| Popis: |
In recent years changing agricultural conditions are leading to insect pests becoming a more serious threat to UK crop production for a variety of local and global factors. Two important factors in these changes are: (i) increasing public intolerance of the use of exogenously applied chemical insecticides which can have negative effects both in terms of public health (through exposure to residues on food crops) and the environment; and (ii) changes in the distribution and abundance of insect pests as a result of increased mean temperatures resulting from global climate change, which will alter the pest breeding habits and periods of damage. In terms of environmental effects, are the effects on non-target organisms including beneficial insects that play a role in the suppression of insect pest populations. To control pests, the development of novel pesticides should focus on non-chemical alternatives derived from natural sources. In the present study venom proteins were isolated and purified from the parasitoid wasp Eulophus pennicornis (Hymenoptera: Eulophidae) and the sea anemone Actinia equina (Actiniaria: Actiniidae) by gel filtration on a 16/60 HR Sephacryl and anion exchange FPLC chromatography (Pharmacia-LKB Mono Q HR 5/5) in order to evaluate their potential toxicity towards lepidopteran (Lacanobia oleracea) and dipteran (Musca domestica) larvae. Characterisation of the parasitoid venom by DNA sequencing of a venom gland cDNA library demonstrated the presence of trypsin-like serine proteases (NM_001170877), insulin-like growth factors (NP _001082137), late trypsin (XP _001604401), and metalloproteases (XP _001599927.1). Venom proteins from the sea anemone were identified by mass spectrometry (MALDI TOF/TOF) as actinoporins (Q9Y1U9) and Na+ and K+ channel inhibitors which are known to act as neurotoxins (P61541, Q9NDA5, P81791). Genes encoding purified parasitoid proteins were subsequently expressed in Pichia pastoris as a microbial expression system and the late trypsin recombinant protein was tested for insecticidal activity. Sea anemone toxins, on the other hand, were purified from source for subsequent bioassay. The recombinant proteins and protein fractions were then tested for their insecticidal activity by injection into L. oleracea and M domestica larvae. Anti-moulting factor and juvenile hormone esterase inhibition factor purified from the parasitoid venom were shown to cause between 70-100% and 50-90% mortality respectively in L. oleracea after a period of 14 days, with observable physiological changes beginning on day 7; the recombinant late trypsin protein (XP _ 001604401) was shown to cause 70% mortality in L. oleracea larvae after 8 days. Out of 30 fractions isolated from sea anemone acrorhagi using gel. |
