Effects of mannitol ingestion on adult fruit flies

My very first, first-author publication (coauthored with the incredible Katherine Fiocca) came out last month in PLoS One (an open access journal, which means anyone can view the article for free, found here). The article is titled “Mannitol ingestion causes concentration-dependent, sex-biased mortality in adults of the fruit fly (Drosophila melanogaster)”; I’m excited to have the opportunity to share some of the main findings of this work with all of you! But first – what even is mannitol?

Image result for mannitolMannitol is a “sugar alcohol” (don’t worry, it won’t get you drunk). It’s found naturally in fruits and fermented products and (like erythritol and other sugar alcohols) is often used as a low-calorie sweetener. Basically, it tastes sweet to your tongue without increasing your blood sugar levels dramatically. #lifehacks Mannitol is also cool because it’s used medically to reduce pressure around the brain following traumatic injury.

It turns out that when you feed mannitol to different groups of insects you get vastly different responses. This is particularly interesting because mannitol is naturally occurring in some of the stuff these organisms might regularly eat (especially flies that eat a lot of rotting fruits). In some insects, eating mannitol is lethal – in others, mannitol is a carbohydrate they can break down and use for nutrients! Crazy.

Madboy74 CC0 (Wikipedia)

In fact, even within a species mannitol seems to have varying effects. We were interested in following up on a previous study from our lab that showed, at one concentration, that mannitol is more lethal to female than male fruit flies. We aimed to replicate this sex-biased mortality effect, and see if it would be consistent across concentrations (dose-dependence provides strength to a cause-and-effect relationship). We also noted in the previous data set that flies housed in vials with the opposite sex might have been dying more than flies housed in single-sex vials and wanted to follow up on this observation more carefully. Finally, we wondered if this sex-biased effect was consistent at the larval (developmental) stage – or only adults.

Ultimately, we wondered – why is mannitol sex-biased? Is this effect consistent across concentrations? Across life stages? Is it consistent across culturing (single vs. mixed sex conditions) and mating conditions? Understanding the answers to these questions can begin to help scientists understand the mechanisms behind mannitol’s lethality – and perhaps why it affects different insects in different ways – providing us with a tool for studying the biological pathways that underpin insect development, reproduction, and nutrition.

Result 1: Concentration-Dependent, Sex-Biased Adult Mortality

Figure 2 in the paper; stars indicate a difference in survival between males and females.

Mannitol’s lethality was found to be concentration-dependent for both adult males and females (i.e. the more they eat, the faster they die); but females died at lower concentrations and faster than males. However, this was not true in larvae – male and female larvae saw equally reduced survival on mannitol foods! This indicates that the cause of mannitol’s sex-biased lethality is not genetic (i.e. it’s not inherently to do with being a female fruit fly). Instead, it’s something about being an ADULT female fruit fly – perhaps, something to do with the demands of reproduction.

Adult female fruit flies can lay 60 eggs a day after only mating once. Producing all those eggs requires a lot of protein – much more than it takes for male fruit flies to make sperm. To get all this protein, adult females are constantly eating, and when there’s mannitol in the food, this increased food consumption could lead to increased self-dosing with mannitol and thus increased mortality. To support this hypothesis that increased eating might underlie increased female fly mortality, we tested two things:

  1. Do female flies actually eat more than male flies?
  2. If female flies are not mated (and thus lay fewer eggs and have lower reproductive demands), does that increase their survival compared to mated female fruit flies?

Result 2: Differences in Food Consumption between Male and Female Flies

To measure how much flies eat, we put liquid food in a tube and (over five hours) measured the change in volume of liquid food per tube (compared to evaporation controls with no flies). Vials of male flies ate significantly less mannitol food than vials of female flies, showing that female flies do eat more food per hour than males.

Result 3: Mortality Depends on Mating Status and Culturing Condition

Fig 5 in the paper; letters that are different from one another indicate a significant difference in survival. Longevity is just another way of viewing survival (instead of average # dead, it’s average # of days survived).

Is female fly survival in mixed-sex vials lower than female fly survival in single-sex vials because the females in these vials are mated – or because male flies are present? To answer this, we tracked survival in vials of unmated females housed only with other females, vials of females that mated once but were then housed only with other females, and vials of mated females and males together. For female flies, mating with males once reduced longevity on mannitol foods, even if they were housed with only other females after mating – indicating that the increased demands of reproduction (leading to increased food consumption) may be responsible for reductions in female survival.

What it means?

Overall, we found that mannitol’s sex-biased effects on fruit flies are not genetically-based (no larval stage sex-biased mortality). Instead, like most other drugs, lethality is concentration-dependent and based on the ‘dose’ the individuals take. In the case of female flies, eating more food to meet the protein demands of egg laying means they dose more, and die more, than males. Preventing them from mating increases their survival, by reducing the nutritional demand of laying eggs. Understanding this mechanism of sex-biased mortality may help us figure out why different species, and different individuals within each species, are differently impacted by mannitol.

Next steps?

More follow-up work on larvae (which had delayed development on mannitol foods) would be a great future direction (and stay tuned…). Additionally, while we know what’s causing the sex-bias in mortality, we still don’t understand the cause of mortality itself. There’s some evidence that polyols cause flies to regurgitate to a lethal extent and dehydrate, which fits with our observation of mannitol encrusted on dead fly mouths. Mannitol could also be accumulating in the body cavity if it cannot be broken down, starving the flies by taking up all the available space for nutritive food. More work needs to be done to understand how mannitol acts as a lethal agent to adults, which will lead to a better understanding of how mannitol affects different insects SO differently!

