Researchers from the Department of Medical Entomology at Sydney’s Westmead Hospital share some of their latest research into bed bug resistance. 

If you’re still finding bed bugs a difficult pest to control, or you’ve got a few hardy survivors that refuse to die, you’re certainly not alone. Recent research is shedding a light on exactly why this might be.

You will have no doubt heard of insecticide resistance being a key reason for the bed bugs’ resurgence, with the modern bugs able to withstand significantly higher doses of insecticide that would normally kill a susceptible population. This is particularly well established around the world with the pyrethroids (the most widely available group of insecticides), and has also recently been confirmed in the US with the neonicotinoids (a group that includes compounds such as imidacloprid and acetamiprid).

However, when we talk about ‘insecticide resistance’, what might be causing that resistance – the ‘mechanism’ – could actually be one of several different biological processes within the insect. In bed bugs, insecticide resistance is likely to result from a combination of up to three resistance mechanisms:

  • kdr-type target site insensitivity
  • metabolic detoxification
  • or reduced penetration.

So, why does this matter? Primarily, it’s because we have so few insecticides left today that are reliably effective, and we want to protect them for as long as we can. But to do so, we have to understand what mechanisms of insecticide resistance are present within the bed bug populations we’re dealing with today.

This was a challenge we faced in Australia recently when, after collecting a field strain of bed bugs from the suburb of Parramatta (within Sydney), we found it to be the most insecticide resistant strain we’d ever encountered. However, we also noticed that when we forcibly kept the bugs on a recently insecticide-treated surface, some of the bugs would succumb after a relatively short time, some would die after a few hours, and some simply wouldn’t die at all.

Knowing this, our lab set about investigating what mechanisms may be contributing to the observed resistance. Firstly, Dr Kai Dang determined that the strain uniformly possessed kdr-type mutations that meant that pyrethroids wouldn’t bind to their target site correctly. However, we also had other strains that possessed the same mutation that weren’t as highly resistant, so something more had to be going on.

The next step was to examine for metabolic detoxification and, in particular, two types of enzymes that might be breaking down the insecticide before it has a chance to work. These enzymes are broadly called ‘oxidases’ and ‘esterases’. Beyond knowing that a strain possesses metabolic detoxification, it is useful to know what type of enzyme might be present as each type has the potential to confer cross-resistance to more than one insecticide group. For instance, oxidases detoxify pyrethroids, but may also have the potential to work against neonicotinoids and could explain why resistance to that group has developed so quickly.

One way to test for enzyme activity is to use chemicals called ‘synergists’. Synergists can inhibit, or lower, the levels of enzymes and by using different types of synergists we can understand which enzymes type is present. One of the best-known synergists is a chemical called piperonyl butoxide, or ‘PBO’. PBO is very useful as it can inhibit both esterases and oxidases, but that ability in turn makes it hard to determine which enzyme type is contributing to any perceived resistance. However, a new synergist known as ‘EN16/5-1’ had recently been developed that only inhibits esterases (as opposed to oxidases). This meant that by using both synergists in combination, there was a unique opportunity to investigate the role of metabolic resistance and to determine which enzyme type was responsible.

Our studies investigated the Parramatta strain, as well as a range of bed bug strains originating from cities across Australia. This included an additional strain from Sydney (New South Wales), two from Melbourne (Victoria) and one from Alice Springs (Northern Territory).

The results indicated that metabolic detoxification played a major part in conferring resistance to our field strains, but also that there appeared to be a mix of both oxidase and esterase dominated strains (two strains each). The Parramatta strain’s resistance could almost (almost…) be ‘turned off’ by the addition of the synergists – the use of the both PBO and EN16/5-1 indicated that esterases were likely to be playing a major part in that strain. However, there still remained the problem of some bugs that we just couldn’t kill – these were the best of the best at resisting insecticides, and we needed to know why!

So the final part of our study was to look at the cuticle thickness, and examine it for any differences between bugs that died early versus those that were the hardy survivors within the same strain. Interestingly, older bed bugs have thicker cuticles – the cuticle is laid down much in the same way a tree ring is, except on a daily basis.

As such, we had to carefully age the bed bugs, to ensure no bias was introduced.

We then repeated the earlier insecticide forced exposure experiment, monitored how quickly they were affected, and then separated them into respective groups of ‘intolerant’ (died within 2 hours), ‘tolerant’ (still alive at 4 hours), and ‘resistant’ (still alive at 24 hours). Some careful manipulating under an electron microscope then enabled us to measure the cuticle thickness in the bed bugs’ legs (Figure 1).

Chart showing increasing bed bug cuticle thickness leads to increased tolerance to insecticide
Figure 1: Increasing cuticle thickness leads to increased tolerance to insecticide

The results clearly indicated that the susceptibility to insecticide decreased as thickness of the cuticle increased, with bed bugs with the thickest cuticles showing the highest level of resistance. When this finding is combined with our knowledge about the presence of target-site and metabolic detoxification in this strain, it means that these ‘super-resistant’ bugs are perfectly evolved to resist most pyrethroid insecticides available to pest managers (Figure 2).

Chart showing that the resistant Parramatta bed bug strain had a significantly thicker cuticle than the susceptible Monheim bed bug strain
Figure 2: The resistant Parramatta strain had a significantly thicker cuticle than the susceptible Monheim strain

The important implication of this is that when attempting control of bed bugs in a field situation, you’re likely to be up against some bugs that are just naturally ‘super-resistant’ compared to all the other bugs in the infestation. Miss those bugs and they’ll either start the infestation anew, or potentially spread it to a new location. Either way, these will be even more difficult to control as you have selected the ‘super-duper’ bugs!

Fortunately, there are two main ways to partially overcome this super-resistance. The first would be to use non- chemical means (no matter how resistant these bugs are they can’t withstand a vacuum!), and the second is to ensure you’re using an insecticide either with piperonyl butoxide (PBO) or one that is a combination product with pyrethroid and neonicotinoid (such a imidacloprid), or use a silicate based product as resistance does not occur to this group. Both methods significantly improve the effectiveness of control methods, and thus reduce the likelihood of call-backs.

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David Lilly, Cameron Webb and Stephen Doggett, Department of Medical Entomology, Westmead Hospital, Sydney

David Lilly is a PhD Student at the University of Sydney based at the Department of Medical Entomology, Westmead Hospital, Westmead. He is a recipient of an Australian Postgraduate Award that is generously supported by an Industry Top-Up Grant from Bayer CropScience, Australia.