Fewer infections found in forest fragments

As human populations expand, we are converting ecosystems from one state to another.  In the case of tropical forests, conversion of forest to cropland may leave behind fragments of relatively undisturbed forest surrounded by a matrix of cropland or other forms of development.  Conservation ecologists are exploring whether ecological processes and ecosystem structure in these fragments work pretty much like normal forested regions, or whether fragments behave differently.  To do this, in a few locations around the world such as the Wog Wog Fragmentation Experiment in New South Wales, Australia, researchers have systematically created forest fragments of various sizes.  They can then ask a variety of questions comparing fragments vs. intact forest. For example,  how does species diversity, or how do processes such as competition, predation and mutualism differ in the two landscapes?

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Aerial photo of Wog Wog Fragmentation Experiment at the time the experiment began in 1987. Credit: Chris Margules.

Julian Resasco was working as a postdoctoral associate in Kendi Davies’ lab at the University of Colorado on a study that looked at changes in invertebrate communities in response to fragmentation at Wog Wog. Beginning in 1985, researchers had set up a network of pitfall traps, which are cups that are buried with their tops level to the ground, so that any careless organism that wanders in will be trapped in the cup.  Some pale-flecked garden skinks, Lampropholis guichenoti, also had the misfortune to become entrapped and became subjects for the study. The invertebrates, and the 186 unfortunate skinks were preserved in alcohol and stored as part of the Australian National Wildlife Collection.

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Skink museum specimens at the Australian National Wildlife Collection. Credit: Julian Resasco.

Much later, Resasco arrived and began dissecting skink guts to analyze the prey items for a study that looked at how the skinks shifted prey consumption (their feeding niche) in response to fragmentation. While dissecting the skink guts, he noticed that some of the skinks had worms (nematodes) inside their guts.  These nematodes were relatively common among skinks from continuous eucalypt forests, rare among skinks from eucalypt fragments, and absent from skinks in the cleared, pine plantation matrix.

ResascoFig1

Top. The study area in southeast Australia, showing location of continuous forest, forest fragments and surrounding matrix.  Dots indicate locations of pitfall traps. The matrix was planted in pine seedlings soon after fragmentation.  Bottom. The pale-flecked garden sunskink Lampropholis guichenotti. Credit: Jules Farquar

As it turned out, the nematode was a new species, which Resasco and a colleague (Hugh Jones) named Hedruris wogwogensis. Nematodes in the genus Hedruris use crustaceans as intermediate hosts, which alerted Resasco and his colleagues that the terrestrial amphipod Arcitalitrus sylvaticus, which was very common in the pitfall traps, was probably an important intermediate host.  When amphipods from pitfall traps were examined microscopically, a small portion of them were infected with Hedruris wogwogensis. The researchers concluded that amphipods became infected when they ate plants that harbored nematode eggs or young nematodes, which then developed in amphipod guts, and were passed on to skinks that ate the amphipods.  Thus somewhat inadvertently, one aspect of the study transitioned into the question of how fragmentation can influence the transmission of parasites.

After concluding their skink dissections, Resasco and his colleagues discovered that skinks in continuous forest had five times the infection rate as did skinks in fragmented forest.  In addition, no skinks collected in the matrix were infected. Infected skinks harbored a similar number of nematodes, whether they lived in continuous forest or fragments (see the Table below). Lastly, amphipods were considerably more common in skink guts and pitfall traps from continuous forest, less so in fragments, and least in the matrix.

ResascoTab1good

Summary of data collected by Resasco and his colleagues. Nematode intensity is the mean number of nematodes per infected skink. Nematode abundance is the mean number of nematodes per skink (infected and uninfected). 

The researchers put these findings together in a structural equation model.  The boxes in the model below represent the variables, while the numbers in smaller boxes over the arrows are the regression coefficients, with larger positive numbers (in black) indicating stronger positive effects, and larger negative numbers (in red) indicating stronger negative effects.  The model revealed three important findings.  First, habitat fragmentation strongly reduced amphipod abundance.  High amphipod abundance was associated with high nematode abundance (that is the +0.20 in the model), so lower amphipod abundance from fragmentation reduced nematode abundance. Second, habitat fragmentation positively affected skink abundance – more skinks were captured in fragments than in intact forest, but this increase had no effect on nematode abundance in skinks.  Finally, note the direct arrows connecting “Fragmentation” to “Log nematode abundance in skinks”.  This indicates that other variables (beside amphipod abundance) are reducing infection rates in skinks that live in fragments and the matrix.

