The Food Search Box assay – where do we go from here?

A blog post highlighting the article by N. Tsvetkov, B. Madani, L. Krimus, S. E. MacDonald, and A. Zayed in Insectes Sociaux

By Armo Zayed

 

Assay for spatial learning and memory

Assay for spatial learning and memory.

 

As central place foragers, honey bees have an amazing ability to fly several kilometers away from their colony to forage and then beeline back to their home without getting lost. Honeybee foragers can perceive and communicate spatial information via the famous waggle dance. But how are these traits encoded in the honey bee genome?

When Nadia Tsvetkov joined my lab in 2012, she was keenly interested in studying the genetics of spatial learning and memory in bees. She spent several months training bees to fly through mazes. The maze experiments were fun but took too long, and our sample sizes were far fewer than the hundreds of bees needed to tease out the likely subtle genetic effects on learning and memory. What we needed was an assay that was as fast and as easy to standardize as the proboscis extension reflex assay – the workhorse of insect olfactory learning and memory studies. We tried some different approaches (one involved a very beautiful but unwieldy maze constructed out of Christmas balls) until colleague Dr. Suzanne MacDonald, a vertebrate biologist at York University’s Department of Psychology, suggested that we try the food search task paradigm. The paradigm is commonly used to study spatial learning and memory in primates and rodents. A common protocol entails hiding toys or food in boxes within a testing arena that animals are allowed to explore. Over time, the animals learn and can recall the location of boxes that contain rewards.

So we set out to try a similar assay on bees. For prototyping, we used some common and inexpensive items; we made the testing arena of clear Tupperware containers and we employed several artificial flowers made out of Q-tips. After a few pilot assays, we decided that a small arena containing four flowers was the best compromise between complexity and length of the experiment. Nadia worked out the testing protocol that involved placing bees into the arena where one of the artificial flowers had a sucrose reward. Once a bee found and fed from the rewarding flower, it was removed and tested again for a total of three training trials. Finally, the bee entered the arena where none of the flowers had a sugar reward. This time, the bee had to rely solely on its memory to find the focal flower. Our data analysis showed that bees subjected to this test exhibited two telltale signs of learning and memory: they improved their ability to find the rewarding flower during training, and they were able to recall the location of the rewarding flower after training. The Food Search Box (FSB) was born.

We carried out two more experiments to test the utility of the FSB for studying spatial learning and memory in bees. We first compared the performance of nurses (young honeybee workers that nurse the brood) and foragers (older workers that forage outside the colony) in the FSB paradigm. While both nurses and foragers did equally well in the training trials, foragers did substantially better than nurses in the memory test. So, is it age (young vs. old) or behavioral state (nurse vs. forager) that is influencing spatial memory in the FSB? To answer this question, we carried out another study on same-aged workers. We treated these workers with either cGMP, which causes precocious foraging, or cAMP, which does not alter behavioral state. The cGMP-treated bees performed similarly to foragers in the FSB, while cAMP and the control bees performed like nurses in the assay. Taken together, the results of these two experiments indicate that behavioural state (nurse vs. foragers) is primarily associated with differences in spatial memory in the FSB.

We are very excited by the results of the FSB; we were able to test bees quickly without much attrition. It is feasible to screen hundreds of bees within a short period, opening up the door for genetic and genomic studies of spatial learning and memory in honeybees. While it is certainly possible to improve on the design to enhance automation (i.e., RFID readers or tactile sensors to passively record visits to artificial flowers), the low-tech version presented in the paper is very easy to set up and perform. We are looking forward to feedback from the community on the test, and we hope it will provide a useful tool for studying spatial learning and memory in honeybees and other insects.

Interview with a social insect scientist: Luke Holman

Square Luke

IS: Who are you and what do you do?

