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Using dung to figure out how herbivores respond to drought

Blog written by Joel Abraham. Read the full paper here.

Droughts are expected to increase in frequency and severity as a result of human-caused climate change, which makes understanding how droughts impact ecosystems essential. Unfortunately, studying droughts can be a major challenge. Because droughts are unpredictable, studying them often requires quick thinking and improvisation.

So, when my co-author Carla Staver and I heard in 2015 that a severe drought was happening at her long-term field site in Kruger National Park in South Africa, we decided to capitalize on the opportunity to learn more about how droughts work. A lot of savanna animals eat mostly plants (they’re herbivores) so we thought they might be negatively affected by drought, but their responses to drought represent a big – and important – unknown. You can, to a large extent, approximate drought conditions for plants in an experimental setting. But herbivores, especially the large mammals that inhabit African savannas, are too big and move around too much to study in a lab. This meant that a large-scale approach was needed to capture their responses.

Kruger happens to be the perfect place to work at large scales. It’s a massive savanna reserve –about the size of New Jersey – making it one of the largest protected areas in the world. What makes this especially interesting is that the 2014-2016 drought affected different parts of Kruger differently – some places were hit hard, whereas others were pretty much normal. This meant that we could compare what animals were doing in a range of different drought conditions.

Counting dung in Kruger
Fieldwork in Kruger during the drought – this photo shows a part of Kruger that wasn’t affected much by the drought, in contrast to parts of the park that were severely affected (see below).

To do our work, we took advantage of the fact that animal dung is a great way to understand what animals are up to in tropical savannas. To quote a well-known children’s book, everybody poops, and every species poops in slightly different ways. Zebra dung (my favorite) is formed into beautiful, sleek pellets the shape of kidney beans, whereas elephants poop out what are essentially massive piles of mulch – largely undigested twigs, leaves, and grass. Also, animals can only poop in places they’ve been, so counting where animal dung is and how much is there actually gives you a pretty good sense of how animals are distributed. You can also use dung (nice and fresh!) to figure out what animals are eating, via lab techniques to analyze the plant contents of dung. Armed with this dung-based approach, we counted animal dung at more than 150 sites throughout Kruger and collected nearly 600 dung samples over the course of two years to see how animal behavior changed during the drought. This whole process – counting and collecting thousands of piles of animal dung in total – made me fall in love with just how powerful poop can be!

Some fresh zebra dung
Fresh zebra dung – that’s some pretty good looking poop!

Herbivores responded to the drought in different ways. Animals capable of eating both grass and trees (we call these ‘mixed feeders’; elephants are a great example) ended up eating more trees than usual during the drought, particularly where the drought was worse. But, animals that eat only grass (‘grazers’, including zebras) couldn’t switch what they were eating, and so instead moved to parts of Kruger where the drought wasn’t as bad. Meanwhile, tree eaters (aka ‘browsers’, like giraffe) didn’t change their behavior much at all. What’s so interesting about these results is that they’re actually not all that different from what these animals do during normal years. During normal dry seasons, mixed feeders switch to eating more trees as grass gets used up. Similarly, it is widely known that grazers can travel really far distances even in normal conditions – think the mass migrations of wildebeest in the Serengeti. So, these drought behaviors are actually just amplifications of behaviors that these animals already have!

A Kruger wildebeest during drought
Mixed feeders switch to eating still-green trees during drought, whereas grazers (like this wildebeest) either go hungry or move to greener pastures.

These results have some important consequences for savannas going forward, too. Other research by my lab mate Maddy Case is showing us that the effects of droughts on trees can be severe, too, halting tree growth and causing widespread die-offs in some tree species. The increased reliance of some animals on trees during drought will only make things worse for trees, which could prove very bad as droughts become more severe and more frequent.

Also, we found that grazers relied on movement to survive drought. But moving only helps you if there are places to go. This drought was patchy, and in a large park like Kruger, there’s lots of space to move. But many savanna reserves are small and cut off from other parks by fences, which prevents animals from moving. These fences, which we have erected to protect animals, may very well pose a problem as droughts become more frequent.