ResascoFig3

Structural equation model showing effects of fragmentation on nematode infection in skinks. Amphipods are the intermediate host.  Black arrows indicate significant positive effects of one variable on the other, while red arrows indicate significant negative effects. Solid lines represent fragments compared to controls and dashed lines represent the matrix compared to controls. Thicker lines are stronger effects.

At this point, we still have an incomplete understanding of the system.  We know that fragmentation reduces amphipods, which require a moist and shaded environment to thrive.  Reduced amphipod abundance leads to lower nematode infection rates in skinks.  But we know that other variables are important as well; perhaps nematodes survive more poorly in fragment and matrix soils. Interestingly, pine trees were planted in the matrix and are beginning to mature and shade out the matrix environment. Amphipod abundances are on the rise, so the researchers predict that nematode infection rates will begin to increase accordingly.  Those studies have begun.

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Eucalypt forest canopy at Wog Wog. Credit: Julian Resasco.

Looking at the bigger picture, it is clear that fragmentation may decrease (as in this study) or increase the abundance of an intermediate host. As an example of fragmentation increasing intermediate host abundance, the researchers describe a study in which fragmentation increased the abundance of the white footed mouse, an intermediate host for black-legged ticks (that host the bacteria that causes Lyme disease). We need to unravel the connections between landscape factors and the various species they influence, so we can begin to understand how human changes to the landscape can influence the transmission of diseases.

note: the paper that describes this research is from the journal Ecology. The reference is Resasco, J.,  Bitters, M. E.,  Cunningham, S. A.,  Jones, H. I.,  McKenzie, V. J., and  Davies, K. F..  2019. Experimental habitat fragmentation disrupts nematode infections in Australian skinks. Ecology  100( 1):e02547. 10.1002/ecy.2547. Thanks to the Ecological Society of America for allowing me to use figures from the paper. Copyright © 2019 by the Ecological Society of America. All rights reserved.

Invasive crayfish depress dragonflies and boost mosquitoes

Paradoxically, obliviousness and intense focus can be two sides of the same coin, as the following story highlights.  As a new graduate student at the University of Minnesota, I took a field ecology course at the University’s field station at Lake Itasca (famously known as headwaters of the Mississippi River).  One afternoon we watched dragonflies at a small pond; the male dragonflies were obviously patrolling territories and behaving thuggishly whenever intruders came by, and amorously whenever females of their species approached.  Surprisingly, territorial males chased off male intruders of any species, even though they posed no reproductive threat to them.  Why, I wondered…  So I sat there for many hours and kept very careful track of who chased whom, and for how long.  Big focus time. Ultimately, these observations blossomed into my doctoral dissertation.  Unfortunately, these observations also blossomed into the most virulent case of poison ivy known to humanity, as my intense focus on dragonflies obliviousized me to the luxurious patch of poison ivy, which served as my observation perch.

Anax junius Henry Hartley

Anax junius dragonflies in copula.  The male has the bright blue abdomen.  Credit: Henry Hartley.

Despite this ignoble incident, dragonflies remain one of my favorite animal groups.  They are strikingly beautiful, brilliant flyers, and fun to try to catch. In addition, they have so many wonderful adaptations, including males with penises that are shaped to scoop out sperm (previously introduced by another male) from their mate’s spermatheca, and females who go to extremes to avoid repeated copulation attempts, for example, by playing dead when approached by a male. Thus I was delighted to come across research by Gary Bucciarelli and his colleagues that highlighted the important role dragonflies play in stream ecosystems just west of Los Angeles, California.

Back Camera

Captured dragonfly nymph.  Dragonflies require from one to four years to develop in aquatic systems, before they metamorphose into terrestrial winged adults. As nymphs, they are fearsome predators on aquatic invertebrates. As adults, they specialize on winged insects, though there are stories of them killing small birds. Credit: Gary Bucciarelli

Bucciarelli and his colleagues came up with their research question as a result of working in local streams with students on a different project.  They wanted to know if invasive non-native crayfish (Procambarus clarkii) affect the composition of stream invertebrates and whether removal of crayfish could lead to rapid recovery of these invertebrate communities.

