Mar 15, 2005

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Transgenic fructane potatoes – possible effects on the potato beetle

(2001 – 2004) Justus Liebig University Giessen, Institute of General and Special Zoology, Working Group: Cellular recognition and defence processes (ZEAP)

Topic

Fructane potatoes have a modified carbohydrate metabolism. They produce a higher content of soluble sugars with new sugar components.

The altered carbohydrate situation in the potato presents a different nutritional quality for pests. That means changes in the feed quality of fructane potatoes can be assumed and that the condition and fitness of eating and suckling pest insects can be affected.

In this project the fitness of the potato beetle in particular, as the most important pest in potatoes, is to be examined.

Summary

The data available so far show now noticeable change in the trophic (feeding) relationships between the various transgenic fructane potatoes and the Potato beetle (a herbivore). The physiology of the potato beetle does not appear to be substantially altered by eating the transgenic fructane potatoes. Therefore, no increased risk is to be expected from a change in pest numbers.

Experiment description

The fitness of the Colorado beetle (Leptinotarsa decemlineata) are tested accorded to parameters of reproduction and egg production, as well as resistance to disease. Reproduction and disease resistance are physiologically processes which take a lot of energy. A change in the quality of the feed can lead to a reduced or increased rate of reproduction and/or to a weakening, or strengthening, of the immune system against microbes and parasites.

Larvae and newly emerged beetles eating

In 2003 and 2004 tests glasshouse tests were carried out combined with work on the field test site of the Federal Biological Research Centre for Agriculture and Forestry (BBA) in Kleinmachnow.

Rearing the animals. The research was carried out on various stages of development of the Colorado beetle (larvae, pupae and imagos (fully formed insects). As feed, for comparison purposes, leaf material from transgenic fructane potatoes and conventional varieties was made available. The beetles were reared in the glasshouse on an isogenic potato line for the laboratory tests. Insects from the final larval stage as well as freshly emerged beetles and sexually mature beetles were used for the tests.

Results

Blood analysis. Insects possess an open circulation system, in which blood and circulatory fluid (haemolymph) can circulate between the organs freely. Nutrients are transported and foreign bodies immunologically killed, digested (phagocyted) or engulfed. The blood analysis of the individuals tested gives information on the nutritional situation (sugar value) as well as on the immune reaction to pathogens.

Fig.1 Plasmatocyte (Haemocyte (Blood corpuscle) with cell runners) are at up to 95% the greatest proportion of the total haemocyte population in haemolymph (blood and circulatory fluid) of the Colorado beetle.

Fig.2 Where there is antibacterial activity in the haemolymph tests of infected animals so-called hemolysis is formed in the agar (arrow).

Fig.3 Plasmatocytes absorb the haemoloymph by means of phagocytosis of bacteria. In phagocytosis, bacteria or fungal spores are taken up by mobile cells, digested and so rendered harmless.

Fig.4 Bacterium culture plates with reductions from the intestine

Fig.5 Egg skin with an egg cell from a Colorado beetle female almost ready to lay.

Fig.6 Fractionalized hemolymph samples to display vitellogenins in female potato beetles.

Haemocytes (blood corpuscles) are easy to take from the Colorado beetle and show a noticeable change in composition after infection with pathogens. With the help of tissue tests and cell biology analyse the different haemocyte types of the Colorado beetle can be distinguished and, in part, allotted a specific function. So far no difference in the blood analysis between beetles fed with conventional or genetically modified potato plants could be found.

Number of haemocytes (blood corpuscles). In the final larval stages until the beginning of the pupal stage a continual growth in the number of haemocytes can be observed. The number of haemocytes again falls sharply in the pupal phase. After injection with bacteria (Micrococcus luteus) the number of haemocytes changes. It is completely different in infected animals to in untreated control animals. A comparison of the haemocyte count in larvae, which have been reared on various transgenic lines and wild varieties, showed no differences. However the total blood cell count in animals on another conventional variety of potato was significantly higher than the haemocyte count in the larvae from transgenic plants and the wild type.

Assessment of antibacterial activity. The defence potential against bacteria and fungi was assessed using the haemolymph protein spectrum. For this purpose larvae and imagos were taken from conventional and the genetically modified fructane potatoes, infected with chosen test bacteria and fungi and after a defined time their haemolymph tested for antimicrobial activity.

As the antimicrobial factors are, as a rule, proteins, an induced defensive reaction can be recognised by the change in the haemolymph protein spectrum.

In the haemolymph of the Colorado beetle antibacterial activity against various plant-associated bacteria could be shown 24 hours after bacterial infection (with Micrococcus luteus).

The research has so far shown no sign of differences in the antibacterial activity of the haemolymph between animals on conventional or genetically modified potato plants.

Phagocytosis activity . The engulfing of foreign bodies is an important cellular reaction in the defence mechanism of insects against infection. In phagocytosis, bacteria or fungal spores are taken up by mobile cells, digested and so rendered harmless. The phagocytosis performance can be tested using simple injection tests with marked microbes. The two haemocyte types plasmocyte and granular cells are responsible for the cellular immune response of the Colorado beetle. Plasmocytes can eliminate bacteria using phagocytosis (see figure 3).

Tests of gut flora. The composition of the bacterial gut flora was assessed in correlation with feeding on the potato lines and isolated bacteria were tested for their sensitivity to the antibacterial factors. So far no difference between the test beetles on transgenic and isogenic potato lines has been found.

Changes in the reproductive performance of the Colorado beetle. To assess the reproductive states the level of the maturity of the ovaries and the yolk protein (vitellogenin) content of the haemolymphs from female beetles was established. These parameters are clear and easy to assess. The tests were carried out on female beetles from climatic chamber tests as well as from field tests. No changes in protein content resulting from the genetic modification of the feed plant were found.