Team:KU Leuven/Project/Aphid Background

Synopsis

During a long period of co-evolution, plants and aphids have established a complex interaction . On the one hand, aphids have established complex life cycles involving extensive phenotype plasticity with rapid population growth (Blackman and Eastop, 1984) and a very short generation time (Morrison and Peairs, 1998). On the other hand, plants have also evolved delicate and complicated defence systems comprising constitutive defence traits and defence pathways induced upon aphid attack (Chen, 2008). Breeders and growers are still struggling to find an efficient strategy for aphid control in major crop plants, as the damage can run in the millions. The KU Leuven iGEM project has an answer to this problem. Our project consists of a genetically engineered bacteria that can reduce the aphid damage on a sustainable way by hacking into their communication system.

Basic aphid biology

Aphids are an extremely successful group of insects that can be found throughout the world, with the greatest number of species in temperate regions such as Europe, North America and Asia. An individual aphid is small and unnoticeable, however, they can become so numerous that they can damage whole fields or orchards. Their complex life cycles and polymorphisms enables them to exploit their host plants and to respond quickly to changes in their environment (Dixon, 1973). Aphids are small, mostly soft-bodied insects of the super family Aphidoidea (Minks and Harrewijn, 1987). Their body can be reddish, yellow, green, black brown, or almost colourless (Dixon, 1973). About 4400 species of 10 families are known and around 10% of these species are serious pests for agriculture and forestry. Worldwide, the annual crop losses due to aphids are estimated at hundreds of millions of dollars (Morrison and Pears 1998).

How aphids feed on plants

Aphid damage to plants can occur in various ways, mainly by stealing nutrients . Aphids use modified mouthparts, called stylets , to obtain their food by sucking plant juices (Minks and Harrewijn, 1987). Normally, aphids passively feed on phloem sap of their host plants, incidentally they can pierce a xylem vessle. Once a phloem vessel, which is under high pressure, is punctured, the sap is forced into the aphid’s stylet. Phloem sap is rich in sugars, but poor in amino acids, which are essential for growth. Therefore, aphids ingest a very large amount of food in order to acquire a sufficient amount of proteins. The residual digested food, which consists mainly of sugars, is excreted into the exterior in the form of a droplet of honeydew (Dixon, 1973). This sugary liquid is produced through a ‘tail’ at the rear end of the aphid, called a cauda. Most aphids also have paired tubular structures found at the end of the abdomen, called cornicles. Through these cornicles, aphids excrete droplets of a quick-hardening defensive fluid containing triacylglycerols, the cornicle wax. Other defensive compounds, such as alarm pheromones , can also be secreted by the cornicles (Minks and Harrewijn, 1987).

Life cycles

Aphids have a various patterns of complicated life cycles and occur in various types of polymorphisms . These polymorphisms are in relation to two biological characteristics of the aphid life cycle: alternation of sexual and parthenogentic generations and alternation of host plants (Minks and Harrewijn, 1987). Most aphids are parthenogenetic (asexual) and viviparous (give birth to living offsprings) for most of the year, but are also capable of sexual reproduction with the production of eggs during the winter. The annual cycle generally (97% of all species) includes a single sexual generation. These mating females (oviparae) lay eggs, all of which are female. On hatching, each egg gives rise to a wingless offspring that gives birth parthenogenetically to further parthenogenetic females (viviparae). These may be winged or wingless, in response to environmental conditions (Dixon, 1973). Crowding is a particularly strong stimulus to develop aphids of the winged, dispersal stage. Other external factors also play a role in the morph determination: food availability and quality, predators, photoperiod, time of the year and so on (Minks and Harrewijn, 1987).
In addition, aphid species have evolved a wide range of adaptive mechanisms based on the seasonal development of vegetation (Dixon, 1973). Many aphid species are monophagous, meaning they feed on only one plant species. In contrary to the economically important aphids, who have a wider host range and are therefore polyphagous (Blackman and Eastop, 1985). Polyphagous aphids have a primary woody plant during autumn, winter and spring and a secondary herbaceous plant in the summer. During bud burst and leaf development in spring and leaf senescence in autumn, trees are a richer source of food for aphids than in summer, when the leaves are mature. This has resulted in host alternation, in which aphids leave trees in summer and colonize the more recently evolved herbaceous plants that grow and flower throughout summer (Mordvilko, 1928).
In conclusion, aphids can rapidly exploit agricultural landscapes because of their high reproductive potential, endless source of food, dispersal capacities, adaptability to local environment, short generation time and feed on an endless source of plants (Dedryver et al., 2010).