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Invasive crayfish, P. clarkii, sits on the stream bottom. Credit: Gary Bucciarelli

The researchers collected stream invertebrate samples and noticed a dramatic pattern – in all the streams with crayfish there were numerous mosquito larvae, but in all of the streams without crayfish there were no mosquito larvae and much greater numbers of dragonfly nymphs. This led them to formulate and test two related hypotheses. First, dragonfly nymphs (Aeshnaspecies) are more efficient predators on mosquitoes (Anopheles species) than are the invasive crayfish. Second, crayfish interfere with dragonfly predation on mosquitoes in streams where crayfish and dragonflies are both present.

Field Sampling

Student researchers collect stream samples. Credit: Gary Bucciarelli

Bucciarelli and his colleagues systematically sampled 13 streams monthly from March to October 2016 in the Santa Monica Mountains. Eight streams have had crayfish populations since the 1960s, while four streams never had crayfish, and one stream had crayfish removed as part of a restoration effort in 2015. Overall, streams with crayfish had a much lower number of dragonfly nymphs than did streams without crayfish.  In addition, streams with crayfish had substantial populations of Anopheles mosquitoes, while streams without crayfish (but much higher dragonfly populations) had no Anopheles mosquitoes in the samples.

BuccTable1

Number of mosquito larvae (MSQ) and dragonfly nymphs (DF)  by month in streams with crayfish (CF – top row of data) or without crayfish (CF Absent – bottom row)

This field finding supports both of the hypotheses, but the evidence is purely correlational.  So the researchers brought the animals into the laboratory to test predation under more controlled conditions.  They introduced 15 mosquito larvae into tanks, and exposed them to one of four treatments: (1) a single crayfish, (2) a single dragonfly nymph, (3) one crayfish and one dragonfly nymph, or (4) no predators. The researchers counted the numbers of survivors periodically over the three day trials. As the graph below indicates, dragonflies are vastly superior consumers of mosquito larvae compared to crayfish.  However, when forced to share a tank with crayfish, dragonflies stop hunting, either huddling in corners or actually perching on the crayfish.  By 36 hours into the experiment, all of the dragonflies had been eaten by the crayfish.  After three days, mosquito survival was similar when comparing tanks with crayfish alone with tanks that had both a crayfish and a dragonfly.

BuccFig2A

Mean number of surviving mosquito larvae in tanks with a lone dragonfly (DF), a lone crayfish (CF), one crayfish and one dragonfly (CF+DF) in comparison to controls with no predators.

Bucciarelli and his colleagues conclude that dragonfly nymphs are much more efficient predators of mosquito larvae than are crayfish. But when placed together with crayfish, dragonfly foraging efficiency plummeted. Field surveys showed a negative correlation between crayfish abundance and dragonfly larvae, and much greater mosquito larva populations in streams with crayfish.  This supports the conclusion that invasive crayfish cause mosquito populations to increase sharply by depressing dragonfly populations and foraging efficiency.  This is a complex trophic cascade because crayfish increase mosquito populations despite eating a substantial number of mosquito individuals.

The researchers argue that crayfish probably relegate dragonfly larvae to inferior foraging habitats, thereby limiting their efficiency as mosquito predators. As such, ecosystem services provided by dragonflies to humans are greatly diminished.  Recently, several new mosquito species that are disease vectors have moved into California.  Thus the loss of dragonfly predation services could pose a public health threat to the human population.  Bucciarelli and his colleagues recommend removing the invasive crayfish to restore the natural community of predators, including dragonflies, which will then naturally regulate the increased number of potential disease vectors.

note: the paper that describes this research is from the journal Conservation Biology. The reference is Bucciarelli, G. M., Suh, D. , Lamb, A. D., Roberts, D. , Sharpton, D. , Shaffer, H. B., Fisher, R. N. and Kats, L. B. (2019), Assessing effects of non‐native crayfish on mosquito survival. Conservation Biology, 33: 122-131. doi:10.1111/cobi.13198. Thanks to the Society for Conservation Biology for allowing me to use figures from the paper. Copyright © 2019 by the Society for Conservation Biology. All rights reserved.