Our results also challenge long-held beliefs about the flexibility of animal behavior. Here we found that animal behaviors are limited; animals could only use their existing dry-season behaviors to respond to the drought. This contrasts with the commonly held belief amongst ecologist and evolutionary biologists that behavior is quite flexible, and that it is easy for animals to modify their behavior. Instead, our findings reveal that animals are fundamentally creatures of habit, and that we might need to rethink how we view some animal behaviors. Not that behavioral constraints are an entirely new concept. As the saying goes, you can’t teach an old dog new tricks. Or, in this case, you can’t teach a hungry grazer to eat trees.

Messy talons and beaks can tell us what a raptor eats

Blog written by Ryan Bourbour, a PhD student in the Department of Animal Science and Graduate Group in Ecology at the University of California, Davis. Read the full paper here.

Picture an autumn day in coastal California, accompanied by hundreds of migrating songbirds, hawks, and falcons. This influx of avian predators and prey along the Pacific coast happens every September and October in the Marin Headlands of California just north of San Francisco. On peak migration days, migrant raptors seem to swarm the hillsides, ambushing their unsuspecting songbird prey to fuel the next leg of their long journey, leading us to wonder: 1) are these raptors tracking flocks of migrating songbird prey as a plentiful food source, and, most importantly, 2) how can we study the diet of migrating raptors?

A migrant Sharp-shinned Hawk that has visible prey remains on its toes and talons.

Migratory raptors survive in three spatially and temporally distinct realms within their annual cycle: the breeding grounds, non-breeding grounds, and the migratory route, with the latter being one of the most dangerous times of a raptor’s life1. Much of the available information on the diet of migratory raptors primarily comes from studies focused on the breeding and non-breeding grounds when conventional diet study methods can be feasibly carried out, (e.g. observations, nest cameras, and analysis of pellets and prey remains), leaving gaps in our knowledge on the foraging ecology of a migrant raptor actively on migration. For raptors that primarily prey on songbirds, such as accipiters and falcons, hunting almost daily during migration is necessary in order to survive the long journey. Understanding what resources are critical for raptors to survive their migratory journey may shed light on the co-evolution of migration strategies between migrant predators and prey2,3, and also has conservation implications in the face of anthropogenic climate change and mismatch of cues4,5 if there are disruptions in trophic interactions along a migratory route. Our primary motivation for our migration research was to develop a new research technique to study raptor diet when direct observations are nearly impossible and then implement this technique to investigate exactly what fuels raptor migration.

We collect prey DNA from the exterior of the raptor’s beak (left) and talons (right) using a nylon swab tip.

Previous research on the foraging ecology of migrating raptors has highlighted correlations between peak migratory movements of migrant raptors and migrant prey6,7, which suggests migrating raptors may be keying in on energetically taxed migrant prey and possibly tracking this food source to increase hunting opportunities2,3. A more modern approach to studying diet was used in New Mexico by collecting visible songbird prey feathers from the talons of migrating Sharp-shinned Hawks (Accipiter striatus), which allowed researchers to identify 50 songbird prey items over 5 years8. However, in order to study the diet of migrant raptors in depth, we wanted to develop a method that could provide a robust sample size within a single migration season. So before we set out to study the foraging ecology of migrant raptors, we had to think outside the box.

The inspiration for our idea emerged while we were banding migrating raptors at migration monitoring stations. We noticed that these migrant raptors often have visible prey blood and tissue (sometimes fresh, sometimes not) on their beaks and talons. Swabbing that “messy beak” or those “messy talons” would mean we could be sampling prey DNA, so naturally, we wanted to test this idea out. We collaborated with the California Raptor Center at the University of California, Davis and designed a controlled study to confirm that when we swabbed a beak or talon we were actually sampling what the raptor had eaten. We sampled three resident raptors that had different diets. We tested for the presence of chicken DNA and only found positive detections on raptors that were fed chicken, which confirmed that our method was ready to try in the wild. The most important and exciting part was that we were still able to detect whether chicken had been eaten even when beaks and talons were visibly “clean”. This led us to believe we would be able to detect prey DNA on any talon or beak, regardless of whether there was visible prey remains or not.

