Electron microscopy to the rescue 

Once the production method had been developed and the insecticidal properties established, the two researchers sought to understand how the spores worked on the mosquito larvae. "Regarding terrestrial insects, the spore of an entomopathogenic fungus usually adheres to the animal's cuticle. It literally 'sticks' to it but this is simply a nonspecific passive interaction. It is only as a result of a recognition mechanism of the host that the spore eventually germinates. It then produces an infectious filament that penetrates the insect's body thanks to physical and enzymatic pressure. Once inside, the fungus produces toxic metabolites aimed at destroying the immune system. Tissues suffer necrosis and are destroyed by the filaments' proliferation. The animal eventually dies. However, in this case, we aren't talking about adult insects, but larvae that live in a strictly aquatic environment! I therefore wanted to identify how the spores enter and exert their toxicity over the Culex quinquefasciatus. Is it the usual route? Or do other entry routes come into play?"

To answer these questions, Thomas Bawin went to Philippe Compère, senior research associate at ULg's Functional and Evolutionary Morphology Laboratory. There, he infected his larvae with Aspergillus clavatus spores, which turned out to be the most virulent. He monitored the spores progress at regular 48-hour intervals as they travelled through the insect's body, using scanning and transmission electron microscopy techniques. The first method allowed him to observe the surface of the larva, while the second provided an inside view of the body through histological sections. "The scanning quickly showed me that the spores didn't adhere to the surface of the larval body, nor to their respiratory siphon, but to the setae around the mouth. An interesting observation since it would suggest that the larvae ingest the spores".

It was transmission microscopy that revealed the spore's action inside the animal. "The comparison of images of the control larvae and the infected larvae clearly showed an increasing presence of the spores in the alimentary bolus, as the hours passed. In fact, these cells are in a state of dormancy. As the hours go by, they become increasingly active, resulting in greater permeability and the secretion of toxic components capable of damaging the digestive epithelium and muscle tissues. This occurs between 8 and 24 hours after infestation". However, there is still a bit more work to do to find out which toxic components are involved. This phase of the work is continuing at Gembloux and should come to an end in the coming months. "It's probably a cocktail of enzymes and metabolites which isn't only toxic to insects, but to other living beings as well... However, we must be cautious: at this point, it's just a hypothesis prompted by the - meagre – scientific literature that exists in this field".

A basic duty of care 

Question: if the fungi spores enter the organism to destroy tissues relatively quickly, is this proof that Aspergillus can be used in biological control? Not so fast! The Culex quinquefasciatus larvae are affected following the ingestion of spores but the same could also be true for many other filter feeders present in the same ecosystem, such as daphnia. So far, there is nothing to say that there is a recognition system specific to this host. Before using these fungus spores against mosquitoes, we have to ensure that the zooplankton, and even the fish, won't suffer as well from such interventions in the environment. "It is likely that a natural organism such as Aspergillus doesn't have the same remanence as a chemical insecticide", the researcher adds. "But that doesn't mean we shouldn't check whether its pathogenicity is truly selective or, at least, whether it is effective on a limited range of hosts. Otherwise, we won't be doing what we set out to do".

The first cautious step would be to only treat small breeding sites with limited spraying. These are usually located in close proximity to human activities: used tyres, tin cans and discarded recipients, tank ends, etc. It might also be possible to maximise the impact of the spores by changing the formulation used. "In my opinion, the best option is to use oils or emulsions. These are probably likely to keep the spores on the surface of the water, without dragging them down, and thereby increasing the surface of the larvae's body that comes into contact with them, while maintaining the pathogenicity. But this is just a hypothesis at this point". There are still many verifications to be carried out but maybe, one day, they will lead to the commercial use of Aspergillus for biological control.