Rewilding tropical forests: dung is the key

Rewilding means different things to different people. Basically, it involves restoring a species, or several species to an area from which they have been extirpated by humans. Conservation biologists might study the population size and distribution of the returned species, ecologists might focus on interactions between the returned species and other species, while anthropologists might investigate how humans in the area are adjusting to having a new species in their lives.  One of the most famous examples of rewilding is the return of gray wolves to the Greater Yellowstone Ecosystem in western U.S.A., which can be looked at from the perspective of how the wolf populations are doing numerically, how they affect their prey (elk) or their prey’s prey (willow and aspen in the case of elk), and how they affect ranchers in the surrounding areas.

Conservation ecologists have begun a major rewilding program in Tijuca National Park in Brazil, introducing agoutis in 2010 and brown howler monkeys (Alouatta guariba clamitans) in 2015. Howler monkeys were extirpated from this park over a century ago, so ecologists worried that the monkeys might interact with the remaining species in unexpected ways.  For example, this forest hosts several species of invasive fruit trees, such as the jackfruit (Artocarpus heterophyllus). Luisa Genes and her colleagues were concerned that howler monkeys might eat fruits from these trees, and poop out the seeds in new forest locations, causing the invasive species to spread more rapidly.

GenesHowler1

Introduced howler monkey holding the second baby born to her in the forest. Credit: L. Genes.

Even a disturbed rainforest such as Tijuca National Park hosts a large number of plant species, so the interactions can be complex and difficult to study.  As is so often the case in ecology, one very important complex of interactions involves poop.  Specifically, howler monkeys eat fruit off of trees, and poop the seeds out, usually at a new location, effectively dispersing the seeds.  But there is a second link in this seed dispersal interaction.  Twenty-one species of dung beetles use howler monkey poop for food for themselves and their offspring, breaking off small sections into balls and rolling the balls to a new location.  This process of secondary dispersal is nice for the beetles, but also for the seeds within the balls, which can now germinate in a new location without competing with the large number of seeds in the original howler monkey pile.

RafaelBrixDungBeetles

Two dung beetles battle over a dung ball. Credit: Rafael Brix.

Genes and her colleagues were interested in two basic questions.  First, were the howler monkeys eating fruit from a few select tree species, or were they eating from many different types of trees, thereby dispersing seeds from many species?  Before releasing the monkeys (two females and two males), they attached radio transmitters to the monkeys so they could easily track them, and note what they ate.  Based on 337 hours of observation, the howler monkeys ate fruit from 60 different tree species out of 330 possible species in the forest (18.2%).  This is an underestimation of actual howler monkey contribution to seed dispersal, because the researchers observed the monkeys for a relatively brief time, and fruit consumption by the monkeys should increase over time as the population of monkeys (and possibly tree diversity), continues to increase.

bugio reintroduzido no PN Tijuca 2015

Male howler monkey released in 2016.  Note the radio transmitter on its right rear leg. Credit: L. Genes.

The second question is whether secondary dispersal by dung beetles was reestablished following reintroduction of howler monkeys.  To answer this question quantitatively, Genes and her colleagues set up an experiment that used plastic beads of various sizes instead of seeds. The researchers set up circular plots of 1m diameter with 70 grams of howler monkey poop in the middle.  Each pile was mixed with seeds (actually beads) of four different sizes (3, 6, 10 and 14 mm diameters) to mimic the range of seed sizes. The researchers measured secondary seed dispersal by returning 24 hours later and counting the remaining beads, reasoning that the rest had been moved by dung beetles (along with the poop) to a new location.

Genes and her colleagues discovered that the median rate of seed dispersal (bead removal) was 69% with larger seeds being moved at a significantly lower rate than smaller seeds.  Thus secondary seed dispersal by dung beetles was still operating in this ecosystem even after howler monkeys had been absent for over 100 years.

GenesdFig2

Removal rate of beads (seed mimics) from dung piles by dung beetles in relation to bead size.  Different letters above treatments indicate statistically significant differences between treatments. 