To implement our new method on wild raptors, we collaborated with the Golden Gate Raptor Observatory and their volunteer raptor bander team to collect samples over the 2015 and 2016 migration seasons from over 600 migrating Sharp-shinned Hawk and Merlin (Falco columbarius) individuals. To confirm our swabbing technique, we randomly selected 19 beak and talon swabs and amplified the prey DNA using COI primers developed for songbirds. These primers isolate a specific region in the mitochondrial DNA, known as a DNA “barcode”. We sequenced the DNA barcodes, referenced them to a public barcode database, and identified our barcode sequences to the species level. We detected only probable songbird prey, which was amazing news! The next steps in our migration diet study are to sequence the prey DNA collected from the beaks and talons of hundreds of migrating raptors using DNA metabarcoding (high-throughput sequencing) to describe migration diet in detail, and investigate how important migrant prey are for fueling raptor migration.

Another exciting thing about our beak and talon swabbing method is that it can also be applied outside the context of migration. We are currently using it to investigate and describe the pathways of anti-coagulant rodenticide exposure in wintering raptors on farms by comparing blood samples to dietary (beak/talon swab) samples. Our beak and talon swabbing technique shows promise in helping researchers study the diet of raptors, and even other inconspicuous predators, when documenting prey selection is logistically difficult with traditional methods.

An immature Red-tailed Hawk with prey blood on its beak (left) and Ryan Bourbour swabbing a beak for prey DNA after a blood sample was taken to test for rodenticide exposure.

Works cited:

  1. Bildstein, K. L. (2006). Migrating raptors of the world: their ecology & conservation. Cornell University Press.
  2. Kerlinger, P. (1989). Flight strategies of migrating hawks. University of Chicago Press.
  3. Ydenberg, R. C., Butler, R. W., & Lank, D. B. (2007). Effects of predator landscapes on the evolutionary ecology of routing, timing and molt by long‐distance migrants. Journal of Avian biology38(5), 523-529.
  4. Jones, T., & Cresswell, W. (2010). The phenology mismatch hypothesis: are declines of migrant birds linked to uneven global climate change?. Journal of Animal Ecology79(1), 98-108.
  5. Buskirk, J. V. (2012). Changes in the annual cycle of North American raptors associated with recent shifts in migration timing. The Auk129(4), 691-698.
  6. Aborn, D. A. (1994). Correlation between raptor and songbird numbers at a migratory stopover site. The Wilson Bulletin, 150-154.
  7. Nicoletti, F. J. (1997). American Kestrel and Merlin migration with green darner movements at Hawk Ridge. Loon68, 216-220.
  8. Delong, J. P., Cox, N. S., Cox, S. W., Hurst, Z. M., & Smith, J. P. (2013). DNA Sequencing Reveals Patterns of Prey Selection in Migrating Sharp-Shinned Hawks. The Condor115(1), 40-46.

Vampire bats have a ‘gut reaction’ to habitat destruction

Blog written by Melissa Ingala, Daniel Becker & Nancy Simmons. Read the full paper here.

Habitat destruction can severely impact wildlife populations, often by restricting access to living space, particular habitat types, and food resources. Other more subtle impacts of habitat fragmentation on animal physiology can be difficult to measure; for example, do such changes in resources influence the symbiotic bacteria in the gut? And do those impacts have physiological fitness consequences for hosts? Some previous studies in primates have shown that degraded habitats are associated with decreased fitness of the gut microbiome. Because we know the microbiome aids in nutrition and helps train the host immune system, such decreased fitness in the gut could translate to less healthy, more infection-prone animals. In spite of this, very few studies link differences in the environment with changes in feeding and downstream changes in microbiotas and immunity in wild hosts. However, such studies could help inform wildlife conservation as well as help with assessment of potential pathogen spillover risks from reservoir hosts of zoonoses.

