number of flowering plant species

known to be threatened stood at 8,084 in 2010 (ICUN, 2010), with a significant driver of species loss

being from habitat change and introduced species (UNEP, 1995).

Generally plant diversity is highest in habitats that have an intermediate level of productivity. The

unimodel (also known as “the humpback”) created by Grimes (1973) explained the relationship

between the productivity of a habitat related to the diversity of plants that could survive. Low

productivity within a habitat (such as sand dunes), means its potential for supporting organisms

decreases due to limited food within the food chain. Similarly the number of species decreases in

the higher productive environments due to the competition for light (Grime, 1979). The intense

competition for light excludes many species because they are less able to compete under these

conditions. Therefore creating a habitat with a productivity that is ample for survival, but is not

excessive, means organisms can co-exist, but there is limited potential for any one species to

become dominant (Lambers et al, 2010).

Habitats with the greater potential for co-existence of species will also have a higher structural

complexity. This higher structural complexity of swards, regularly support a greater abundance and

diversity of species compared to habitats with less complex structural swards (high and low

productivity) (Heck & Wetstone, 1977). Intermediate levels of nutrients available to plants means

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that potential competitive species are unable to competitively exclude other species and therefore

biodiversity levels can be higher (Lambers et al, 2010).

The addition of nutrients into a “low nutrient environment” can limit the ability of species that have

adapted to these lower nutrient conditions to compete (Fisher et al, 2009). The addition of nutrients

promotes dominance from specialised plants to use the available nutrients to grow tall and form

extensive canopies, which can drastically affect the species diversity. Rosette forming plants then

have limited access to light, resulting in a decreased net biomass production under the canopy

(Eriksson et al, 2006). Anthropogenic impacts by nutrient pollution on such delicate environments

include direct nutrient application from farming and the burning of fossil fuels (Nature Education,

2013). The excess volumes of nitrogen and phosphorus in soils from these alter habitats by

creating nutrient enrichment (MEA, 2005).

In 1859, both Charles Darwin and Alfred Wallace, coherently explained that species have the

abilities to adapt to their environments (Greenberger, 2005). Adaptions by animals and plants are

made possible by having variations of characteristics within populations. By having these variations

within the populations, a species can increase the probability of having selective traits that assist

reproduction or survival. Those surviving, or the most fertile individuals are typically suited to their

environment and this drives natural selection (Niklas, 1997). One evolved adaptation of plants

which highlights traits that are suited to their environment, is being carnivorous in a low nutrient

environment.

Terrestrial plants generally acquire the majority of their nutrients via the soil, either from direct

absorption from the roots or indirectly by symbiotic relationships with mycorrhizal fungi (Smith &

Read, 2008). A vast array of non-mycorrhizal plants living in low nutrient- habitats have developed

alternative highly specialised strategies to obtain essential nutrients for survival. One of these

strategies is to become a carnivore (Lambers et al, 2010).

1.2 Carnivorous plants

The discovery and identification of new carnivorous plants continues. Approximations in 1954 stood

at 500 species (Kitchen, 1954) compared to recent estimations from Bathlott et al (2007) of 630

species. These are species that have developed selective traits of a carnivorous nature, which

include attracting and trapping prey. Then the plant proceeds to produce digestive enzymes, so that

the plant can absorb the nutrients from that prey (Kitchen, 1954).

Carnivorous plants have evolved independently of each other, six times in five separate orders of

flowering plants (Albert et al, 1992). Taxonomically, carnivorous plants can be divided into two

divisions; passive (passively ensnaring animals) and non-passive (demonstrating movements to trap

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animals). Both divisions are typically found in very specific habitats and commonly in barren

locations. Environments with poor nutrients and limited soil availability create a requirement for

plants to adapt to traits like carnivory which can supplement their diet (MacPherson, 2010).

Non passive behaviour includes the adaptions of leaves to trap prey, digest prey and then adsorb

nutrients, as seen in Dionaea muscipula. Some plants, such as the Utricularia, have adapted so

much to a carnivorous diet, that the plant now has no reliance on photosynthesis (Adamec, 2006).

For the carnivorous trait to be beneficial, the plant must gain more energy than the energy spent.

The energy spent includes the production of nectars and odours to attract insects to drive evolution

of these carnivorous traits. The whole carnivorous process must gain more nutrients than what was

originally available in the soil (Moran, 1996; Bohn & Federle, 2004). Additional maintenance costs

to attract insects include ultra-violet designs and bright projections (Moran et al, 1999), consumable

trichomes (Merbach et al, 2002) as well as the excretion of mucilage or resin which can also

capture prey.

An example of passive carnivorous plants are the pitcher plants, such as Darlingtonia californica.

The Darlingtonia californica has adapted its leaves into a tubular shape to create a vase structure.

Insects are attracted inside the structure via glands that excrete nectar, which are positioned inside

a hooded formed leaf, which will then close to stop the insect from escaping (Botany Society of

America, 2012). The plant is reliant on those insects passively falling into the leaf structure. At the

base of the tubular leaf, digestive enzymes release the nutrients from insects that are consequently

absorbed by cells similar to those that are found on root systems (Slack, 2010).

One plant species which is still debated as a being a carnivorous plant is the teasel Dipsacus

fullomom (Dipsacaceae). The base of the leaves fill with rain water and this has been observed

capturing falling insects (Christy, 1923). However, the plant does not live in a low nutrient

environment, which would be regularly suggested to have caused the carnivorous trait (Ellison &

Gotelli, 2001). Instead the plant lives in soils which are calcareous and nitrogen enriched, which

would typically be a lethal environment to most carnivorous species (Adamec, 2006). The height of

the plant (ranging from 0.5-2.5m) is also greater then what would be expected (Lloyd, 1942). A

study by Shaw & Shackleton (2011) discovered that Dipsacus fullomom does in fact benefit from the

nutrients of captured insects. Instead of enhancing the growth of the plant, the additional mineral

nutrients from the insects appeared to increase the production and mass of its seeds. However, the

plant does not appear to attract insects, meaning it cannot be described as a fully carnivorous plant.

It is suggested that Dipsacus fullomom is possibly in an intermediate state of becoming fully

carnivorous and therefore is a protocarnivorous plant (Shaw & Shackleton, 2011).

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1.3 The possibility of Capsella bursa-pastoris seeds being carnivorous

Barber (1978) discussed the possibility that C.bursa-pastoris seeds may have the capability of

digesting the proteins of insects through enzymes. It still remains to be proven whether this species

benefits from this potential strategy. It is plausible, that the seeds may be neither passive nor non-

passive. Instead it may be a type of protocarnivorous plant, which is on the evolutional pathway to

becoming a defined carnivorous plant (Schnell, 2002). However, the plant does not display all the

traits (attract, kill and then digest prey), as seen with a true carnivorous plant (Albert et al, 1992).

Plants can benefit indirectly from the attracted insects, which can be seen in the case of the

protocarnivorous plant, Roridula gorgonias. This species captures insects but not to obtain their

nutrients, but to use the captured insects to attract another insect called Pameridea. It then benefits

from the nutrient that the Pameridea leaves whilst visiting via the feces, which fall onto the ground

and are absorbed by the root system (Acton, 2012).

Seed defence against pathogens can be seen throughout plant species via Reactive Oxygen

Species (ROS). These molecules, which are chemically reactive, are produced at various stages of

the seed’s life, from embryogenesis to germination (De-Rafael et al, 2001). Byregulating plant cell

growth, they are able to thicken cell walls, which is a defence against pathogens entering the plant.

It has been suggested by El-Maarouf-Bouteau & Bailly (2008) that seeds regularly have defences

against predation during germination from the ROS. The rise of intracellular ROS production in

germinating seeds, displays a defense reaction against microorganisms. If the ROS production

becomes extracellular, it could be possible to limit bacterial growth in soil and the development of

pathogens. Many seeds can be directly toxic to microorganism (El-Maarouf-Bouteau & Bailly, 2008)

and therefore may have the potential to kill. It also highlights the ability that various seeds have to

adapt to their surrounding natural environmental pressures.

C. bursa-pastrois have mucilaginous seed, which means they produce gel once submerged in

water.The mucilaginous covering of the seed aids the germination process by creating a heat shield

and reducing vital vapour loss (Young & Evans, 1973). The seeds especially benefit from the

mucilaginous covering, when colinizing new habitats that are disturbed and barren. The substance is

also a source of food and water, which enables the seed to be self sufficent and therefore it does not

need to rely on a covering of soil for germination. This is particularly beneficial when colinising a

disturbed habitat (Harper et al, 1965).

Barber (1978) discussed that the pellicles covering C. bursa-pastrois seed provided it with the

ability to release a sticky mucilage. The same ability applies to most of the Brassicaceae family (also

known as the mustard or cabbage family). The mucilage released provides the family with the

potential to attract and capture prey (Ying, et al, 2012). However, earlier studies by Barber et al

(1974) found that the mucilage released by Brassicaceae seeds, was not equal throughout the

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family. The study concludes that this variation in mucilage release within the Brassicaceae family

affected the ability to attract mosquito larvae, between different seed species.