Overall, ecological interactions among howler monkeys, plants, and dung beetles were rapidly reestablished once howler monkeys were reintroduced to the community.  There are plans to introduce five more howler monkeys this year, which should further increase beneficial seed dispersal, and hopefully allow plant diversity to increase as well.  One problematic observation was that howler monkeys also ate invasive jackfruit, which could promote its dispersal within the community.

Luciano_Candisani_38-1

Luisa Genes monitors howler monkeys in the forest. Despite its apparent lushness, the forest still lacks many species and interactions that you would expect to find in an intact forest. Credit: L. Candisani.

The researchers discovered only 21 species of dung beetles, which was somewhat lower than other studies have found.  It is probable that conversion of this land into farmland in the 19thcentury led to the decline and/or demise of some dung beetle species.  With reintroduction of howler monkeys, and the passage of time, Genes and her colleagues expect that this rewilding effort should lead to a more robust ecosystem, with increased howler monkey populations supporting high dung beetle abundance and diversity, and more effective dispersal of many plant species. To understand the overall impact on forests, the researchers recommend that future studies should compare seedling survival and forest regeneration in areas where howler monkeys were reintroduced to areas where howler monkeys are still missing.

note: the paper that describes this research is from the journal Conservation Biology. The reference is Genes, L. , Fernandez, F. A., Vaz‐de‐Mello, F. Z., da Rosa, P. , Fernandez, E. and Pires, A. S. (2019), Effects of howler monkey reintroduction on ecological interactions and processes. Conservation Biology, 33: 88-98. doi:10.1111/cobi.13188. Thanks to the Society for Conservation Biology for allowing me to use figures from the paper. Copyright © 2019 by the Society for Conservation Biology. All rights reserved.

Quoll vs. toad: a toxic brew

A native of Central and South America, the cane toad, Rhinella marina, was introduced to Australia in 1935 with great fanfare. The plan was for the voracious cane toad to eat all of the grey-backed cane beetles that were plaguing sugar cane plantations in northern Australia (a similar introduction had been successful in Puerto Rico).  But the plan failed, in part because there was no cover from predators, so the toads were not enthusiastic about hanging out in sugar cane plantations, and in part because adult beetles live primarily near the tops of sugar cane, and cane toads are poor climbers.

UniToad_BenPhillips

A cane toad. Credit: Ben Philips

So now, northern Australia has a cane toad plague, which is wreaking havoc on ecosystems, and threatening many native species, including the northern quoll, Dasyurus hallucatus. These omnivorous marsupials eat fruit, invertebrates and small vertebrates.  Unfortunately, their long list of food items includes cane toads, which are highly toxic to most consumers, having poison glands that contain bufotoxin, a composite of several very nasty chemicals.  If a northern quoll eats a cane toad, it’s bye bye quoll.

Male captive born northern quoll_EllaKelly

A northern quoll. Credit: Ella Kelly.

Unfortunately most quolls have not gotten the message; huge numbers are dying, and populations are going extinct.  As toads continue their invasion from north to south, more quoll populations, particularly those in northwestern Australia, will be at risk.

KellyFig1

Map of Australia showing past (light shading) and recent (dark shading) northern quoll distribution, and present (solid line) and future (dashed line) cane toad distribution.

Some quolls show “toad-smart” behavior and don’t eat toads. Ella Kelly and Ben Phillips are trying to understand how this happens. This is particularly important because a few quoll populations have managed to survive the cane toad plague by virtue of being toad-smart (though 95% of quoll populations have gone extinct in the wake of the cane toad wave). The researchers reason that if there is a genetic basis to toad-smart behavior, it might be possible to introduce toad-smart individuals into populations that have not yet been overrun by cane toads.  These individuals with toad-smart genes would breed and spread their genes through their adopted population.  This strategy of targeted gene flow would give the recipient population the genetic variation needed, so that some individuals (those with toad-smart genes) would be more likely to survive the cane toad invasion.  Over time toad-smart behavior would spread throughout the population via natural selection.