Damage to plants and solutions

Aphid damage to plants may be caused directly , by nutrient drain, or indirectly by several mechanisms. These indirect mechanisms include plant virus transmission through the aphids’ stylet. Injection of saliva containing phytotoxic components has a disastrous effect on plant growth and harmful fungi growth on the aphids’ honeydew hinders photosynthetic activity. Common damage symptoms on plants are decreased growth rates, mottled leaves, yellowing, stunted growth, curled leaves, gall formation and low yields and death (Dixon, 1973). Different methods can be used to control an aphid infestation. For many crops, insecticides provide a simple and effective strategy for aphid control. However, the application of such chemicals is not desirable in the long term , because of the development of insecticide resistance and the potential negative effects on the population dynamics of other beneficial insects. Aphids are vulnerable to many kinds of predators, including ladybird beetles and parasitoid wasps, and parasites which can also be used to biologically control aphids (Minks and Harrewijn, 1987).

Economical important crops

Aphids represent the most important group of insect pests in agriculture in temperate Europe. They can infest a wide range of arable and horticultural crops in a very short period of time, which is why farmers are advised to treat this aphid pest problem from the very first symptom . The most widespread species in Europe are: green peach aphid (Myzus persicae), Bird cherry-oat aphid (Rhopalosiphum padi), potato aphid (Macrosiphum euphorbiae) and Black bean aphid (Aphis fabae).
In general, aphids reduce crop yield and/or quality, which lead to significant economical losses. 
The major crops infected are wheat, barley, sugar beet, potatoes, lettuce, and beans . The damage caused by aphids depends on the type, for instance the black bean aphid causes damage by direct feeding whereas the green peach aphid causes the most damage by being a virus vector for more than 120 viruses (Rothamsted insect survey).
The average percentage loss caused by different aphid species is variable, ranging from 4,4% on potatoes, since these plants are rather resistant to aphids, compared to 46-43% on field beans . In 1979, aphids caused a potential economic loss of £70 x 106 in Britain. In the US, aphids have an economic impact of more than one-quarter billion dollars in an outbreak year (Larsson et al., 2005).

Research groups

Due to the high impact of aphids on crop damage, several research groups have been trying to tackle this problem by the use of biological pest control.
Rothamsted research group is currently investigating the use of genetically modified crops that produce E-beta-Farnesene in order to repel aphids. They are carrying out a two year experiment, the Rothamsted wheat trial.
Other research groups we worked together with is Biobest and pcfruit. Biobest offers a whole series of parasitoids and predators against aphids for the purpose of biological control of pests. Biobest green lab investigates the relationship between plant and aphid in search of a method to protect plants from this pest. Pcfruit has a similar mission, but more focused on fruit trees. The advice that Biobest and pc fruit provides is very well appreciated amongst the farmers.
APPEAL, an organisation that stands for ‘Assessment and valuation of Pest suppression Potential through biological control in European Agricultural Landscapes’, is controlling aphids by providing natural enemies in order to create a natural ecosystem service has an immense economic value.

Chemical control of aphids - aphicide

Several factors need to be taken into consideration when choosing a good aphicide. An aphicide should be selectively toxic to aphids so that predators and parasitoids of aphids, as well as pollinating insects, are not killed in the process. This is important especially in fruit growing. Systemic action of an aphicide is essential, in order to reduce the number of treatments, since aphids can colonise a plant almost continuously. This means that application of the aphicide could be on the foliage, in the soil or to the seed, and would still be able to carry out its function. Residual activity is favoured to treat crops that are often re-infested by aphids but this is not favoured for food crops, as the aphicide should be decomposed into harmless compounds before harvest. In order to prevent transmission of plant viruses, aphicides should act rapidly. Besides this aphicides should not harm the plant any further; low phytotoxicity (Schepers, 1989).
It is difficult for one chemical compound to comply with all these requirements and on top of that, the induction of aphid resistance should be avoided. Pest resistance to a pesticide is commonly managed through pesticide rotation, which involves alternating among pesticide classes with different modes of action to delay the onset of or mitigate existing pest resistance. The use of pesticides has many disadvantages to which the BanAphids should provide a solution.