Hi! I’m a Senior Lecturer working at the University of Melbourne. I did my undergrad and PhD in Sheffield, UK. My PhD was on sperm morphology in fruit flies, and I began working on social insects during my first postdoc at the University of Copenhagen with Prof. Patrizia d’Ettorre. Since discovering a bunch of ant, bee, and wasp queen pheromones in 2010-14, a lot of my research has focused on working out how queen pheromones evolve, how they work, and what they can tell us about the origins of eusociality. Currently, I also work a lot on sexual selection and ‘meta-science’ (including topics like p-hacking, research methods, and the gender gap in the science workforce), and I teach undergraduate genetics and evolution.

IS: How did you develop an interest in your research?

In short, I read most of The Selfish Gene during high school, and realised evolutionary biology was incredible! It was earthshattering to realise that we could make sense of nature’s astounding complexity and weirdness using simple, logical theory. Initially my research focused on puzzling traits that seem to defy conventional evolutionary logic: my PhD was on a group of flies that produce thousands of specialised, infertile sperm (apparently on purpose). Are these sterile sperm a worker caste that help the fertile sperm somehow? Or are they casualties of intragenomic conflict? We still don’t really know, but I was hooked.

IS: What is your favourite social insect and why?

Probably the black garden ant, Lasius niger, for changing my life by making my career in science possible. If it weren’t for their reliably massive mating flights in the parking lot outside my office in Copenhagen, I would not have had such a successful first postdoc. Each year I could collect up to 900 queens in a couple of hours, enough for a whole summer of experiments, simply by strolling around on summer evenings. A close second is the ant Lasius flavus, because they’re bright orange, very gentle, and have a nice simple cuticular hydrocarbon profile that’s easy to analyse by GC-MS.

IS: What is the best moment/discovery in your research so far? What made it so memorable?

Probably the day I isolated the queen pheromone of Lasius niger. I moved to Denmark in summer 2008, and on Patrizia’s advice I began my first experiment with synthetic pheromones, on boxes of ants kept in my living room (they needed to be treated twice daily with pheromones for a month). At the same time I was working day and night on a Marie Curie fellowship application, and exploring the new city. I ran the whole experiment blind, so for ~2 months I had no idea if the putative pheromones were doing anything. After collecting all the blind-coded data and making the graphs and statistics, I took them to Patrizia’s office for decoding, and we realised that we had isolated arguably the first ant queen pheromone. That felt great! As well as being a significant discovery, it was a lucky break that set the stage for a productive postdoc.

IS: Do you teach or do outreach/science communication? How do you incorporate your research into these areas?

I have written a little for The Conversation, which is a great website – for those that don’t know it, it hosts science journalism explained in plain English by actual researchers. I’d encourage your readers to contribute to it: it’s a non-profit enterprise with a large readership that provides a good antidote to mainstream science journalism, which is hit-and-miss. In my lecturing, I certainly touch on my own research interests, but I have also been surprised by how much my teaching has helped my science. Many times, I have been writing lectures and realised that I didn’t properly understand something, or I have noticed papers or knowledge gaps that lead me to a new research project (particularly when I have been asked to teach something outside my comfort zone).

IS: What do you think are some of the important current questions in social insect research and what’s important for future research?

The question that springs to mind is “Does methylation matter?” There are many review papers arguing that social insects use DNA methylation to regulate caste polyphenism, but precious little data, and most of the data that we do have is non-experimental and uninformative (e.g. “We looked at the methylome of one queen and one worker, and found some differences”). There’s a widely-cited 2008 Science paper showing that experimental manipulation of DNA methylation causes larvae to develop into queens, and we could settle the question by replicating or expanding this approach.

More broadly, I think social insect research would benefit by incorporating recommendations from the ongoing “Reproducible Research” movement: open data, good experimental design, transparent statistical analysis, etc. In a recent meta-analysis, I showed that 70% of queen pheromone experiments were not conducted blind, and that non-blind experiments had hugely inflated effect sizes (presumably due to observer bias). Sample sizes were very often strikingly small (e.g. n=6, barely enough to analyse), making these studies almost entirely uninformative. Given that social insects are known for being numerous, I don’t think it’s unreasonable to insist on large, blind, well-designed experiments. I have also found that many flat-out social insect scientists refuse to share or archive their raw data, hindering research synthesis and ensuring that their results are unavailable for re-use or fact checking.