The differing quanitities of mucilage released was directly linked to the odours which affected the

attraction of the larvae, therefore altering the larvae’s behavior. The variations of mucilage

released also affected their capability to capture mosquito larvae. Larvae which were in contact

with the C. bursa-pastrois seeds appeared to be attached by their mouths to the seeds (Barber,

1978). The cellulose element of the muscilage created the ability for the muscilage to become

sticky. Therefore, increased volumes of cellulose within the mucilage gel also increased its ability to

capture larva. The attachment of larvae, which was created by the cellulose within the muscilage

gel, appeared to kill the larvae by only attachment, stopping the larvae from feeding. Further studies

by Page and Barber (1975), concluded that the volumes of cellulose also affected chemotaxis.

Again the seeds with greater volumes of cellulose within the mucilage layer displayed greater

chemotaxis. The attraction of larvae to the seeds was notable only after being submerged in water.

Moreover the Brassicaceae seeds with less cellulose only showed similar abilities to attract larvae

after a prolonged period. Barber and Page (1974) also highlighted that the larvae died at a faster

rate when attached to the seeds compared to larvae without contact. The findings of Barber and

Page (1976) differed from the earlier studies of Barber et al (1974) because the latter concluded that

the larvae died due to toxins released within the aqueous attachment. However, both explained that

the larvae appeared to express no signs of struggle or stress.

Later studies from Barber and Page (1976), suggested that C. bursa-pastoris seeds may pocess the

ability to take up nutrients from dead larvae. This may be achieved by the hydrolysis of proteins

during the germination period. Amino acids can then be produced, which could be absorbed for the

benefit of growth. This was supported by a previous study by Nelson et al (1961), who concluded

that the imbibition of protein was possible via the release of protease in the mucilage during the

germination period of C. bursa-pastoris seeds. Barber (1978) concluded that the seed had displayed

features of being carnivorous, and this was more obvious in further studies using nematodes,

protozoans and bacteria. Even though the study concluded that the seeds attracted and captured

organisms, it was still unclear whether the plant would benefit from this process.

C. bursa-pastoris is widespread on the majority of continents and is also common in man-made

habitats (Neuffer, 2011). It has diverse characteristics within its genetic make-up, which are

influenced by multiple interacting factors (Hurka & Neuffer, 1997). Baker (1965) suggested that the

plant has a germination strategy, which can be described as a “general purpose genotype”. The

“general purpose genotype” is a germination strategy that plants can employ so they can utilize a

wide range of phenotypic plasticity. The ability to change its characteristics / traits to its new

environment provides the plant with the opportunity to often be a colonizer. The plant then has a

clear advantage, when arriving on cleared land (be that arable or urban) and also on disturbed soils,

where growing conditions can vary considerably between sites (Agate, 1998).

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1.4 The changing countryside of the United Kingdom

The biodiversity of the United Kingdom (UK) has been intrinsically linked to the agricultural practices

that have shaped the landscape due modernisation of agriculture. Consequently, agricultural land

also has the ability to generate and support the biodiversity in the UK (Commons, 2007). Recent

decades have seen the UK agricultural industry intensify which is having a detrimental effect on the

farmland biodiversity (MacDonald et al, 2012).

Plant species that have evolved to be habitat specialists have struggled to survive due to the altering

habitat conditions. Many species that evolved traits that were specific for their habitat requirements

have been disadvantaged by an altered habitat. The decline in species that were habitat specialists

has seen a trend of species that are habitat generalists becoming the common taxa. The habitat

generalist is capable of acommodating several different habitat conditions and therefore is more

adaptable to changing habitat conditions (Robinson & Sutherland, 2002).

1.5 The importance of understanding the biology ofCapsella bursa-pastoris seeds

This increases the importance of understanding the biology of C. bursa-pastoris, which is suggested

to be a habitat specialist (carnivorous traits). Interestingly, the plant also appears to display

characteristics of being a habitat generalist, due to its ability to colonise various locations. Species

that are lost through nutrient enrichment are those with these specialised adaptations to a low nutrient

environment.

Therefore understanding plants with carnivorous adaptations can be vital to understanding the low

nutrient environment. Developing knowledge will assist management of biodiversity in these areas,

especially when nutrient enrichment is so freely available in the modern world.

However, C. bursa-pastoris is not a rare plant and therefore has limited benefits for biodiversity

purposes. But it is vital to understand the ecological interactions within our habitats, to understand the

ecosystem services they provide. If the ecological interactions of common species are still not fully

understood, then we are in danger of losing mechanisms from the ecological interactions of rare

plants before they are known. Moreover, the benefits that come from these ecosystem services will be

lost.

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2. Research questions

I. Are Capsella bursa-pastoris seeds able to positively attract insect larvae?

II. Are Capsella bursa-pastoris seeds capable of increasing mortality of insect larvae within

close proximity?

III. Does Capsella bursa-pastoris benefit in the early stages of growth from the presence of

deceased larvae?

3. Methods and materials

3.1 Materials

All Brassicaceae family seeds (except Lepidium sativum) were purchased from a Emorsgate seeds

Ltd (Shepherds purse Capsella bursa-pastoris, Garlic mustard Alliaria petiolata and Hairy bitter cress

Cardamine hirsute). The company collects wild seeds, which enabled my study to have a reasonable

reflection of wild ecological interactions and seed traits. Lycopersicum Var. cerasiforme and Lepidium

sativum, were commercially cultivated and purchased local to Aberystwyth, Wales.

Aberystwyth University provided: bench lamps (40watt bulbs), 24hr plug timer switches, plastic Petri

dishes (100mm and 50mm), Whatman grade 1 circular inserts (85mm and 50mm), distilled water,

Fruit fly Drosophila melanogaster larvae and a low energy microscope (Leica zoom 2000, 240v).

Data analysis was performed in SPSS (IBM SPSS 20 statistical package).

3.2 Experimental insect - Fruit fly Drosophila melanogaster

The orignal studies by Barber (1978) and Reeves and Garcia (1969), which explored the potential of

carnivorous traits in C. bursa-pastoris seeds, used Mosquito larvae. Mosquito larvae are mainly

water dwellers, which means they would be unlikely to interact with C. bursa-pastoris seeds (Silver,

2007).

Barber (1978) used nematodes, protozoans and bacteria as their experimental insects which also

found an enhanced mortality rate in the presence of C. bursa-pastoris seeds. These three were not

freely available , which was the same for other soil organisms, like worms , which might also come

into contact with of C. bursa-pastoris seeds in the soil environment. For this reason, Drosophila

melanogaster larvae were chosen. The Drosophila melanogaster larvae were the most freely

available animal within the University, which are similar morphologically to mosquito larvae. Also,

Drosophila melanogaster larvae have the potential of having a natural ecological interaction with C.

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bursa-pastoris seeds in the soil habitat in the UK. The colonies were also regularly checked for fungi

and infections, making the larvae a reliable cultivated test animal.

Larvae were reared in a media consisting of 80% maize flour, 10% yeast, 8% bacteriological agar,

10% glucose syrup, 10% propionic acid and 0.2% Nipigen. The colonies were maintained in a dry

incubator at the temperature of 24.5oC

The selected Drosophila melanogaster larvae were in their 1st instar larval stage of development. By

selecting individuals in the same developmental stage, it provided a minimum of four days of larval

activity until the pre-pupa stage (Ashburner & Thompson, 1978). Moreover, the experiments

monitoring the larvae’s behaviour were conducted for a length of 6 days.

3.3 The range of test plant seeds

The seeds of plant species were chosen because of their attributes for potential effects on larvae

behaviour and mortality. Four species from the Brassicaceae family were chosen, which were Garlic

Mustard Alliaria petiolata, Cress Lepidium sativum, Hairy Bitter cress Cardamine hirsute and

Shepherds Purse C. bursa-pastoris. The reason for the selection was to explore the possibility that

all the Brassicaceae family have the potential to exhibit carnivorous traits, as suggested by Barber

(1978).

To test if the growth of the plants or gases released during the germination affected the larvae,

Lepidium sativum and Alliaria petiolata were tested. Lepidium sativum is known for its reliable

growth at room temperature, whereas Alliaria petiolata was unlikely to germinate at all, as it was a

bulb. By using both of these, plus a control, it was then possible to test if plant growth or seed

presence, had an effect on the larvae.

To test if carnivorous traits are “cause and effect” (a trait caused to be developed by the presence of

external pressures), Cardamine hirsute was chosen. In the UK, the habitats of C. bursa-pastoris are

shared with the Cardamine hirsute. Their seed sizes are also similar (approximately 0.009grams),

which can be a driver for expressing a need for additional nutrition in early growth, as small seed are

unable to store large volumes of endogenous food (Barber, 1978).