Human health

Pesticides can enter the human body through inhalation of aerosols, dust and vapour that contain pesticides; through oral exposure by consuming food and water; and through dermal exposure by direct contact of pesticides with skin. Pesticides are sprayed onto food, especially fruits and vegetables, they secrete pesticides into soils and groundwater, which can end up in drinking water, and pesticide spray can drift and pollute the air. For all pesticides to be effective they must be toxic. Insecticides cause the greatest number of pesticide poisonings in the United States (Lorenz, 2009), but their toxicity can also be harmful to other animals, organisms and the environment.
The effects of pesticides on human health are more harmful based on the toxicity of the chemical and the length and magnitude of exposure; Hazard = Toxicity x Exposure (Lorenz, 2009). Insecticides targeted to disrupt insects can have harmful effects on the nervous systems of mammals, due to similarities of vertebrate structure. Organophosphate insecticides inhibit the enzyme cholinesterase causing an accumulation of acetylcholine in the body resulting in uncontrolled neuron transmissions. These chemicals can poison all life forms with cholinesterase in their nervous system, such as insects, fish and birds. The biggest concern when using pesticides should be, how long it takes for these chemicals to fully be degraded . These chemicals can bioaccumulate in the body over time as well as bioaccumulate in animals of our food chain such as fish and chicken.

Resistance

Pesticide resistance occurs when a pest population can no longer be controlled effectively by a pesticide that was previously effective at controlling the pest. Pest species evolve pesticide resistance via natural selection: the most resistant organisms are the ones to survive and pass on their genetic traits to their offspring. Their are several ways in which resistance can occur via natural selection (Daly, H., 1998).
First, pest species are usually capable of producing a large number of offspring. This increases the probability of random mutations and rapid collection of pesticide resistant aphids. Secondly, as a result of co-evolution of herbivores and their host plants, numerous physiological capabilities to detoxify or tolerate phytotoxins have been developed. Thirdly, humans often rely almost exclusively on insecticides for pest control. This increases selection pressure towards resistance. Pesticides that have long residual activity contribute to selection for resistant organisms even after they are no longer being applied (Daly, 1998).
This resistance problem is most persistent, widespread and serious in a few aphid species such as Myzus persicae and Aphis gossypii. This seems to be associated with situations where a large proportion of the population is subjected to strong selection pressure for example, their occurrence in green houses means they reproduce continuously throughout the year and are therefore heavily selected with insecticides. The biochemical nature of resistance in Myzus persicae was found to be a high copy number of esterase genes according to their degree of resistance (Field et al., 1988).

Destruction of beneficial insects – biological pest control

The indiscriminate use of insecticides can destroy biodiversity by killing off non-target organisms. Biological pest control is a commonly used alternative to pesticides today. This means that the natural predator or parasitoid of a pest insect is released into the area of infestation in order to control the pest population. For example, natural predators such as the green lacewing or the ladybug could be used to control an aphid population. However when insecticides are also sprayed on these areas, it could kill both the pest and the beneficial predators or parasitoids. Muckenfuss et al. showed that the use of insecticides on diamondback moths (a cabbage pest) created a rebounded pest population due to the loss of its natural predators. This example can be compared to the misuse of antibiotics. Faulty use of antibiotics will kill off the intended bacteria population but allow the growth of mutated bacteria that was normally suppressed by the intended bacteria population. This phenomenon, wherein the population of a pest species rebounds to equal or greater numbers than it had before pesticide use, is called pest resurgence and can be linked to elimination of predators and other natural enemies of the pest. A related phenomenon is called secondary pest outbreaks, an increase in problems from species which were not originally very damaging pests due to loss of their predators or parasites (Daly et al., 1998).