IS: What research questions generate the biggest debate in social insect research at the moment?

Unfortunately, the answer is probably still the continuing fallout over the 2010 Nature paper by Nowak, Tarnita and Wilson! For my part, I am baffled by both sides. The original paper made various obviously untenable claims, e.g. that kin selection theory has made only a “meagre” contribution. However I also find it odd how much effort has been invested in rebutting this paper: there are dozens of papers replying to it, and I have sat through multiple angry conference talks. At some point I think we just need to shrug and get on with it. An uncomfortable truth is that all science is somewhat wrong, and I’m not convinced that doing science “at” a particular person/group is a useful way to advance knowledge.

IS: What is the last book you read? Would you recommend it? Why or why not?

I Contain Multitudes by Ed Yong, about the microbiome. Absolutely fascinating read with Ed’s characteristically playful, funny style. A sample factoid: some researchers think that humans and bacteriophages have co-evolved. Our gut is lined with mucus that just so happens to be a great habitat for phages to lurk and take out bacteria that try to get through the gut lining. I never thought that I was involved in a mutualism with a virus!

IS: Outside of science, what are your favourite activities, hobbies or sports?

I like travel, hiking, rock climbing, yoga, and meditation. In the past I was cripplingly addicted to juggling, and spent many hours a day throwing small sacks of seeds in the air; my record was 8 balls for about a second, or 5 balls for about a minute. I also spend plenty of time reading the news and fretting, though I’m trying to cut down.

IS: How do you keep going when things get tough?

The last 2-3 years have been very stressful, as I made the transition from postdoc to lecturer. Instead of doing less science to make room for my new responsibilities, I just worked harder, which was a bad idea as I ended up burning out. I now try to leave work on time and keep active, but years on fixed-term contracts has left me with a loud inner voice that shouts, “You should be publishing!”. I’ve been lucky to have lots of supportive friends and family to lean on, but I think the best way to manage in tough times is to make sure you’re not already exhausted by your normal work. Maybe the answer is to sit down and plan for your mental health and well-being, just as you would plan for a field season.

IS: If you were to go live on an uninhabited island and could only bring three things, what would you bring? Why?

Swiss army knife, water purifier, and a boat to get off the island.

IS: Who do you think has had the greatest influence on your science career?

Probably WD Hamilton. Reading his 1964 papers, it’s amazing how many future branches of evolutionary ecology he foreshadows in throwaway sentences. Also, I likely wouldn’t have become a scientist if his work wasn’t evangelised so well in The Selfish Gene. As for people I’ve actually met, I pick my second postdoc advisor, Prof. Hanna Kokko. I try to emulate her incisive way of thinking, her efficient work practices, her skill in balancing work and life, and her kindly enthusiasm for everything.

IS: What advice would you give to a young person hoping to be a social insect researcher in the future?

I’d recommend keeping up with research outside as well as inside the social insect world! To its detriment, a lot of social insect research is focused on narrow, taxon-specific issues, and only uses methods that have previously been applied to social insects. This slows down the pace of development in social insect science, and reduces its appeal to researchers from other fields. Some of my best papers involve applying a widely-used concept or method to social insects in a novel way. For example, dozens of non-social insect people work on ‘intralocus sexual conflict’, the concept that there is antagonistic pleiotropy for male and female fitness, leading to maladaptation in both sexes. I pointed out that the same thing applies to queens and workers: basically, it’s hard to make a perfect worker and a perfect queen using the same genome, so each caste ends up a little bit maladapted. Another example involves the analysis of gene expression data. Most social insect transcriptomics studies test each gene for differential expression one at a time, but in other fields it has long been commonplace to also test for differential expression in ‘modules’ of strongly co-expressed genes.