Targeted gene flow requires the trait to be influenced by genes.  To test for a genetic basis to the toad-smart trait, Kelly and Phillips designed a common-garden experiment, capturing some quolls that had survived the cane toad invasion (toad-exposed), and others from regions that had not yet been exposed (toad-naïve).  At Territory Wildlife Park, Northern Territory, Australia, the researchers bred these quolls to create three lines of offspring: Toad-exposed x toad-exposed, toad-exposed x toad-naïve (hybrids), and toad-naïve x toad-naïve.  They raised these three lines under identical conditions at the park. Kelly and Phillips then asked, are there behavioral differences in how these three lines respond to cane toads?

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Northern quoll captured in Northern Territory, Australia. Credit: Ella Kelly.

The researchers set up two experiments.  First they asked, which would a quoll (that had never before experienced a cane toad) prefer to investigate if given the choice: a dead cane toad or a dead mouse? It turned out that the quoll offspring with two toad-exposed parents were somewhat more interested in mice than in cane toads.  The same was true for the hybrids.  However, the toads with two toad-naïve parents showed little preference.

Second, and more important, the researchers gave quolls from the three lines the opportunity to eat a toad leg (which does not have enough poison to harm the quoll). The results of this experiment were striking; offspring of toad-naïve parents were twice as likely to eat the toad leg than were offspring of toad-exposed parents, or hybrids with one parent of each type.

KellyFig4

Proportion of toad-naive (both parents toad-naive), hybrid and toad-exposed (both parents toad-exposed) quoll offspring that ate a cane toad leg. Error bar = +/- 1 SE.

Kelly and Phillips conclude that toad-smart behavior is a genetically-based trait that has been under strong natural selection in populations of quolls that survived the cane toad invasion.  Hybrid offspring behave similarly to the offspring of two toad-exposed parents, suggesting that toad-smart behavior has a dominance inheritance pattern. The researchers propose using targeted gene flow, in this case introducing toad-adapted individuals into populations prior to the arrival of cane toads. Recently, Kelly and Phillips released 54 offspring with toad-smart genetic backgrounds onto Indian Island, which is about 40 km from Darwin.  The island has a large cane toad population, so the researchers will follow the introduced quoll population to see whether it is genetically equipped to survive in the presence of the cane toad scourge.

note: the paper that describes this research is from the journal Conservation Biology. The reference is Kelly, E. and Phillips, B. L. (2019), Targeted gene flow and rapid adaptation in an endangered marsupial. Conservation Biology, 33: 112-121. doi:10.1111/cobi.13149. Thanks to the Society for Conservation Biology for allowing me to use figures from the paper. Copyright © 2019 by the Society for Conservation Biology. All rights reserved.

The complex life of the pea

As a behavioral ecologist, I’m spending a surprising amount of time reading and writing about plants these days.  It turns out that plants are amazingly complex and interactive; you just need to know where and how to look.  Today we will discuss the humble pea plant, how it is infected with a virus that is carried by an aphid that sucks its xylem, and how a herbivorous weevil fits into the whole system.   The virus is the pea enation mosaic virus (PEMV), which causes pea leaves to yellow and wither, and also creates enations (scaly tissue) to develop on a leaf’s undersides. The aphid vector (a vector is the organism that carries a disease) is Acrythosiphon pisum, while the herbivorous weevil is Sitona lineatus.

Picture1

Pea plant infected with pea enation mosaic virus. Credit Paul Chisholm.

David Crowder has been studying plant/insect interactions for many years, and knew that most researchers who studied interactions between plants and insect vectors focused their attention on the plants, insects and the disease, but did not consider how other species in the community might affect this relationship. Paul Chisholm was a PhD student in Crowder’s lab; and working with two other researchers, they explored whether S. lineatus, an abundant herbivore of peas, influenced viral transmission.  They expected that if the pea was first attacked by the weevil, it might be more susceptible to subsequent viral infection.  Conversely, if the pea was infected by the virus, it might be less able to chemically defend itself against subsequent herbivory by the weevil.