To test outside the Brassicaceae family, Tomato seeds Lycopersicum Var. cerasiforme were

selected. It has been suggested that this plant has the ability to capture aphids via sticky hairs on

the stem (Smith, 2009 & Simons, 1981). Similar to the Dipsacus fullomom, the Lycopersicum var.

cerasiforme also has the potential to absorb those nutrients indirectly from insects, through the root

system (Simons, 1981). Therefore, Lycopersicum var. cerasiforme, may also be a protocarnivorous

plant.

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3.4 Breaking seed dormancy

Cardamine hirsute, C. bursa-pastoris seeds and Alliaria petiolata bulbs were stored in refrigeration

(4oC) for four days and in complete darkness, to optimise the viability (Lannetta et al, 2007). This

was not necessary for the Lycopersicum var. cerasiforme & Lepidium sativum seeds, due to the

seeds being modified from their natural state by commercial cultivation.

3.5 Germination and growth conditions

Germination was assumed to begin once the dry

seeds were in contact with water. For the seeds in

this study, moist conditions were deemed

appropriate conditions for germination (Woodstock,

1988). A pilot study revealed that 10ml (for 10cm

Petri dishes) and 5ml (for 5cm Petri dishes) of

distilled water was the minimum required to be

added to the samples every 24hr. The moist

environment created preferable conditions that were

suitable for the imbibition by the seeds to begin

(Robert et al , 2008).

To replicate spring/ autumn conditions which suit the

germination of C. bursa-pasoris seed (Utah State

University, 2014), the temperature ranged between

6oC (night temperature) and 26

oC (daytime

temperatures), which was also ample for all seeds in

this study. The daytime temperature also allowed the

fact that Drosophila melanogaster larvae develop optimally at 25oC in laboratory conditions

(Ashburner & Thompson, 1978). Light was provided for ten hours every day, as seen in Figure 1.

Figure 1. A photo capturing the growing and

germination conditions provided for this

study.

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4. The experimental designs

4.1 Experimental design one

To test: Are Capsella bursa-pastoris seeds able to positively attract insect larvae?

The rate at which larvae were attracted to each seed was tested within 100mm Petri dishes. The

circular paper inserts, were divided by a single line separating the twenty seeds from the twenty

larvae (50:50 ratio). All seeds and larvae were placed at least 20mm from the dividing line to create a

40mm buffer zone (as seen in Figure 2). Counts of migrating larvae into the seed zone were

recorded every 24h, over a six day period. Each seed species had five replicates for this experiment.

Figure 2. An illustration of the set up for experiment one, to monitor larvae’s attraction and migration

towards the seed species. A buffer zone of 40mm separated the seeds from the larvae. Larvae that

past over the line into the territory of the seeds were recorded as counted data.

Buffer zone

Dividing line

separating

zones

20 larvae 20 seeds

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4.2 Experimental design two

To test: Are Capsella bursa-pastoris seeds capable of increasing the mortality of insect

larvae within close proximity?

Smaller Petri dishes (50mm) were used, to promote close proximity between the larvae and seeds,

enhancing the interaction between the twenty seeds and twenty larvae (50:50 ratio), as seen in

Figure 3. This was monitored for 6 days, every 24h, and then counted the active, pupated and

deceased larvae using a microscope. Each seed species had five replicates for this experiment.

Figure 3. An enlarged illustration of the mix of larvae and seeds within a 50mm Petri dish. All larvae

were free to migrate around the dish and recordings of counted dead larvae were taken every 24hrs.

4.3 Data analysis for experiment one and two

Due to the nature of the count data collected an unbalanced analysis was required. Using a Kruskal

Wallis test it was possible to analyse the significance of the difference within an equal distribution.

Larvae were kept as the dependant variable throughout and seeds were the independent variable

between each set of samples.

20 larvae/ 20

seeds mixed

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4.4 Experimental design three

To test: Do Capsella bursa-pastoris, benefit in the early stages of growth from the presence

of deceased larvae?

Each C. bursa-pastoris seed was paired alongside a dead adult larva, thus creating an environment

that provided the seed with the direct opportunity to absorb nutrients from the dead larvae (refer to

Figure 4). Placing the pair together, also directly challenged Barber and Page’s (1976) suggestions

that C. bursa-pastoris seeds have the capability to absorb nutrients from dead larvae.

Figure 4. An illustration of the set up for the experiment pairing all seeds alongside a dead larva. To

boost germinating seeds and recorded data, fifty seeds and fifty larvae were used in this experiment.

As mentioned previously, carnivorous traits are typically caused by external pressures, like a low

nutrient environment (MacPherson, 2010). Therefore, this experiment created four growing

environments for the seeds, to test if these affected the seedling growth (refer to Table 1).

The rate at which seedlings grew was tested within 100mm Petri dishes. To replicate a growing

environment where nutrients are freely available, topsoil was mixed with distilled water (50:50 ratio)

to release the nutrients. Once the liquid had settled, this was applied to the samples every 24h, in the

same quantity as distilled water (10ml minimum per dish). To replicate nutrient deprived conditions

the seeds were grown in isolation, except for water (Simons, 1981).Each treatment to C. bursa-

pastoris seeds was replicated 5 times.

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Samples Nutrients available

Control - No nutrient

environment

Just seeds and distilled

water

Low nutrient

environment without

insects

Soil nutrients only

Low nutrient

environment

with insects

Larval nutrients only

Diverse nutrient

availability

Soil and larval nutrients

Table 1.This table explains the four treatments that C. bursa-pastoris seeds were grown in, to

determine whether the seeds benefited from larval nutrients.

Due to difficulties prompting the growth of the Capsella seeds, the volumes of each sample

increased to fifty larvae and fifty seeds. Dry weight determinations were attempted in a pilot study for

all seeds, but the growth of all the seeds was not sufficient to produce the data required for this

study. Therefore, Capsella seedlings were deemed the sole focus for measurements of the growth.

Measurements, with a ruler, recorded the seedling’s height from the border of the seed to the top of

the main plant stem. In situ measurements reduced the damage to seedlings.

The samples were grown for fourteen days. Data counts were taken every day but statistically

analysed on the seventh day (mid-point) and fourteenth day (end of growth period).

4.5 Data analysis for experiment three

The amount of seeds that germinated throughout this experiment was not equal between the

samples. Therefore an unbalanced ANOVA statistical test was carried out using a general linear

model. All growth within each Petri dish was averaged (mean) and then placed within this statistical

test (seen in Table 2).

Page | 18

Soil

+ -

+

Soil / Larvae Larvae

-

Soil Control

(No soil / no

larvae)

Table 2. The diagram above shows the four treatments which were used to grow the C. bursa-

pastoris seeds. It also shows the combinations of analyses used in the two way ANOVA analysis,

and the results can be seen in Tables 4 and 5.

5. Results

5.1 Experiment One

In experiment one the amount of larvae attracted to all seeds differs significantly (P=0.005) and this

is also the same when including the control samples (P=0.008). After the sixth day, C. bursa-pastoris

seeds, attracted significantly more than all other seeds and the control (refer to Figure five). The

enlarged error bars of C. bursa-pastoris group data do not overlap the error bars from all over seed

groups and the control. The results show that C. bursa-pastoris do positively attract the insect larvae.

It is also apparent that larvae appeared to favour both C. hirsute and Lycopersicum var. cerasiforme

compared to the control and L.sativum. The mean amount of larvae attracted to C. hirsute and

Lycopersicum var. cerasiforme is greater but the variance and this indicates that the range of data

overlaps with the range of data from A. petiolata. However, the larvae attracted to L. sativum is equal

to the control, which indicates the L. sativum seeds had no effect on attracting the larvae.

Larvae

Page | 19

Figure 5. The histogram above shows the mean amounts of larvae attracted to each seed group. The

error bars are + /- standard error in each sample. The rate of attraction towards the seeds and the

control was significantly different after six days. The graph illustrates this significance coming from the

larvae that were attracted to the C.bursa pastoris seeds (for raw data, see appendix).

*The control group has been carried out in this experiment at a later date.

5.2 Experiment two

During the first 24hr ,the rate of mortality differed significantly between the seeds (P=0.003) and also

with the inclusion of control (P=0.001). C. bursa-pastoris and C. hirsute seeds both produced greater

mean mortalities compared to the other groups. The variances of the two seed groups were also

larger, indicating that the sample data within these seed groups varied considerably. The mean

number of larvae killed in the presence of C. bursa-pastoris seeds, shows that the larvae mortality

rate was higher in the first 24hr compared to all other seeds samples. In Figure 6, it is clear that C.

bursa-pastoris seeds are having a significant effect on the mortality of the larvae in the first 24hrs

compared to all groups except for the Cardamine hirsute seeds. In this first 24hrs, it also worth

mentioning that L. sativum and the control, did not affect the mortality of larvae. Whereas,

Page | 20

Lycopersicum var. cerasiforme and A. petiolata appeared to increase the mortality rate, but to a far

lesser extent than C.bursa-pastoris and C. hirsute seeds.

