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Natural Born Killers

Wax moth larvae infected with juvenile Heterorhabditis nematodes to allow nematodes to grow into their adult forms. Adult nematodes are collected from the water surrounding the moth larvae and used in further experimentation.

Client Natural Insect Control
Professor(s) Charles Nasmith
Program Biotechnology Advance Program
Students Dara Palmer and Autumn Henry

Project Description:

Pests can do a large amount of damage to important food crops and one of the most damaging pests is insects. The current method for dealing with insect pests is to spray crops with chemical pesticides. Unfortunately, pesticides are environmentally damaging, expensive, and dangerous to both human and animal health. Thus, the study of using Entomopathogenic nematodes (EPNs) as biological control agents can provide a safer, cheaper and more environmentally friendly alternative to traditional pesticide use. Insect larvae are part of the life cycle of nematodes as juvenile nematodes will enter the pores of insect larvae and once inside the larvae, they will release bacteria. This bacteria kills the larvae allowing the nematodes to grow into their adult forms inside the larvae. Once the nematodes have reached full maturity, they will recollect the bacteria and exit the dead larvae. As the life cycle of nematodes is dependent on an insect host, scientists have begun to use this cycle as a biological control agent for food crops. However, research on nematodes is lacking and more must be completed before nematodes can be seen as efficient insect control agents.
For our experiment, we received four nematode species/populations from the Natural Insect Control (NIC) Company, which included Steinernema carpocapsae, Steinernema feltiae, Heterorhabditis bacteriophora and an unknown species. We focused on identifying differences between the four nematode species/populations and determining their optimal storage/survival temperature. Since nematodes have very few visual differences between them, they have to be identified using their DNA structures. In order to complete this, DNA was extracted from each species/population and analysed to determine any differences between their DNA sequences. Overall, we were able to identify genetic differences between the DNA of the four species/populations and determine that the unknown population of nematodes is likely to be another Heterorhabditis (i.e we received two populations of Heterorhabditis) population due to its similar DNA structure.
Once each species/population had been identified, their optimal storage and survival condition was determined. All four species/populations were split into 3 smaller sample sizes and these were stored in water at three different temperature points, 4°C, 10°C and 19°C respectively. They were kept in these temperature for 4 weeks with their viability (amount of living nematodes compared to dead nematodes) percentage being calculated each week. After the four weeks were completed, we determined that the two Steinernema populations survived effectively in all three temperatures as their population numbers did not diminish significantly over the test period. In comparison, the two Heterorhabditis populations survived at the 10°C and 19°C temperatures but they rapidly lost viability each week at the 4°C temperature point until the fourth week where both populations had nearly no surviving nematodes.
Using our DNA identification of the species/populations and the data from our determined optimal storage and survival temperature, the NIC Company can promote and identify their products with greater accuracy. This allows for both the company and clients to use their nematode products more effectively as insect biological control agents.


Short Description:

Our research aims to DNA fingerprint nematode species and find their optimal survival temperature to create a more capable insect pest control agent.



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Video Presentation


Gallery

24 hours after Galleria (Wax moth) larvae have been infected with juvenile nematode strains. Dark purple and dark brown larvae in the image are dead, light brown and yellow larvae are alive. After another 24 hours, all the moth larvae will be killed by the nematodes. After larvae have all died, they are placed on a system called a water trap, which involves placing the larvae cadavers on top of a filter paper with the edges of the paper sitting in water. The nematodes inside the cadavers will begin to move out of the larvae in search of the water.
This image shows the adult nematodes collected from the water traps. These nematodes are usually less than 1 mm in size, so they can only be viewed under a microscope. This image is an electrophoresis gel, which allows for the comparison of DNA. Each line on the gel represents a specific size of DNA, different species/populations will have different DNA sizes which causes their lines to appear differently.
This graph represents the viability of each nematode species after 4 weeks of incubating in three separate temperatures to determine what the optimal storage and survival temperature of each species is.


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