ChisholmFigAppendixBC

(Left) Pea aphids. Credit: Shipher Wu under a Creative Commons Attribution 4.0 International Public License. (Right) Very adult pea leaf weevils, Sitona lineatus. Credit: Gail Hampshire under a Creative Commons Attribution 4.0 International Public License

It is easy to visually distinguish between PEMV-infected and uninfected plants, so the researchers could assess whether infected plants tended to suffer more defoliation by weevils than did uninfected plants.  They visited 12 different fields in northern Washington and western Idaho, USA, and measured defoliation by counting the number of feeding notches left by the weevils after feeding on 3 – 10 infected pea plants and an equal number of nearby uninfected plants on each field (more feeding notches = more defoliation).  They discovered that PEMV-infected plants tended to suffer substantially higher herbivory than did uninfected plants

ChisholmFig1A

Herbivory (as measured by number of feeding notches) caused by weevils on paired uninfected (black bars) and PEMV-infected (blue bars) pea plants sampled from 12 different fields. Error bars for all figures represent 1 SE.

Given the correlation between herbivory and infection, Chisholm and his colleagues then explored whether (1) the weevil preferred to feed on infected plants, and/or whether (2) infective aphids preferred to feed on plants that had been damaged by herbivorous weevils.  Both questions were answered with behavioral choice assays done in a greenhouse. First, the researchers created two groups of pea plants.  The first group, sham infected, were fed on by aphids not carrying PEMV for 48 hours, while the second group of plants were fed on by PEMV-infected aphids for the same duration. Aphids were removed and PEMV infection developed within 15 days in the PEMV infected plants.  The researchers then set up one sham infected and one PEMV-infected plant in a test cage, and released two weevils equidistant from the plants, allowing them to feed for six days.  They discovered that aphids fed much more voraciously on the PEMV-infected plants.

ChisholmFig1B

Mean leaf area removed from sham infected and PEMV-infected pea plants.

For the second experiment, the researchers again created two types of plants: undamaged – no herbivory, and damaged – 48 hours of weevil herbivory.  Weevils were then removed, and one leaf from each plant was connected to each end of a tube, while still attached to each plant.

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Experimental setup with tube attached to one leaf of each experimental plant.  Aphids were introduced into the central tube. Credit Paul Chisholm. 

The researchers added either 25 infectious or 25 non-infectious aphids, and allowed them 3 hours to choose a leaf.  PEMV-infected aphids preferred damaged leaves, while uninfected aphids showed no preference.

ChisholmFig1C

PEMV-infected aphid preference for undamaged (no herbivory) or damaged (weevil herbivory) leaves.

Chisholm and his colleagues then turned their attention to whether weevil herbivory made pea plants more susceptible to PEMV infection.  In one experiment they allowed PEMV-infected aphids to feed on plants for 3 days, and then introduced 0, 1 or 3 weevils who fed on the plants for another 6 days.  They used a protein assay to estimate the PEMV-titer (concentration) of each plant and discovered that the plants that were exposed to greatest herbivory had the highest PEMV titer (see graph below).  In a second experiment the researchers allowed weevil herbivory before adding the aphids, and found no effect of prior herbivory on PEMV titer.

ChisholmFig2A

Relative PEMV-titer of infected leaves after they were subjected to herbivory by zero, one or three weevils for six days. different letters above bars indicate significant differences between treatments.

What causes these plant responses to challenges by PEMV and weevils?  The researchers discovered that levels of three important plant hormones increased either in response to PEMV infection, weevil herbivory or both.  At this point it is not clear how these different hormone levels interact to bring about the changes we’ve described.

The researchers conclude that weevil behavior has a profound influence on the interactions between aphids, the viruses they carry and the pea plants they feed on (and infect).  The weevil is not a vector for the virus, yet it affects the virus directly by altering plant behavior and physiology and indirectly by altering the behavior of the vector (the aphids).  PEMV outbreaks are more likely when weevils are abundant, as aphids prefer damaged plants, and feeding by weevils increased the PEMV titer in infected plants.  Crowder argues that interactions in which a non-vector species influences the relationship between a host and its vector (and the pathogen it carries) are probably extraordinarily common in crop systems.  So if we want to understand crop susceptibility to pathogens we need to cast a broad net and consider both the direct and indirect effects of a community of species that can influence how the crop responds to infection.

note: the paper that describes this research is from the journal Ecology. The reference is Chisholm, P. J., Sertsuvalkul, N. , Casteel, C. L. and Crowder, D. W. (2018), Reciprocal plant‐mediated interactions between a virus and a non‐vector herbivore. Ecology, 99: 2139-2144. doi:10.1002/ecy.2449. Thanks to the Ecological Society of America for allowing me to use figures from the paper. Copyright © 2018 by the Ecological Society of America. All rights reserved.