In our study, we sought to elucidate links between environmental habitat fragmentation, host diet, microbiome structure, and immunity by studying common vampire bats (Desmodus rotundus) in a forest fragment and a protected archaeological reserve in Belize. Common vampire bats are specialized to feed on mammal blood, and usually feed on tropical forest mammals in undisturbed habitats. In Belize, rapid agricultural intensification has transformed the landscape from mostly seasonally dry forest to a matrix of forest remnants and cattle pastures, which provisions vampire bats with abundant food resources and could create opportunities for pathogen transmission between the bats, cattle, and humans. Because of this, we predicted that the vampire bats living in the fragmented forest would forage mostly on cattle, and as a result, would have lower microbial diversity compared with the vampire bats in the protected mature forest. We further wished to test if the relative abundances of gut microbes were related to host immune defense. To test these hypotheses, we sampled hair to measure diet (via stable isotope analyses), fecal swabs to characterize the gut microbiome, and blood plasma to test for links between the microbiome and innate immune function using a bacterial killing assay (which quantifies complement-mediated defense).

Kakabish
Aerial photo of fragmented forest at Ka’kabish. Note the cattle herd visible at the top right margin of the forest. Photo credit: Burton Lim

We found that the vampire bats from the fragment had isotopic signatures that suggest they primarily feed on livestock, while the diets of the bats from the protected forest were more variable. There was a great degree of variation in livestock consumption within sites, suggesting that even some of the bats from the protected forest leave to forage on livestock in adjacent pastures. This is probably because cattle are a predictable resource confined to an open area and thus require less foraging effort to find than, say, the collared peccaries or Brocket deer dispersed over the densely wooded area of the reserve.

Surprisingly, we did not detect differences in overall microbial community composition between the two sites, but we did find that the bats from the fragment had more heterogeneous microbiomes. An interesting feature of microbiomes is that they can behave much like pool balls if disturbed; if “healthy” microbiomes are the pool balls racked neatly at the center of the table, the strike of the cue ball (i.e. a perturbation) results in increased dispersion rather than some deterministic change. That is to say, disturbed microbiomes need not be defined by directional shifts in structure or composition, but rather a dynamic instability that would not exist in the absence of the perturbation. Using an enrichment analysis designed to measure log-fold changes in the abundance of bacterial taxa, we also showed that while overall community composition was the same across sites, individual bats feeding on higher amounts of livestock showed enrichment or depletion of several core bacterial taxa. This suggests that changes in vampire bat diet may be a mechanism behind the increased microbial dispersion we measured.

When we tested for associations between these core bacteria and the ability of vampire bat plasma to kill bacteria (specifically, Escherichia coli), we found that the relative abundance of individual core bacteria had either a positive (in the case of Edwardsiella and Mycosplasma) or negative (in the case of Staphyloccocus) relationship with host immune defense. This suggests that changes in the relative abundance of these bacteria may be associated with decreased resistance to infection in hosts, but more detailed work on the relationships between these gut bacteria and host immunity is needed.

Our study demonstrates the value of an integrative approach for studying the consequences of habitat destruction in tropical forests.  By combining metagenomic, immunological, and ecological methods, we can achieve a deeper understanding of how human activities alter animal physiology and zoonotic disease cycles. In the future, we plan to expand this work to integrate GPS technologies and short-term diet analyses to better understand how vampire bats are using this fragmented landscape, how changes in diet and foraging area impact rabies spillover risk, and how both of these issues are related to the microbiome. Our hope is that by using such a systems-level approach, we will be able to advance both regional conservation and public health goals.

Main photo: The common vampire bat (D. rotundus); photo credit: Brock and Sherri Fenton

Pesticide causes bumblebee flight to fall short

Blog written by Daniel Kenna & Richard Gill. Read the full paper here.

For social bees, such as bumblebees, colonies rely on the foraging behaviour of worker individuals to collect nectar and pollen. Efficient foraging is underpinned by flight performance, hence any environmental stressor affecting the dynamics of flight could translate to reduced colony success. Studies revealing what factors affect bee flight, therefore, can help us to understand why bee populations across the world are in decline and mitigate the threat to the pollination service these vital insects provide.

The growing challenge of feeding the human population across the globe has seen conventional farming practices becoming heavily reliant on the application of chemical pesticides for crop protection. The use of these pesticides in agricultural landscapes has been implicated as a contributing driver of bee declines, but knowledge gaps remain as to the mechanism by which pesticides pose a risk. While acute exposure to pesticide residue is unlikely to kill a bee, we are becoming increasingly aware that sublethal effects on bee behaviour can have detrimental knock-on effects to colony functioning and health.

