Hello Sriram,

These are very interesting questions! Who knows? I don't know if anyone has ever looked into this in more detail?! The main focus in differentiation is probably more on small molecules (or combinations of such) rather than on ultrasound, because exclusion-experiments suggested for it. Even though some scientists believe that certain forms of differentiation could be triggered by anything - even maybe by hits (like hits with a hammer). Therefore your suggestion may be interesting. Differentiation is not my research topic, so I may confuse some things. However, as far as I know, scientists started looking for factors that are involved in certain differentiation events for quite some time now, with marginal success - you can e.g. force differentiation from blood streams to insect forms with acetate/cis-aconitate. Differentiation, however, is a bit more complicated than just switching from VSGs to procyclins; morphology and metabolism are very different between and among the bloodstream forms and the insect forms (notice my use of the plural form when I use the term "forms"). Trypanosomes begin to differentiate from proliferative long slender forms (LSF) to cell cycle arrested short stumpy forms (SSF) already in the bloodstream - they are also clearly distinguishable by eye. This differentiation has been demonstrated to occur in a density-dependent manner and is believed to be triggered by a small - not yet identified - compound; the SIF (stupy induction factor). The SIF is therefore believed to be secreted by the cells themselves and is very likely amplified via a positive feed-back loop (quorum sensing). During this process, intermediate form cells (IF) gradually change their morphology (and metabolism). Short stumpies are widely accepted to be the form that is pre-adapted for the survival within the fly. It is also widely accepted that they are the only cells that show long-term survival under the very different conditions inside the fly. During a blood meal cells can be exchanged both, from the blood to the fly and vice versa. Through some processes, the short stumpies lose their cell cycle arrest inside the fly and regain their proliferative ability. Some trigger mechanisms have to take place that transform them into the first of several insect forms, the procyclic trypomastigotes - I have no idea how that may happen.

In the fly, the cells undergo a rather complicated journey through its various compartments and a variety of specialized forms can be found at different places - most of these forms are again distinguishable by eye and obviously very likely also differ significantly in their metabolism. Many bottle-neck barriers exist inside the fly alone that force or require differentiation. There are, I think as many as ten or even more, described forms in the tsetse fly alone and this may not even be the complete picture. In the salivary glands, which is the first and the last step of the parasites' journey through the fly, you would again find another cell cycle arrested insect form (metacyclic procyclic form), which in turns is pre-adapted for the mammalian blood stream – they are thought to give raise to the long slenders once a factor in the blood triggers this. And there you have yourself some idea about the rather complicated life cycle of the trypanosomes with many, many bottle-necks and maybe as many - if not more - differentiation factors / -inducers. And I’m sure, the full image is still very incomplete, also since inside the mammalian host the parasite not only seem to proliferate in the blood but also in other regions (CNS, fat, lymphatic vessels,…). You would have to look at what happens to the different blood stream cells (LSF, IF, SSF) and maybe also some insect cells when they are exposed to these ultra sound patterns. One of the problems certainly is, that cultivated (monomorphic) cells differ greatly from cells extracted from the blood or from flies. Once you would have establish a culture, the question would be; do they show a distinct growth phenotype in comparison to the untreated control? Can you see change in any expression level (RNA, Protein)? This would itself be a HUGE project! And don't forget to make triplicates :-)

To your other question for which I have absolute no background knowledge: According to my 2 minutes literature research, I think most of the ultrasounds produced by glossina are linked to their behavior or seem to be a communication method – which of course does not exclude an involvement in (facilitated) differentiation. “Could Ultrasound be used in Tsetse fly infested forests to confuse the tsetse fly's signals?” Sounds again like an extremely complicated task! The first question is, does the sound have a function or is it just the result of something? Then would it be the received sound or maybe rather the resulting vibrations that would have the function? In any case how would you want to produce that sound (or vibration) - with a piezzo, a loudspeaker, something vibrating? And what would be the range in which it would be effective? And how densely dispersed are the flies in nature anyways? And, if too intense, would they not rather escape from these produced sounds/frequencies/vibrations to a place, somewhere you can't reach them? Maybe ultrasound could be a method to kill the trypanosomes in the host?! But then again, you would have to be super specific with it and you had to make sure that you target only the trypanosomes. I really like your ideas but it would certainly require A LOT of research before these questions could be answered.

Best,

Martin

Thanks for the detailed answer.