Figure 6. The histogram above shows the mean mortality rate of larvae whilst in the presence of the

seeds and control samples after 24 hrs. There is a significant difference on the effect of each group of

seeds on the mortality rate of larvae. The histogram illustrates this difference coming from the higher

rate of mortality caused by C.bursa pastoris and C. hirsute seeds. The error bars are + /- standard

error in each sample. (for raw data, see appendix).

*The control group has been carried out in this experiment at a later date.

After 48hrs, there is not a significant difference (P = 0.055) between the mortality rate of the larvae in

all the seed groups. The histogram in Figure 7, indicates that there is a trend of C.bursa-pastoris and

C. hirsute seeds still are causing a higher mortality rate than the other seeds . Observations also

noticed the larvae becoming engulfed by both seeds in the first 48hrs, which can be seen in Figure 8.

The error bars of these two are also separate to all other seed’s variances which indicates that even

though it is not a significant difference, the trend is still apparent. If the C. hirsute was taken out of the

Page | 21

graph, it is clear that C .bursa-pastoris was having a higher rate of mortality on the larvae. However,

there is a highly significant difference when the control is included (P=0.02). All the seeds appear to

have produced a greater mortality rate of larvae compared when the seeds were absent (control).

Figure 7. The histogram above shows the mean mortality rate of larvae whilst in the presence of the

seeds and control samples after 48 hrs. C.bursa-pastoris and C. hirsute seeds appear to have a trend

which follows the first 24hr period, where the mortality of larvae is higher. This is not a significant

difference compared to the other seeds in this experiment. The error bars are + /- standard error in

each sample.

*The control group has been carried out in this experiment at a later date.

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Figure 8. The photos above are observations of C.bursa-pastoris (Left photo) and C. hirsute seeds

(right photo), engulfing larvae with the mucilaginous substance released during their germination. The

red rings circle the seeds that are exhibiting this behaviour of capturing the larvae.

After 72hrs, there was no significant difference between the seeds, the significance ranged from

P=0.205 to P=0.368. C.bursa-pastoris seeds still gave the highest mean mortality rate of larvae

throughout the six days, but not at a significant rate. After the sixth day, Figure 9 indicates that C.

bursa-pastoris seeds were still not significantly different to the rest of the seeds (P=0.308). Using

Figure 10, it is possible to see that the mortality rate of larvae and variance rises with all seed groups.

The rate of mortality and variance of all seeds, are becoming closer to the rate of C.bursa-pastoris

seeds as the time progresses but A.petiolata mortality effect and variance is still visibly lower than that

of C.bursa-pastoris.

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Figure 9. The histogram above shows the mean mortality rate of larvae whilst in the presence of the

seeds and control samples after the sixth day. Compared to the first 24hrs (seen in Figure 6), the

means and variances are becoming less pronounced except for the control. The error bars are + /-

standard error in each sample.

*The control group has been carried out in this experiment at a later date.

When the control is included within the statistical analysis, there is a significant difference between

the six groups (P=0.008) after six days. When the control is included, the result is significant

throughout the experiment, ranging from P=0.002 to P=0.009 (refer to Table 3). Therefore the seeds

were having a significant effect on the mortality of larvae compared to the control which did not have

the seeds present (seen in Figure 10).

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Table 3. The table contains all the P values throughout the experiment, showing the significance

between the samples. It is clear to see that the main significant difference between the seeds’ effects

on mortality rate of larvae is in the first 24h period. Whereas, when the control is present, the

significance is constant throughout the six days.

*The control group has been carried out in this experiment at a later date.

Figure10. The Interpolation line and error bars above show the mean mortality rate of larvae and

standard errors, whilst in the presence of the seeds and control samples throughout the experiment. It

is possible to see from the graph that the mortality rate of the larvae became similar over time, as

evidenced by the condensed error bars. The control effect on the larvae was however always

significantly lower throughout. The error bars are + /- standard error in each sample.

*The control group has been carried out in this experiment at a later date.

Days 1 2 3 4 5 6

With

control*

0.001 0.002 0.007 0.006 0.009 0.008

Just

seeds

0.003 0.055 0.261 .205 .368 .308

Page | 25

Whatever was causing the mortality differences during the first 48hrs, did not appear to be having a

lasting effect on the capability of Drosophila melanogaster to emerge from pupation. There is no

significant difference (P=0.398) between the amount of larvae that emerged as flies once they had

pupated (seen in Figure 11). C.bursa-pastoris seeds did have the lowest mean amount of larvae that

have pupated to emerge, but the variance of this data was also similar to the variance ranges of

Lycopersicum var. cerasiforme, A. petiolata and C. hirsute seeds . However, L. sativum did appear to

have more emerging flies in its population of pupated larvae compared to the larvae in the presence

of C.bursa-pastoris seeds.

Figure 11. The histogram above shows the mean number of flies which emerged after pupation. The

C. bursa pastoris did appear to lower the mean number of larvae that emerged from pupation, but this

is not significantly different to the other seeds in this experiment. The control was unable to be used in

this experiment due to timing. (For raw data, see appendix).

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5.3 Experiment three

According to the results from the Two Way ANOVA test, there was not a significant difference

between the four treatments (seen in Table 2). After seven days the growth of C. bursa-pastoris was

similar in each treatment compared to the control (soil - P=0.365, larvae- P=0.392, soil and larvae

P=0.392 reported in Table 4).

Figure 12. The histogram above shows the four treatments and the mean growth of C. bursa-

pastoris after seven days. The two way ANOVA analysis compared all four treatments against each

other and also in the absence of one another. (For raw data, see appendix).

Page | 27

Table 4. The table above shows the output from the two way ANOVA analysis from data recorded on

the seventh day. From this table it is possible to see the results of the level of significant difference in

the growth measured in height, between the treatments. (For raw data, see appendix).

After fourteen days, the growth of seedlings was again not significantly different to the control (soil -

P=0.147, larvae - P=0.549, soil and larvae - P=0.826, refer to Table 5). Using Figure 12 and 13, it is

possible to see that the error bars are enlarged when the seeds were grown in the presence of both

soil and larvae. The enlarged error bars were present in both the seventh and fourteenth day growing

periods, showing there was a larger variance of plant heights in the samples in this treatment.

Tests of Between-Subjects Effects

Dependent Variable: plant height

Source Type III Sum of

Squares

Df Mean Square F Sig.

Corrected Model 10.182a 3 3.394 .708 .574

Intercept 430.954 1 430.954 89.858 .000

Soil 4.420 1 4.420 .922 .365

Larvae 3.920 1 3.920 .817 .392

soil * larvae 3.920 1 3.920 .817 .392

Error 38.368 8 4.796

Total 564.690 12

Corrected Total 48.549 11

a. R Squared = .210 (Adjusted R Squared = -.087)

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Figure 13. The histogram above shows the four treatments and the mean growth of C. bursa-pastoris

after fourteen days. The two way ANOVA analysis compared all four treatments against each other

and also in the absence of one another. (For raw data, see appendix).

Page | 29

Tests of Between-Subjects Effects

Dependent Variable: plant_height

Source Type III Sum of

Squares

Df Mean Square F Sig.

Corrected Model 17.799a 3 5.933 .862 .487

Intercept 796.548 1 796.548 115.718 .000

Soil 16.576 1 16.576 2.408 .147

Larvae 2.615 1 2.615 .380 .549

soil * larvae .350 1 .350 .051 .826

Error 82.602 12 6.884

Total 953.479 16

Corrected Total 100.401 15

a. R Squared = .177 (Adjusted R Squared = -.028)

Table 5. The table above shows the output from the two way ANOVA analysis from data recorded on

the fourteenth day. From this table it is possible to see the results of the level of significant difference

in the growth measured in height, between the treatments. (For raw data, see appendix).

6. Discussion

This study set out to explore the possibility of carnivorous traits in C. bursa-pastoris seeds, as

mentioned by Barber (1978). Three experiments were implemented to question different aspects of

a true carnivorous plant. These being; do the seeds attract larvae? Do they kill the larvae? Plus

finally are the C. bursa-pastoris seeds able to benefit from the nutrients from deceased larvae? The

results from these experiments found that the C. bursa-pastoris seeds do attract more larvae at a

significantly different rate compared to the four other seeds tested. The C. bursa-pastoris seeds also

possessed the ability to increase the mortality rate of larvae at a significant rate in the first 24hrs. But

this study was not able to conclude whether C .bursa-pastoris was able to benefit during early stages

of growth from the presence of the deceased larvae.

6.1 The attraction

The results are similar to some of the findings from Barber and Page (1975), who found that C. bursa-

pastoris seeds caused a positive chemotaxis of larvae. Barber and Page (1975) concluded that

certain mucilaginous seeds once submerged in water strongly attracted larvae. Moreover, results from

experiment one in my study found only the C. bursa-pastoris and C. hirsute significantly attracted

larvae compared to the other mucilaginous seeds. A possible reason for the greater attraction of

larvae to C. bursa-pastoris seeds may relate to the discussion from Barber et al ( 1974) & Ying, et al(

Page | 30

2012) regarding the mass of mucilage that each seed could release. There is evidence from Young &

Evans (1973) that the coating of the C. bursa-pastoris seeds contain approximately 25% mucilaginous

substance. Whereas, for instance the L. sativum seeds only contain approximately between 6.5 -15%

(Divekar et al, 2005) which correlated with (Barber et al, 1974) that the amount of available mucilage

affected the potential to attract larvae.