Previous studies on bumblebees exposed to the neonicotinoid class of insecticide have reported that workers not only take longer to forage, but also bring back less food to the colony, which can lead to reduced colony growth. One potential explanation for this reported impairment to foraging efficiency is that certain aspects of flight capability, such as flight endurance and speed, are affected by neonicotinoid exposure. We set out to test this by feeding workers of Bombus terrestris audax (the Buff-tailed bumblebee) a field-realistic dose of neonicotinoid (imidacloprid), and investigated the possible effects on flight performance using a tethered flight mill setup (Figure 1).

Figure 1: Left panel: one of the flight mills used in our experiment; right panel: bumblebee worker attached to the arm of the flight mill via a metal tag glued to the thorax. Images by Danny Kenna

Modifying a previous flight mill design used on moths and honeybees, we were able to let bumblebees fly inside a lab while attached by a magnet to a revolving arm of a flight mill. This led to the tested bees effectively flying around in circles, in which we could record the time it took for every full circuit to be completed. We were able to calculate the length of time each bee flew, and by considering the circumference of each circuit could also measure the speed of each circuit and total distance flown, allowing us to investigate the dynamics of flight for neonicotinoid-exposed and unexposed workers.

Our experiment revealed that exposure to the neonicotinoid had a striking negative effect on flight endurance, with exposed workers only managing to fly around a third of the distance and duration of non-exposed workers on average. This equated to a 1 km decrease in flight distance, and if taken in the strictest sense, exposure to neonicotinoids at a field-realistic concentration therefore has the potential to reduce the total foraging area of a bumblebee colony by over 80%. Consequently, neurotoxic pesticide exposure will place increased stress on bumblebee colonies. For example, exposed foraging bees may find themselves unable to reach previously accessible resources, or incapable of returning to the nest following exposure to contaminated flowers. Not only would this reduce the abundance, diversity, and nutritional quality of food available to a colony, but could also limit the pollination service a colony is able to provide.

Interestingly, analysis of flight velocity revealed a response by the exposed workers that possibly explains, at least in part, why their flight was terminated prematurely. We found that exposed workers flew significantly faster in the initial part of the flight test, with average velocity only converging with unexposed workers once most of the exposed bees had dropped out of the flight trial (Figure 2). Our findings suggest that neonicotinoid exposure resulted in a hyperactive-like state causing exposed workers to fly faster than expected. As neonicotinoids are similar to nicotine and act by stimulating neurons, this ‘rush’ or hyperactive burst of activity does thus make sense. However, our results suggest there may be a cost to this initial rapid flight, potentially through increased energy expenditure or a lack of motivation, in the form of reduced flight endurance.

Figure 5 - flight velocity over time - subset
Average velocity (m/s) flown by each treatment group (control = solid red, pesticide = dashed blue) plotted for each consecutive circuit for the first 2,500 circuits. Numbers at the bottom of the graph refer to the number of bees still flying on the corresponding circuit.

Our findings take on an interesting parallel to the story of the ‘Tortoise and the Hare’. As the famous fable states, ‘slow and steady wins the race’. Little did Aesop know that this motto may be true for bumblebees in agricultural landscapes. Just like the Hare, being speedier does not always mean you reach your goal quicker, and in the case of bumblebees, exposure to neonicotinoids may provide a hyperactive ‘buzz’ but ultimately impairs individual endurance.

 

 

Lizards respond to the chemical call to arms of plants

Blog written by Jay Keche Goldberg. Read the full paper here.

Compared to our fast paced animal lives plants often appear inanimate, but they live dynamic lives and respond to their environment in curiously creative ways.  One of their more intriguing responses is the production of herbivore induced volatiles. These small airborne molecules are referred to as a plant’s “cry for help” since they are produced when a plant is under attack and often attract the enemies of the attacker to rescue the plant from being eaten. This phenomenon – known as indirect defense – was discovered in the 1980s and has since been an ongoing source of fascination and scientific inquiry.