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The Pesticide (maybe) in Your Coffee

Insecticides are a huge industry in the United States – whether we’re talking the small-scale can of Raid for your kitchen counter ants or the much larger scale agricultural market. But what if there was something already on your kitchen counter that might take care of those ants for you?

Erythritol is the main compound found in Truvia, a common artificial sweetener that many people use for baking or their morning Cup o’ Joe. Erythritol is a non-nutritive sugar alcohol – so while it sweetens your food, it can’t be digested by your body. The fact that it is sweet (like sucrose or other sugars) makes it attractive to insects such as Drosophila melanogaster, one species of small fruit fly that is a very common organism for scientific study. In this case, attractive can also mean deadly.

Figure 1. Drosophila melanogaster raised on food containing Truvia show decreased longevity. Truvia is red, Purevia is green, control nutritive sugars are dark blue, and other non-nutritive sugars are light blue. Graph shows percentage of living adult flies raised on food containing different nutritive and non-nutritive sweeteners over time. Note significant decrease in longevity of adult flies raised on food containing Truvia compared to other food.
Figure 1. Drosophila melanogaster raised on food containing Truvia show decreased longevity. Truvia is red, Purevia is green, control nutritive sugars are dark blue, and other non-nutritive sugars are light blue. Graph shows percentage of living adult flies raised on food containing different nutritive and non-nutritive sweeteners over time. Note significant decrease in longevity of adult flies raised on food containing Truvia compared to other food.

My lab published its first ground-breaking (what, can’t a girl brag?) paper on erythritol in PLoS One, entitled “Erythritol, a Non-Nutritive Sugar Alcohol Sweetener and the Main Component of Truvia, is a Palatable Ingested Insecticide” (Baudier et al 2014, before I arrived). As you can see on the graph to the left, flies that ate Truvia had significantly decreased longevity as compared to flies fed PureVia, Sweet ‘N Low, Sucrose, Equal, Splenda, or Corn Syrup. It’s a pretty drastic split. They also ran an experiment confirming which compound in Truvia was the killer compound (spoilers above: it’s erythritol).

Figure 6. CAFE experiments show Drosophila melanogaster actively consume erythritol over time. Upper graph shows prandial behavior of 10 individually housed flies fed 5% erythritol (red columns) and 10 individually housed flies fed 5% sucrose (blue columns) over a 6 hour period. Average intake per fly per hour is graphed for each treatment and separated by sex. Lower graph shows prandial behavior of 10 individually house flies when presented with a choice between 5% erythritol (red columns) and 5% sucrose (blue columns). Average intake per fly per hour is graphed for each treatment and is separated by sex. Note the significant increase in erythritol intake compared to sucrose intake for both sexes.
Figure 6. CAFE experiments show Drosophila melanogaster actively consume erythritol over time. Upper graph shows prandial behavior of 10 individually housed flies fed 5% erythritol (red columns) and 10 individually housed flies fed 5% sucrose (blue columns) over a 6 hour period. Average intake per fly per hour is graphed for each treatment and separated by sex. Lower graph shows prandial behavior of 10 individually house flies when presented with a choice between 5% erythritol (red columns) and 5% sucrose (blue columns). Average intake per fly per hour is graphed for each treatment and is separated by sex. Note the significant increase in erythritol intake compared to sucrose intake for both sexes.

 

 

 

But it doesn’t matter if erythritol kills the flies if they won’t choose to eat it! So Baudier et al. ran several CAFE experiments; one gave the flies access to both sucrose and erythritol of the same concentration and measured how much of each solution the flies ate over time (bottom graph of the figure to the right). As you can see, the red bars are much higher than the blue for both sexes – the flies, when presented with a choice, ate more erythritol than sucrose. If that trend were to hold in the wild, that would be very good news – the flies would self-select to eat the pesticide over other available foods containing non-lethal sucrose!

 

 

 

 

 

 

 

 

While this paper looked at a few more things, the last piece of the puzzle I want to talk about here is the effects of higher or lower doses of erythritol on fly longevity. The graph below shows that flies fed two molar erythritol all died within 48 hours! That’s incredibly fast-acting for a pretty tame pesticide.

Figure 4. Increasing concentrations of erythritol show decreased longevity in Drosophila melanogaster. Graph shows percentage of living adult flies raised on food containing different concentrations of erythritol. Control food is 0.5 M sucrose (blue line), 2 M erythritol (red line), 1 M erythritol (orange line), 0.5 M erythritol (green line), and 0.1 M erythritol (black line) were used. Note significant decrease in longevity of adult flies as concentration of erythritol is increased.
Figure 4. Increasing concentrations of erythritol show decreased longevity in Drosophila melanogaster. Graph shows percentage of living adult flies raised on food containing different concentrations of erythritol. Control food is 0.5 M sucrose (blue line), 2 M erythritol (red line), 1 M erythritol (orange line), 0.5 M erythritol (green line), and 0.1 M erythritol (black line) were used. Note significant decrease in longevity of adult flies as concentration of erythritol is increased.

I hear you saying: “Okay, Meghan, but this is all about flies. Didn’t you promise me that I could take out ants with this stuff?” A recent study by another lab has shown that erythritol works against Solenopsis invicta – the red imported fire ant that causes so much trouble in the United States and abroad. While that probably isn’t the species of ant you have on your counter, it is a promising sign that this stuff may just work on many different groups of insects – from flies to ants, perhaps beyond.

And because erythritol is found in a sweetener meant for human consumption it has been rigorously tested by the FDA and is known to be human-safe (though if you eat a lot, and I mean a lot, of it all at once it may have a laxative effect). In other words, you can feel better about spraying this stuff onto your countertops than Raid. Compared to neurotoxins and other nasty chemical pesticides, erythritol is also thought to be more environmentally friendly too!

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