Barber and Page (1975) explained that seeds that produced mucilage which was “sticky”, had a better

ability to attract larvae. Whereas, the C. hirsute in experiment one, did appear to have a similar ability

to “stick” to larvae similar to C. bursa-pastoris, but did not attract more larvae than Lycopersicum var.

cerasiforme. The Lycopersicum var. cerasiforme seeds didn’t appear to have any ability to stick to the

larvae, but still attracted more than C. hirsute.

It would be beneficial in future experiments to use a chemical analyse of the seeds mucilage to

determine the potential attractant. Panizzi & Parr (2012) found that Drosophila melanogaster larvae

do have a preference to food selection which is mediated by allochemicals. Using the observations of

the larvae feeding on Lycopersicum var. cerasiforme but not on C. bursa-pastoris seeds, it would

interesting to analysis what allochemicals are present and if C. bursa-pastoris is manipulating

chemicals to attract.

The differing effects of each seed group to attract the larvae in my study, could be because of

specific plant / insect relationships. More than 80% herbivore insects are specialist feeders on only a

singular plant family or specie (Loon et al, 2000).

Therefore, the significant results from experiment do demonstrate that Drosophila melanogaster

larvae are positively attracted to C. bursa-pastoris seeds. This may not be enough to conclude that

no insects are attracted to the other four seed groups but a need to develop this experiment with

other insect species, nemotodes or protozoans. The additional insects could also investigate whether

C. bursa-pastoris seeds attract a variety of insect species.

6.2 Mortality rates

The results seen in experiment two are demonstrating that all seeds had some affect on the mortality

of the larvae over a prolong period. The same conclusion came from the studies of Barber and Page

(1975), but the result from my study show this not exclusive to the Brassicaceae family. What is

clear is that there is a significant difference between the different seeds ability to affect the mortality

in the first 24hrs. Larvae in contact with C. bursa-pastoris seeds did die at a much faster rate.

The early studies from Barber et al (1974) suggested that the cellulose element of the mucilage gel

that ouzed from the seeds affected the capability its to stick to the larvae. Thus, stopping the larvae

from feeding. Although larave do have the ability to survive without food for a length of time due to

utilisation of their internal food reserves (Pomerai, 1990). In this case, the control sample displays the

Page | 31

larvaes ability to exhaust its internal reserves, which the mortality rate is still signiifcantly lower to all of

the seeds groups on the sixth day. Observation seen in experiment two, saw no signs of struggle

which mirrors Barber et al (1974) and Barber and Page (1976) studies, but the experiment does

reveal that the cause of higher mortality rate was probably not caused by food deprivation as

suggested by Barber et al (1974) .

The Brassicales plants are known to produce glucosinolates within their tissue (Chen et al, 2007) and

these secondary metabolites are a defense mechanism towards insect herbivores to limit its

predation (Winde & Wittstock, 2011). Specialist feeders such as the larvae of the butterflies from the

Pieridae family, feed exclusively on Brassicales plants and have specialist adaptations to deal with

the toxic glucosinolates consumed (Loxdale et al, 2011). Another possible group of chemical

compounds called Saponins could also be at work in the seeds. These compounds are also

abundant throughout the plant kingdom and serve as a protection from microorganism, fungi and are

toxic to insects in high concentrations. It would be interesting to analyse the quanities of

glucosinolates and saponins inside the seeds and determine whether there is a correlation to

mortality rates of larvae (Kuzina et al, 2009). Especially as the advances in technology since the

1970’s look beyond the cellulose level and possibly could give a clearer indication of the chemical

properties within the seeds causing the mortalities, expanding on Barber and Page’s (1976)

conclusion that toxins are at work to cause higher levels of mortalities in larvae. But it does appear

that whatever is causing the increased mortality is not effecting the larvae’s developement

throughout the pupation period ( refer to Figure 11).

Cardamine hirsute and C. bursa-pastoris seeds both had a similar trend to cause an enhanced

mortality of larvae compared to other seeds in the first 24hrs and into the 48hrs period. If the killing is

a form of defence rather than a mechanism to gain nutrients, it might explain the relations as

suggested by Loon et al (2000) that plants in the same family can share defence mechanisms.

Studies by Haak et al (2013) have revealed there is “cause and effect” in relation to environmental

pressures such as local predation from herbivores. Their study focused on the genus Lycopersicum

and found that there is a range of abilities within the genus to kill insects depending on the species

and their environmental pressures. Interestingly, the plants are capable of allocating toxins into the

plant tissue when damaged, creating a physical barrier, which can then lead to direct mortality of

small insects (Eriksson A. , 2007). It is therefore feasible to think that wild Lycopersicum may be

capable of allocating toxins as a defence mechanism, to its seeds, to deter animals. Therefore the

commercially cultivated Lycopersicum var. cerasiforme used in this study, may not exhibit the full

ability of its genus to kill insects because of the toxic trait being bred out. In hindsight, using

Lycopersicum var. cerasiforme as the non-Brassica control plant may not have been appropriate

because they have known toxicity in the genus. Also this could be another line of experiments, as the

genus Lycopersicum might have protocarnivorous traits in certain conditions.

Page | 32

But the “cause and effect” may explain why both C. hirsute and C. bursa-pastoris seeds had similar

effects on the mortality rates of larvae. Due to the similarities in the seed sizes, shared habitats and

germination strategies.

6.3 Have benefits been seen?

The results from experiment three indicate that imbibition seeds are not benefiting from the additional

larvae nutrients. Between the four treatments the height of new growth of C. bursa-pastoris was not

significantly different when grown in the different conditions. The results from each treatment are

similar after seven or fourteen days, with the C. bursa-pastoris seedlings are not demonstrating any

benefit with the additional nutrients available from soil or larva. A common theme is the enlarged

variance in the height within the treatment which contains both larvae and soil on both seven and

fourteen days.

Nelson et al (1961) explained that it was possible for C. bursa-pastoris seeds to gain the protein

released from the deceased larvae. If the seedlings were obtaining the protein then the benefit was

not being expressed during the fourteen days of early growth.

Therefore, this study can only state the same as Barber (1978) studies did by stating that it is still

unclear if the plant benefits from the process.

The results from experiment three are limited and only focus on the height of the plant in the early

stages of growth. The results do not show if carnivorous traits are present in the plant throughout its

stages of growth, just the young growth and its height. For instance, the experiment does not provide

the opportunity for the C. bursa-pastoris plants to express other benefits from obtain protein such as

an increased establishment rate or reproductive capabilities. These two possible benefits could only

be assessed fairly if the plant was grown to maturity. The advantage of additional protein may only

be seen after the nutrient from the seed has been completed depleted (Raven et al, 2005). The

beginning of establishment phase, is a particularly vulnerable stage of the plants life cycle and

therefore the additional available proteins maybe particularly beneficial at this at this stage. This

vulnerability also applies to pathogens and predator attacks within and above the soil which again

might see the advantage of a defence mechanism particularly useful (Sheldon, 1974). The seedling

will also be open to abiotic factors such as temperature and moisture, therefore the ability to grow

strong will be a distinct advantage. The absorption of additional proteins would be advantageous at

this stage to create a defence of such conditions (Bradley et al, 2002).

As seen in the study of Dipsacus fullomom by Shaw & Shackleton (2011), the additional nutrients

from insect maybe only be when the plant matures and in this case ,it was to benefit the reproductive

process. Dipsacus fullomom actually used the additional nutrients from the captured insects to

increase the production and mass of seeds (Shaw & Shackleton, 2011). C. bursa-pastoris lives in a

variety of soils, which are not always depleted of nutrients and in fact are nutrient rich in agricultural

Page | 33

habitats. Both Dipsacus fullomom and C. bursa-pastoris are commonly found on disturbed ground

and seen as earlier colonisers of land (Harris & Harris, 1997). Therefore the ability to have a

reproductive advantage could be key to their survival in a new habitat.

The enlarged variance recorded in height data when both larvae and soil was present may also need

to be explored. The variance in height data may reflect the “general purpose genotype” which Baker

(1965) previously explained that C. bursa-pastoris deploys to utilize a wide range of habitats and

conditions. Therefore, there are possibly a variety of individuals within the species that thrive in

differing conditions. Moreover the individuals that thrive in low nutrient conditions and limited soils

availability are the individuals within a species that may express carnivorous traits to supplement their

diet (MacPherson, 2010). So to test this idea, it would be appropriate to alter abiotic factors and

provide a variety of soils to explore whether individuals within the C. bursa-pastoris species would use

the available insect nutrients in certain conditions. Therefore, creating the right conditions to activate

possible genotypes, which utilise a carnivorous trait.