Indirect plant defenses – unlike direct defenses like noxious chemicals or physical deterrents such as thorns – recruit a third party to protect the plant, rather than directly impacting the plant’s enemy. Perhaps the most well studied forms of indirect defenses are the domatia and extrafloral nectaries that Acacia trees use to house and feed the ants that protect them, but the body of literature regarding the production and perception of herbivore induced volatiles is rapidly catching up.

Since their initial discovery, herbivore induced volatiles have been shown to be produced by a diverse array of plant species – with some compounds in particular being nearly ubiquitous among plants. Unsurprisingly, they have also been shown to be responsible for attracting a wide range of predators, parasitoids, and even pathogens to the location of herbivores.  The vast majority of these animals are invertebrates – parasitoid wasps, predatory bugs, and even pathogenic nematodes – and only recently have researchers begun to examine the role that vertebrates might play in mediating this phenomenon.

Ornithologists have thus far led the charge in studying the relationship between predatory vertebrates and the plants that host their herbivorous prey. For decades researchers assumed that birds barely used their sense of smell, opting to locate prey using their acute vision instead; however, recent findings are challenging this view. Great tits (Parus major) will use herbivore induced plant volatiles to identify herbivore-infested trees, although other bird species will ignore these same prey-associated cues. Birds have been the focus of research on plant-vertebrate interactions, even though lizards and snakes have long been known to use olfaction when foraging for prey.

Anyone who has watched a lizard or snake has surely observed their tendency to tongue-flick the object around them. This uncanny behavior allows these animals to smell the world around them using a specialized organ known as the vomeronasal organ. By observing the rate at which reptiles perform tongue-flicks herpetologists have been able to study the role of different chemical cues in mediating the lives of lizards and snakes. Researchers have been able to identify the pheromones that reptiles use to find and assess the quality of potential mates, determine the prey-derived chemicals that are used while foraging, and how different species and clades differ in their reliance on chemical versus visual cues while performing these tasks.

Herpetologists have identified two distinct foraging strategies that are used by predatory lizards: active and ambush. Active foragers are constantly on the move, tongue-flicking the environment frequently as they go, while they search for prey. Ambush foragers on the other hand, sit in a location and wait for their prey to move close to them. They tongue-flick infrequently, especially when compared to active foraging species. This has led researchers to consider them primarily visually-oriented and largely ignore food-associated chemical cues, although this paradigm begins to break down when omnivorous and herbivorous lizards are studied as they are known to use chemical cues to locate plant-based foods – even when they are a member of an otherwise ambush foraging clade. Despite having known that some lizards will respond to plant-derived chemicals for years, no one had examined the response of predatory lizards to herbivore-associated plant volatiles until now.

Lizard photo
Photos of study species. (a) Striped Plateau Lizard (photo credit: Genevieve Pintel) (b) Chihuahuan Spotted Whiptail (photo credit: wikimedia commons)

During the summer of 2016 my colleagues and I decided to tackle this gap in the literature and examine the responses of lizards to isolated herbivore associated volatiles. Given the exploratory nature of our study, we selected two well-studied chemicals that are already known not be associated with herbivores and attractive to invertebrate predators: trans-2-hexenal and hexanoic acid. Trans-2-hexenal is member of the green leaf volatile class of compounds – these small lightweight compounds are nearly ubiquitously produced by damaged plants with some being produced by mechanical damage and others being exclusively associated with herbivores. Hexanoic acid is a common component of herbivore body odor and is often derived from compounds present in the herbivore’s diet. These two compounds allowed us to compare the response of lizards to herbivore-associated chemical cues that are derived from the prey itself and the prey’s host plant.  We presented these compounds to two species of lizards – one that forages actively and another that waits in ambush – on cotton swabs, as is standard in studies of lizard olfaction.

We found that our active forager responded to insect body odor, an unsurprising finding given that previous studies have found that close relatives of our study species will not only tongue-flick at prey chemicals, but even try to eat scented cotton swabs. We expected our ambush foraging species to not respond to either chemical given that it is considered a visually-oriented predator but were surprised to find that it responded to our selected herbivore-induced plant volatile – indicating that it may contribute to indirect plant defenses. We hope that future studies may expand upon ours and determine if lizards are attracted to these compounds or even analyze the response of whole families of lizards to better understand how these responses change over evolutionary time.

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