Yesterday, Dr Alan Stewart and I headed back to the Knepp wildland project, which you may remember from my earlier post describing the astonishing amounts of spittle from nymphs of one (or more) of the Aphrophora froghopper species. There are around 18 species of xylem-feeding froghopper and leafhopper species in the UK that have the potential to act as vectors for the Xylella bacterium, should it arrive here. One of the aims of the BRIGIT research project is to sequence the DNA of each of these species and make the data available to scientists all over the world who are working to combat this devastating plant disease. Our job for the day then, was to collect specimens of Aphrophora adults to send off for sequencing. When we saw the nymphs back in May, we couldn't be sure which species they were, but judging from previous years, Alan expected us to find both Aphrophora alni and the less widely-distributed Aphrophora salicina.
To begin with, things didn't seem to be going quite according to plan. There just didn't seem to be that many froghoppers in the trees where we had seen all that spittle only a few weeks ago. Where on earth had they gone?! I seemed to be finding almost every other insect group but them! This included many pairs of one of the 'delightfully' named Flesh Fly (Sarcophaga) species "enjoying each other's company", lots and lots of bumblebees, and an incredibly handsome Forest Shieldbug (Pentatoma rufipes).
And so it continued. I joked to Alan that I might need to change to an Orthoptera (grasshopper and cricket)-based research project as I was finding so many of them. Between us, we saw Field, Meadow and Common Green Grasshoppers, as well as Dark, Oak, Speckled and Roesel's Bush Crickets! For those that don't know, an easy way to tell grasshoppers and crickets apart is to look at the length of their antennae. Crickets (like this very splendid Roesel's Bush Cricket (Metrioptera roeselii) on the left), tend to have very long antennae, sometimes even longer than the rest of their body, whereas grasshoppers (like this nymph likely to be a Field Grasshopper (Chorthippus brunneus) on the right) have relatively short antennae.
But we persisted. And you can imagine my delight when we finally started to find reasonable numbers of Aphrophora!
Once we'd finally found them, we were able to collect good numbers of both Aphrophora alni and Aphrophora salicina. But as we were heading back to the car, Alan's attention was caught by an individual that didn't look quite right. Could it possibly be a third species?? Alan thought it might just be the relatively much rarer Aphrophora pectoralis, but we were going to have to wait until we got back to the University and examined it under a microscope to find out!
But we had one more stop on the way back to the lab. As we were so close, we decided to swing by my in-laws' house as they have a huge number of Rhododendron plants in their garden. We were on the lookout for the extremely colourful Rhododendron Leafhopper, Graphocephala fennahi, a species that was introduced to Europe from the USA in the 1900s which feeds more-or-less exclusively on Rhododendron xylem fluid. Graphocephala obviously like it there, as I found plenty of nymphs (pictured on the left below) on my first sweep and then on a number of other bushes. We agreed that I would head back there again in a couple of weeks once they have become adults, but then on one final bush standing in full sunlight - success! Adults!
Now at last we could head back to the lab to see if we really had found Aphrophora pectoralis. With all of our specimens, we need to tell the clever people doing the DNA stuff which species they're looking at and whether each individual is male or female. To do this, we sedate them with a quick blast of carbon dioxide and take a look with a hand lens or under the microscope.
After confirming that we did indeed have a good mix of both male and female Aphrophora alni and Aphrophora salicina, we got to the 'mystery' specimen. Superficially, Aphrophora pectoralis looks very similar to Aphrophora salicina, but A. pectoralis has a yellow-ish band on the forewings, with a sometimes rather indistinct black band behind it. Did our specimen have this? Yes! See for yourself - Aphorophora pectoralis is on the left below, next to Aphrophora salicina.
A final confirmation was to take a close up look at the face. Both species have 'punctures' (small dots) running either side of the midline of their face, but Aphrophora salicina only has one row on each side, where as Aphrophora pectoralis is said to have two rows. You might have to take my word for this, but our mystery specimen has two:
In combination, pretty convincing evidence that we had indeed, rather unexpectedly, located a third Aphrophora species at our collection site!
Our final job for the day was to prepare the specimens to be sent away for DNA sequencing. Unfortunately, this does of course require us to kill the individuals, but we are careful to only collect as many as we actually need, and to handle them as humanely as possible at all times. We process the samples by putting each specimen into an individual eppendorf tube whilst they are sedated with carbon dioxide, and then dropping the tubes into liquid nitrogen. This "snap freezing" has the double benefit of preserving the DNA for analysis whilst killing the individuals instantly and painlessly.
So all in all, a very successful day. We'd headed out with the expectation of collecting two froghopper species and in fact made it back with a third, as well as our first leafhopper collection of the project. All processed and ready to be sent off for analysis. Time to head home for a well-deserved cuppa!