7. Conclusion

The conclusion of this study is that C. bursa-pastoris have shown the ability to demonstrate two

carnivorous traits; to attract and to kill. But the study was not able to conclude the third and conclusive

carnivorous trait; to benefit from the deceased animal, meaning the study cannot declare that C.

bursa-pastoris seeds are carnivorous. However, the findings from this study and evidence from the

literature have suggested that whether the plant benefits from the interaction needs to be further

explored.

Moreover, findings suggest that all seeds in this experiment had some effect on the larvae’s mortality

rate. As discussed previously, this is one aspect of a carnivorous trait and with the expansion of the

investigation to include microorganisms, bacteria and other insects, there may be an attraction and

benefit for other plant species too. To fully understand if the plant is expressing benefits from the

animal interaction, it may be beneficial in future experiments to observe plants throughout their life

cycle instead of just the new growth.

Carnivorous traits might be prevalent throughout the plant communities. Without thorough

investigations into each species, we are not able to truly understand the ecological interactions within

the habitats we are trying to manage. In a landscape that is increasingly being intensely managed for

ecosystem services, due to the demand for land, it is crucial that we understand what we are

managing.

Word count: 7480

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8. Acknowledgements

I would like to acknowledge the people that have made a real difference in my progress up to

finishing this dissertation. First of all I would like to give thanks to my dissertation tutor, Dr John

Warren, for his attentive guidance and support throughout. A special thanks goes out to all of those

that have changed my academic potential and help me overcome my challenges with dyslexia.

Starting at Easton College with Kristine Robinson, Jerry Kingsley, Deborah Carpenter, Hannah Booty

and throughout my dissertation at Aberystwyth University, a special thanks goes to Alison Nash. Last

but no means least, thanks to all the support from a special group of friends in Aberystwyth and

family in Norwich.

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9. Reference

Acton, A. (2012). Issues in Global Environment: Biology and Geoscience: 2011 Edition. Atlanta:

Scloarly Editions.

Adamec, L. (1997). Mineral nutrition of carnivorous plants: a review. Botany Review, 63, 273–299.

Adamec, L. (2006). Respiration and photosynthesis of bladders and leaves of aquatic Utricularia

species. Plant Biology, 8, 765-769.

Agate, E. (1998). The Urban Handbook: A Practical Guide to Community Environmental Work.

Doncaster: British Trust for Conservation Volunteers.

Albert, A., Williams, S., & Chase, M. (1992). Carnivorous plants: Phylogeny and structural evolution.

Science, 257, 1491-1495.

Ashburner, M., & Thompson, J. (1978). The Laboratory culture of Drosophila. The genetics & biology

of Drosophila, 2, 1-81.

Atwell, B., Kriedemann, P., & Turnbull, C. (1999). Plants in Action; Adaptation in Nature. South Yarra:

Macmillan Education Australia PTY Ltd.

Baker, H. (1965). Characteristics and modes of origin of weeds. London: Academic Press.

Barber, J., & Page, C. (1976). Whats New in Plant Physiol. 8(6), 1-5.

Barber, T. (1978). Capsella bursa-pastoris seeds. Are they "carnivorous"? 39-42.

Barber, T., Page, C., & Felscot, A. (1974). Mosq News. 8, 394-398.

Barthlott, W., Porembski, S., Seine, R., & Theisen, I. (2007). A Comprehensive Guide to Their Biology

and Cultivation. Portland: Timber Press.

Behrouziana, F., Razavia, S., & Phillips, G. (2014). Cress seed (Lepidium sativum) mucilage, an

overview. Bioactive Carnhydrates and Dietary Fibre, 3(1), 17-28.

Bohn, H., & Federle, W. (2004). Insect aquaplaning: Nepenthes pitcher plants capture prey with the

peristome, a fully wettable water-lubricated anisomatic surfaces. Proceedings of the

National Academy of Sciences USA, 101, 14138–14143.

Botany Society of America. (2012). Darlingtonia. Retrieved March 19, 2014, from Botany:

http://botany.org/Carnivorous_Plants/Darlingtonia.php

Bradley, I., Gale, P., & Winterbottom, M. (2002). The Heinemann Science Scheme, Book 3. London:

Heinemann.

Chase, M., Christenhusz, M., Sanders, D., & Fay, M. (2009). Murderous plants: Victorian Gothic,

Darwin and modern insights into vegetable carnivory. Botanical Journal of the Linnean

society, 161, 329-356.

Page | 36

Chen, H., Wang, H., & Li, Z. (2007). Production and genetic analysis of partial hybrids in intertribal

crosses between Brassica species (B. rapa, B. napus) and Capsella bursa-pastoris. Plant Cell

Rep., 26(10), 1791-1800.

Christy, M. (1923). The common teasel as a carnivorous plant. Journal of Botany, 61, 33–45.

Commons, H. o. (2007). The UK Government's "Vision for The Common Agricultural Policy"fourth

report of session 2006-07, Vol. 2: Oral and written evidence. London: The Stationery office.

Darwin, C. (1896). Insectivorous plants. New York: Appleton and Co.

De Cauwer, B., Devos, D., Claerhout, S., Bulcke, R., & Reheul, D. (2013). Seed dormancy, germination,

emergence and seed longevity in Galinsoga parviflora and G. quadriradiata. The

International Journal of Weed Biology, Ecology and Vegetation Management, 54, 38-47.

De Rafael, M., Valle, T., Babiano, M., & Corchete, P. (2001). Correlation of resistance and H2O2

production in Ulmus pumila and Ulmus campestris cell suspension cultures inoculated with

Ophiostoma novo-ulmi. Physiol Plant, 111, 512-518.

Deng, W., Iannetta, P., Hallett, P., Toorop, P., Squire, G., & Jeng, D. (2013). The rheological properties

of the seed coat mucilage of Capsella bursa-pastoris L. Medik. (shepherd's purse).

Biorheology, 50(1), 57-67.

DiTomaso, J., & Healy, E. (2007). Weeds of California and Other Western States. Richmond: UCANR

Publications.

Divekar, V., Kalaskar, M., & Redasani, V. (2005). Isolation and characterization of mucilage from

Lepidium sativum Linn seeds. International Journal of Pharmaceutical Research and

Development, 2(1), 1-5.

Duke, J. (2000). Handbook of Edible Weeds: Herbal Reference Library. Florida: CRC Press.

Ellison, A., & Gotelli, E. (2001). Evolutionary ecology of carnivorous plants. Trends Ecology Evolution,

16, 623-623.

El-Maarouf-Bouteau, H., & Bailly, C. (2008). Oxidative signaling in seed germination and dormancy.

Plant Signalling & Behavior, 3(3), 175-182.

Emorsgate . (2013). Wildflower and Grass seeds. Retrieved March 13, 2014, from Emorsgate seeds:

http://wildseed.co.uk/

Eriksson, A. (2007). Induced defense in tomato damaged by Spodoptera littoralis larvae. Alnarp:

Swedish University of Agricultural Sciences.

Eriksson, B., Rubach, A., & Hillebrand, H. (2006). Biotic habitat complexity controls species diversity

and nutrient effects on net biomass production. Ecology, 87(1), 246-254.

Fisher, J., Loneragen, W., Dixon, K., Delaney, J., & Veneklass, E. (2009). Altered vegetation structure

and composition linked to fire frequency and plant invasion in a biodiverse woodland.

Biological Conservation, 142, 2270-2281.

Page | 37

Givnish, T., Burkhardt, E., Happel, R., & Weintraub, J. (1984). Carnivory in bromeliad Brocchinia

reducta, with a cost / benefit model for the general restriction of carnivorous plants to

sunny, moist, nutrient-poor habitats. American Naturalist, 124, 479-497.

Greenberger, R. (2005). Darwin and the Theory of Evolution. New York: The Rosen Publishing Group

Inc.

Grime, J. (1973). Competitive exclusion in herbaceous vegetation. Nature, 242, 344–347.

Grime, J. (1979). Plant strategies and vegetation processes. Chichester: John Wiley.

Haak, D., & Moyle, L. (2013). Ecological Genomics of plant defense in wild tomatoes. Botany 2013 (p.

Abstract). Bloomington: Indiana University.

Haak, D., Moyle, L., & Hahn, M. (2013, December 5). Staple of recipe favorites--the tomato--reveals

processes that maintain biodiversity. Retrieved March 21, 2014, from National Science

Foundation: http://www.nsf.gov/discoveries/disc_summ.jsp?cntn_id=129676

Harper, J., Williams, J., & Sager, G. (1965). The behaviour of seeds in soil. Journal of Ecology, 273-

286.

Harris, E., & Harris, J. (1997). Wildlife conservation in managed woodlands and forests. London:

Nature Publishing Group.

Heck, K., & Wetstone, G. (1977). Habitat complexity and invertebrate species richness and

abundance in tropical seagrass meadows. Journal of Biogeography, 4, 135-142.