Ants colonise bird nests and raise broods in them

A blog post highlighting the article by M. Maziarz, R. K. Broughton, G. Hebda, and T. Wesołowski in Insectes Sociaux

By Marta Maziarz

As an ornithologist, I have focused on the reproduction of birds but often overlooked the fact that bird nests can also be home to many invertebrates that find shelter, food or a suitable microclimate within them. When we discovered ant workers and their larvae inside nests of the wood warbler Phylloscopus sibilatrix, curiosity drove us to study this phenomenon.

An initial literature review revealed just a handful of published records of ant broods found inside bird nests, including blue tits Cyanistes caeruleus breeding in nest-boxes in Corsica (Lambrechts et al. 2008), and great tits Parus major and marsh tits Poecile palustris occupying tree cavities in primeval stands of the Białowieża Forest, Poland (Mitrus et al. 2015). Blem and Blem (1994) reported ant colonies on the side of nests in nest-boxes used by prothonotary warblers Protonotaria citre but gave no further details. This surprising scarcity of observations of ants in songbird nests suggested that this phenomenon may be exceptional and occur only among cavity-nesting species.

Our discovery of ant workers and their larvae in wood warbler nests, which are domed structures composed of dry grass, moss, and leaves and situated on the forest floor, challenged this view. We made the original finding during long-term studies of wood warbler ecology in 2004-2015 in Białowieża Forest (Eastern Poland), which prompted us to document this phenomenon systematically during 2016-2017. In 2017, we also contacted researchers in Switzerland and the UK to ask them to inspect nests for the presence of ants and their broods. We wanted to find the frequency of ants colonising wood warbler nests, and whether ants are present in wood warbler nests elsewhere in the species’ breeding range.

During our systematic observations in 2016-2017, we found adult ants in 43% of warbler nests, and one-third of nests also contained ant larvae or pupae. These ant broods were situated within the sidewalls of the nests, at or just above ground level. The most frequent species were Myrmica ruginodisor M. rubra, and occasionally Lasius niger, L. platythoraxor L. brunneus. These numbers, compared to 30% of nests containing adult ants and 20% containing broods during the earlier (2004-2015) period, indicated a long-term association between the ants and the birds. The findings from Białowieża Forest contrasted with those from Switzerland and the UK, where we only found single cases of adult ants and their broods. The different frequencies of ant presence between regions could be due to varying densities of bird or ant nests between woodlands transformed by humans to a different degree, but further studies would be necessary to confirm this.

These first records of adult ants and their broods in wood warbler nests showed that occupation of bird nests by ants can be a locally common phenomenon, which may have been overlooked previously in this and other songbirds. Systematic examination of nests belonging to different bird species would be valuable in understanding this further.

Furthermore, the occurrence of ant broods in the walls of wood warbler nests showed that ants colonised these structures following their construction by birds. Why they do this remains unclear; are the ants attracted to the nests by their structure, the presence of other invertebrates as a source of protein, or by heat generated by the birds? More work is underway to answer these questions, but it seems that these potential ant-bird interactions could be much more widespread than has been suspected.

Picture1

Wood warbler nests are dome-shaped and constructed of leaves, grass, and moss. They are usually hidden among low herb vegetation, under a tussock of grass or sedge, or wedged under fallen branches or logs. Such structure and locations could promote their occupation by ants, for example, Myrmicaspp., which raise their broods in similar places.

Picture1

Numerous ant Myrmicaspp. larvae and two larger, well-grown blowfly Protocalliphoraspp. larvae (centre-right) in the wall material of a wood warbler nest

References

Blem CR, Blem LB (1994) Composition and microclimate of Prothonotary warbler nests. Auk 111:197–200.

Lambrechts MM, Schatz B, Bourgault P (2008) Interactions between ants and breeding Paridae in two distinct Corsican oak habitats. Folia Zool 57:264–268.

Mitrus S, Hebda G, Wesołowski T (2015) Cohabitation of tree holes by ants and breeding birds in a temperate deciduous forest. Scand J For Res 31:135–139.