Environment

Pesticides can travel great distances through the environment. When sprayed on crops or in gardens, pesticides can be blown by the wind to other areas. This process is called pesticide drift. Water-soluble pesticides can flow with rainwater into nearby streams or can seep through the soil into ground water. This means that the pesticides may run off to other areas and cause damage to un-targeted animals and plants in other places, as well as contaminating our water supply. Many pesticides are soil contaminants that can have an adverse long-term effect. Due to the loss of organic matter and catabolic microorganisms in the soil, there is a decrease in water retention and catabolism of organic substances (Warsi, 2004). On top of this, insecticides that remain in the soil inhibit nitrogen fixation, which is required for plant growth. Insecticides can also have a direct harmful effect on plants such as poor root hair development, shoot yellowing and reduced plant growth (Rockets, 2007).
Some pesticides can remain in the environment for many years and pass from one organism to another. Fat-soluble pesticides are readily absorbed in insects, fish and other animals, often resulting in retention and persistence of pesticides in food. DDT, for instance, is a fat-soluble pesticide and can accumulate in organisms by a process referred to as bioamplification. A small amount of pesticide can be absorbed into bodies of animals lower in the food chain, the higher the trophic level, the greater the concentration of toxins. The top carnivores hence have a highly concentrated level of toxins and negatively influence this population. As a result, the balance in the ecosystem could be disrupted (Warsi, 2004).

Green peach aphid

In our experiments, we used Myzus persicae or the green peach aphid, a small generalist aphid that can be found worldwide. The green peach aphid attacks plants in the field as well as in greenhouses . Their primary overwintering host is Prunus sp. (peach trees and their hybrids). However, in the summer, they leave their primary host to feed on a wide range of plants including vegetable crops, weeds and ornamentals.
Temperature greatly influences the lifecycle of the green peach aphid, especially cold winters. Aphids overwinter in the egg stage on Prunus trees. As temperatures warm, the aphid may go through multiple generations while still on the tree, but as densities increase, winged adults are produced. These winged forms colonize nearby plants by depositing daughters on one plant and then moving on to the next plant. Each daughter begins asexual reproduction (parthenogenesis) by rapidly giving birth to new daughters, which often already have developing daughter inside them ready to emerge in a few days. As densities increase on the newly colonized plants, and as the plants deteriorate, new winged forms are produced. This cycle repeats as long as the weather is favourable. Eventually, as day length begins to shorten and temperatures change, the winged females search for Prunus trees to colonize. Females arrive on the overwintering locations first and give birth to wingless egg laying forms (oviparae), which mate with winged males.

Adalia bipunctata

In our experiments we used the two-spotted ladybug. Adalia bipunctata, commonly known as the two-spotted ladybug, is a carnivorous beetle of the family Coccinellidae that is found in western and central Europe and North America. The two-spotted ladybeetle feed on aphids and other small insects. The wing covers of adult two-spotted lady beetles are bright-red with one black spot on each side (Marshall, 2000). Its life cycle starts with bright yellow eggs and ladybugs pass through four larval stages. The larvae and the adults are predators. The larvae of older ages move actively on the whole plant for the search of food and also migrate to other plants. The adult individuals keep not far from preys where they mate and lay the eggs. Therefore, ladybugs are frequently used in ornamentals, fruit and vegetable crops as beneficial to control aphids, for example in greenhouses .

Macrolophus pygmaeus

In our experiments, we also used Macrolophus pygmaeus as aphid predator. Macrolophus pygmaeus is a bright green and long-legged predatory. After the birth of the nymphs, they pass through five mobile stages. During the first stages nymphs are yellowish green, but older nymphs are bright green as the adults. In the last two stages the growth of the wings can be seen. Macrolophus predates several pest insects aphids, whiteflies, spider mites and moth eggs. Adult and nymphs search actively for their prey, insert their sucking mouthparts and suck out the contents. Macrolophus pygmaeus is very common in the Mediterranean, therefore it is mostly used as biological control in greenhouses.

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