Hurka, H., & Neuffer, B. (1997). Evolutionary processes in the genus Capsella (Brassicaceae. Plant

Syst. Evol, 2006, 295–316.

ICUN. (2010). Summary statistics table. Retrieved March 7, 2014, from Red List species:

http://www.iucnredlist.org/documents/summarystatistics/2010_3RL_Stats_Table_1.pdf

INRA. (2014). Seedcorn maggot, Bean seed fly. Retrieved April 4, 2014, from INFA Science & Impact:

http://www7.inra.fr/hyppz/RAVAGEUR/6delpla.htm

Kitchen, T. (1954). Insectivorous Plants. Bios, 25(3), 148-159.

Kleijn, D., Rundlof, M., Scheper, J., Smith, H., & Tscharntke, T. (2011). Does conservation on farmland

contribute to halting the biodiversity decline? Trends in Ecology & Evolution, 26(9), 474-481.

Krebs, J., Wilson, J., Bradbury, R., & Siriwardena, G. (1999). The Second Silent Spring? Nature, 400,

611-612.

Krol, E., Plachno, B., Adamec, L., Stolarz, M., Dziubinska, H., & Trebacz, K. (2012). Quite a few reasons

for calling carnivores 'the most wondeful plants in the world'. Annals of Botany, 109, 47-64.

Kuzina, V., Ekstrøm, C., Andersen, S., Nielsen, S., Olsen, E., & Bak, S. (2009). Identification of Defense

Compounds in Barbarea vulgaris against the Herbivore Phyllotreta nemorum by an

Ecometabolomic Approach. Plant Physiol, 151(4), 1977-1990.

Page | 38

Lambers, H., Brundrett, M., Raven, J., & Hooper, S. (2010). Plant mineral nutrition in ancient

landscapes: high plant species diversity on infertile soils is linked to functional diversity for

nutritional strategies. Plant soil, 334, 11-31.

Lancaster County University. (2014). Insects, spiders, mice and more. Retrieved April 2, 2014, from

UNL Extension in Lancaster County:

http://lancaster.unl.edu/pest/resources/pantrypests304.shtml

Lannetta, P., Begg, G., Hawes, C., Young, M., & Squire, G. (2007). Variation in Capsella (shepherd's

purse): an example of intraspecific functional diversity. Physiologia Plantarum, 129, 542-554.

Lloyd, F. (1942). The Carnivorous plants. New York: Ronald Press.

Loon, J., Boer, J., & Dicke, M. (2000). Parasitoid-plant mutualism: parasitoid attack of herbivore

increases plant reproduction. Entomologia Experimentalis et Applicata, 97, 219–227.

Loxdale, H., Lushai, G., & Harvey, J. (2011). The evolutionary improbability of ‘generalism’ in nature,

with special reference to insects. Biological Journal of the Linnean Society, 103(1), 1-18.

MacDonald, M., Maniakowski, M., Cobbold, G., Grice, P., & Anderson, G. (2012). Effects of agri-

environment management for stone curlews on other biodiversity. Biological Conservation,

148(1), 134-145.

MacPherson, S. (2010). Carnivorous Plants and Their Habitats. Poole: Redfern Natural History

Productions Ltd.

Merbach, M., Merbach, D., Maschwitz, U., Booth, W., Fiala, B., & Zizka, G. (2002). Carnivorous

plants: mass march of termites into the deadly trap. Nature, 415, 387-388.

Millenium Ecosystem Assessment. (2005). Synthesis Reports. Retrieved March 6, 2014, from

Overview of the Milliennium Ecosystem Assessment:

http://www.maweb.org/en/About.aspx

Mohlenbrock, R. (1980). Flowering Plants: Willows to Mustards. Illinois: SIU Press.

Moran, J. (1996). Pitcher dimorphism, prey composition and the mechanisms of prey attraction in

the pitcher plant Nepenthes rafflesiana in Borneo. Journal of Ecology, 84, 515-525.

Moran, J., Booth, W., Charles, & J. (1999). Aspects of pitcher morphology and spectral characteristics

of six Bornean Nepenthes pitcher plantspecies: implications for prey capture. Annals of

Botany, 83, 521-528.

Moyes, L. (2004). Adaptatio in plant speciation; evidence for the role of selection in the evolution of

isolating bariers between plant species. Plant adaptation: Molescular Genetics and Ecology,

82-93.

Nature Education. (2013). The nitrogen cycle: Processes, Players and Human Impacts. Retrieved

March 17, 2014, from Knowledge Project:

http://www.nature.com/scitable/knowledge/library/the-nitrogen-cycle-processes-players-

and-human-15644632

Page | 39

Nelson, W., Ciaccio, E., & Hess, G. (1961). Anal. Biochem. 2, 39-44.

Neuffer, B. (2011). Native range variation in Capsella bursa-pastoris (Brassicaceae) along a 2500 km

latitudinal transect. Flora - Morphology, Distribution, Funtional Ecology of Plants, 206(2),

107-119.

Newsham, K., Fitter, A., & Watkinson, A. (1995). Multifunctionality and biodiversity in arbuscular

mycorrhizas. Trends Ecol Evol, 10, 407-411.

Niklas, K. (1997). The Evolutionary Biology of Plants. Chicago: The University Press.

Page, C., & Barber, J. (1974). Proc. Calif. Mosq. Cont. Assoc. 42, 70.

Page, C., & Barber, J. (1975). Mosq News. 35, 47-54.

Panizzi, A., & Parr, J. (2012). Insect Bioecology and Nutrition for Integrated Pest Management. Boca

Raton: CRC Press.

Pomerai, D. (1990). From Gene to Animal: An Introduction to the Molecular Biology of Animal.

Cambridge: Cambridge university Press.

Raven, P., Evert, R., & Eichhorn, S. (2005). Biology of Plants. New York: W.H. Freeman and Company

Publishers.

Reeves, E., & Garcia, C. (1969). Mosquito News. 29, 601-607.

Rice, B. (2011). Carnivorous plants: reversing the roles of predator and prey. A review of carnivory in

the botanical world, 496-518.

Robert, C., Noriega, A., Tocino, A., & Cervantes, E. (2008). Morphological analysis of seed shape in

Arabidopsis thaliana reveals altered polarity in mutants of the ethylene signaling pathway.

Journal of Plant Physiology, 165, 911-919.

Robinson, R., & Sutherland, W. (2002). Post-war changes in arable farming a biodiversity in Great

Britain. Journal of applied ecology, 39, 157-176.

Royal Botanic Gardens. (2014). The environment. Retrieved March 14, 2014, from Kew Royal Botanic

Gardens: http://www.kew.org/science-conservation/environment/challenges/how-many-

flowering-plants

Saxena, K., & Rembold, H. (1984). Attraction of Heliothis armigera (Hübner) larvae by chickpea seed

powder constituents. Journal of applied Entomology, 97(1-5), 145-153.

Schnell, D. (2002). Carnivorous Plants of the United States and Canada. Markham: Timber Press.

Shaw, P., & Shackleton, K. (2011). Carnivory in the Teasel Dipsacus fullonum — The Effect of

Experimental Feeding on Growth and Seed Set. Plos One, 6(3).

Sheldon, J. (1974). The Behaviour of Seeds in Soil: III. The Influence of Seed Morphology and the

Behaviour of Seedlings on the Establishment of Plants from Surface-Lying Seeds. Journal of

Ecology, 64(1), 47-66.

Page | 40

Silver, J. (2007). Mosquito Ecology: Field Sampling Methods. Dordrecht: Springer.

Simons, P. (1981). How exclusive are carnivorous plants? Carnivorous Plant news Letter, 61-81.

Slack, A. (2010). Carnivorous Plants. Massachusetts: MIT Press.

Smith, S., & Read, D. (2008). Mycorrhizal symbiosis (3rd ed.). London: Academic Press and Elsevier.

Stinson, K., Campbell, S., Powell, J., Wolfe, B., & Callaway, R. (2006). Invasive Plant Suppresses the

Growth of Native Tree Seedlings by Disrupting Belowground Mutualisms. Plos One Biology,

4(5).

UNEP. (1995). Global Biodiversity Assessment. Cambridge: Cambridge University Press.

Utah State University. (2014). Weed guide - Mustard family. Retrieved March 26, 2014, from

Extension Utah State University:

http://extension.usu.edu/weedguides/files/uploads/brassicaceae.pdf

Weitbrecht, K., Muller, K., & Leubner, G. (2011). First off the mark: early seed germination. Journal of

Experimental Botany, 1-21.

Winde, I., & Wittstock, U. (2011). Insect herbivore counteradaptations to the plant glucosinolate-

myrosinase system. Phytochemistry, 72(13), 1566-1575.

Woodstock, L. (1988). Seed imbibition: a critical period forsuccessful germination. Journal of Seed

Technology, 12, 1-15.

Ying, A., Dun, Y., Tan, A., Carol, C., Baskin, A., Jerry, M., et al. (2012). Role of mucilage in seed

dispersal and germination of the annual ephemeral Alyssum minus (Brassicaceae).

Australian Journa of Botany, 60(5), 439-449.

Young, J., & Evans, R. (1973). Mucilaginous Seed Coats. Weed Science, 21(1), 52-54.

Page | 41

10. Appendix

Attraction and pupation data

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Seeds A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa

Bittercress sample 1 20 1 0

20 6 0

18 13 2

17 14 3 3 17 17 3 3

0

sample 2 18

2

19 6 1

20 6 0 20 8

0 20 10

0 20 12

0

sample 3 18 1 2

20 5 0

20 7 0

19 8 1 1 18 8 2 1 19 14 1 1

sample 4 19 2 1

19 4 1 1 17 5 3 3 17 7 3 3 18 7 2 2 17 13 3 2

sample 5 20 3 0

19 3 1

17 3 3 1 18 6 2 2 17 8 3 3 14 10 6 3

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Seeds A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa

Shepherds Purse sample 1 16

4

12 5 8 6 11 8 9 9 10 8 10 9 8 8 12 10 10 9 10 10

sample 2 19

1 1 11 3 9 2 11 3 9 6 11 3 10 7 8 3 12 10 6 6 14 14

sample 3 17 1 3 1 15 3 5 3 12 5 8 5 12 7 8 7 7 7 13 12 7 7 13 13

sample 4 14

6

13 3 7 3 11 5 9 7 10 8 10 9 8 8 12 11 7 7 13 13

sample 5 19

1

18 3 2

16 4 4 3 16 9 4 3 15 11 5 4 15 15 5 5

Page | 42

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Seeds A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa

Garlic Mustard sample 1 20 9 0

16 10 4

18 11 2

20 12 0

17 16 3 2 17 17 3 2

sample 2 20 3 0

16 4 4 1 17 6 3 2 15 8 5 4 15 14 5 4 16 16 4 4

sample 3 20 4 0

20 11 0

19 11 1

17 2 3 1 19 3 1 1 19 18 1 1

sample 4 20 1 0

17 1 3 2 16 1 4 3 17 4 3 3 17 7 3 3 17 17 3 3

sample 5 20

0

19 6 1

19 7 1

18 7 1

18 13 2 1 18 4 2 1

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Seeds A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa

Tomato sample 1 20 3 0

17 4 3

16 5 4

18 9 1 1 17 15 3 3 17 16 3 3

sample 2 20

0

16 12 4

15 14 5

16 14 4 3 16 16 4 4 sample 3 20 4 0

14 5 6

18 6 2

17 11 3 1 19 18 1 1 19 19 1 1

sample 4 20 3 0

11 7 9 1 16 8 4 1 15 8 5 4 15 15 5 5 sample 5 20 5 0

19 8 1

14 10 6 6 13 10 7 7 13 12 6 1 13 13 7 7

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Seeds A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa

Cress sample 1 20 4 0

20 9 0

19 11 1

19 17 1 1 18 17 2 1 17 17 3 3

sample 2 20 1 0

20 3 0

19 5 1 1 18 5 2 2 sample 3 18 2 2

19 5 1

19 8 1

19 8 1 1 19 9 1 1 19 18 1 1

sample 4 18 2 2

20 2 0

19 2 1

20 2 0

19 3 1 1 19 19 1 1

sample 5 20 1 0

18

2 1 19 3 1 1 19 3 1 1 19 19 1 1

Page | 43

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Seeds A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa A Pupa B Pupa

Control sample 1 20

0

19

1

17

3

16

4

15

5

18

2

sample 2 20

0

20

0

20

0

20

17

3

19

1 sample 3 19

1

20

0

18

2

17

3

17

3

19

1

sample 4 20

0

19

1

19

1

19

1

20

0

17

3 sample 5 20

0

19

1

19

1

16

4

17

3

19

1

Page | 44

Mortality rates

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Seeds Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died

Shepherds P sample 1 14 2 4 6 6 8 4 8 8 2 10 8 1 10 9 1 10 9

sample 2 16 2 2 10 4 6 5 6 9 1 8 11 0 8 12

8 12

sample 3 7 3 10 6 5 9 3 5 12 0 7 13

13

7 13

sample 4 13 5 2 8 6 6 4 6 10 4 6 10 1 7 12 0 8 12

sample 5 14 3 3 8 5 3 8 7 5 2 8 10 0 8 12

8 12

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Seeds Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died

Bittercress sample 1 9 3 8 7 4 9 5 6 9 3 6 11 2 6 12 1 7 12

sample 2 20 0 0 13 1 6 6 3 11 1 5 14 0 5 15

5 15

sample 3 13 4 2 10 4 6 8 6 6 4 10 6 4 10 6 4 10 6

sample 4 16 1 3 11 4 5 9 6 5 2 9 9 2 9 9 1 10 9

sample 5 8 10 2 7 11 2 6 12 2 3 12 5 3 12 5 2 13 5

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Seeds Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died

Garlic mustard sample 1 17 3 0 14 4 2 9 6 5 8 7 5 5 10 5 5 10 5

sample 2 18 2 0 15 2 3 11 5 4 9 7 4 9 7 4 5 8 7

sample 3 13 3 1 11 3 6 8 3 9 7 3 10 5 5 10 5 5 10

sample 4 18 1 1 15 3 2 11 4 5 6 6 8 4 6 10 4 6 10

sample 5 18 1 1 14 2 4 9 4 7 9 4 7 6 4 10 6 4 10

Page | 45

Day 1

Day 1 Day 2

Day 3

Day 4

Day 5

Day 6

Seeds Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died

Tomato sample 1 18 1 1 13 2 5 8 5 7 6 5 9 5 6 9 4 7 9

sample 2 17 3 0 15 4 1 10 5 5 7 5 8 6 5 9 4 7 9

sample 3 18 2 0 15 2 3 10 2 8 6 6 8 5 7 8 5 7 8

sample 4 20 0 0 16 1 3 8 3 9 6 4 10 2 4 14 1 5 14

sample 5 18 1 1 13 2 5 8 2 10 8 2 10 7 2 11 7 2 11

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Seeds Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died

Cress sample 1 20 0 0 16 2 2 11 4 5 5 5 10 3 5 12 3 5 12

sample 2 18 2 0 14 3 3 9 4 7 6 4 10 5 5 10 4 5 11

sample 3 20 0 0 15 1 4 11 2 7 6 5 9 4 7 9 1 9 10

sample 4 18 2 0 15 3 2 12 4 4 8 5 7 5 6 9 4 7 9

sample 5 20 0 0 16 1 3 11 2 7 8 4 8 4 4 12 3 5 12

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Seeds Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died Alive Pupa Died

Control sample 1 20 0 0 19

1 19

1 19

1 19

1 19

1

sample 2 20

0 19

1 19

1 18

2 18

2 17 1 2

sample 3 20 0 0 20 0 0 11 20 0 20 0 0 20 0 0 17 0 3

sample 4 20 0 0 20 0 0 20 0 0 20 0 0 19 0 1 19 0 1

sample 5 20 0 0 20 0 0 20 0 0 20 0 0 20 0 0 17 0 3

Page | 46

Height records

Day Sap 1 2 3 4 5 6 7 8 9 10 11 12 13 14 SP seed only

sample 1 1 1 4 9 9 10 11 12 12 12 12 12 13

2 sp 2 4

sample 2 1 sp 1 3 5 7 8 8 8 8 8 8 8

2 sp 2

sample 3 1

sample 4 1 3 4 5 7 7 7 7 7 8 8 8 8

2 1 1 1 2 2 3 3 3 3 3 3 3

sample 5 1

1 2 3 4 6 6 7 8 7 7 7 7

Day Sap 1 2 3 4 5 6 7 8 9 10 11 12 13 14 SP w/larvae

sample 1

sample 2 1 1 7 9 9 9

sample 3 1 sp sp 2 2 4

sample 4 1 sp 3 5 6 6 7 7 7 7 7 7

2

sp 3 4 4 6 8 8 8 8 8 8

3

2 3 4 4 4 4 4 4

4 3 5 6 7 8 8 9

sample 5 1 sp 3 5 6 6 7 7 7 7 7

2 sp 3 5 6 6 7 7 7 7 7

Page | 47

Day Sap 1 2 3 4 5 6 7 8 9 10 11 12 13 14

SP w/soil sample 1 1 1 2 4 7 9 10 10 10 11 11 11 11

2 1

sample 2 1 1 3 5 8 9 10 10 10 10 10 10 10

2

sp 3 5 6 7 7 7 7 7 7

3 2 4 4 5

sample 3

4

sample 4 1 1 3 6 8 8 9 9 9 9 9 9

sample 5 1 sp 3 4 7 8 9 9 9 9 9

Day Sap 1 2 3 4 5 6 7 8 9 10 11 12 13 14 SP w/soil+Larvae

Sample 1 1 sp 1 2 5 7 9 11 11 11 11 12

sample 2 sample 3 sample 4 sample 5 1 sp 3 6 9 10 10 10 10 10 10 10

2 2 2 2 3

Sp = sprouting