Females are better speakers and better listeners than males – at least in plants

My age puts me smack dab in the middle of the woo-woo generation, when many people engaged in activities, or shared in belief systems, that were criticized as unscientific, spacey or just plain bizarre.  For example, talking to your plants was purported to make them bigger, greener or more florid.  This hypothesis generated a huge number of science fair projects, but no clear answers (so far as I know – but I admit that I have not done the appropriate research!).  But, it turns out that plants do talk to each other and to some animals.  When attacked by herbivores, many plant species will emit volatile organic compounds (VOCs) into the air that can have two effects.  First, these VOCs can alert nearby plants that herbivores are in the area, and that they should start producing defense compounds in their tissues that will repel these herbivores.  Second, these VOCs can alert predators that herbivores are present, and they should swing by and eat them.

Several studies have shown that female and male plants may differ in several ways that could affect communication.  Females typically invest more in reproduction, grow more slowly and invest more in defense against herbivory. Xoaquin Moreira and his colleagues wondered if sexual dimorphism in defense investment would result in differences between males and female in how they talk to each other. They chose the woody shrub Baccharis salicifolia, in which females grow more slowly but invest more in chemical defense and thus are infested by fewer herbivores than are males.  They focused their study on chemical responses of the plant to the highly-specialized aphid Uroleucon macolai, which only feeds on two Baccharis species.

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Baccharis salicifolia hosting an army of herbivorous aphids. Credit: X. Moreira.

The researchers used greenhouse experiments to explore how Baccharis uses VOCs for communication.  To control aphid movement, each treatment was done in a mesh cage, with one centrally located VOC emitter plant (of either sex), and one female and one male receiver plant equally distant from the central plant. Control emitter plants were untreated, while herbivore-induced emitter plants were given 15 mature aphids, which fed and reproduced on the plants for 15 days.  After 15 days Moreira and his colleagues removed all of the emitter plants and all of the aphids, and then inoculated each receiver plant with two adult aphids.  The researchers measured aphid reproductive rate on the fifth day as their measure of aphid performance, or of plant resistance to aphids.

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Emitter Baccharis salicifolia plant flanked by one male and one female receiver plant. Credit X. Moreira.

Aphids did much more poorly on male and female receiver plants that were associated with male herbivore-induced emitter plants (top graph below).  This implies that these receiver plants became resistant to aphids as a result of their exposure to an airborne substance released by the male emitter plant.  When the researchers used female emitter plants they found something very different.  There was no effect on male receivers, but still a very strong effect on female receivers, which had a much lower aphid reproductive rate than the female plants exposed to untreated female emitter plants (bottom graph below).

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Reproductive performance of aphids raised on control receiver plants (emitter plant with no aphids – clear bars) and herbivore-induced emitter plants (gray bars).  Two left bars show performance on male receiver plants, while two right bars show performance on female receiver plants. Top graph shows data for male emitters and bottom graph shows data for female emitters. Error bars = 1 SE. *** indicates P < 0.001.

Showing differences between sexes in communication is important, but the next step is to figure out how this happens.  In previous research, Moreira and his colleagues identified seven different VOCs that Baccharis emitted after aphid herbivory.  So they explored whether there were differences between males and females in how much of each VOC they emitted in response to aphids.  As before, they subjected some plants (of each sex) to herbivory and others were untreated controls. They then bagged each plant, and passed the collected vapors over a charcoal filter trap at a constant rate for an equal period of time.  After extracting the substances from the charcoal, the researchers used a gas chromatograph to identify and quantify the VOCs.

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Setup for collecting VOCs from Baccharis salicifolia. Credit X. Moreira.

The most impressive finding was a fivefold increase in pinocarvone release by female herbivore-induced plants in comparison to controls.  In contrast, in males there was only a minor pinocarvone effect.

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Relative increase in VOC emission following aphid attack in female (clear triangle) vs. male (filled triangle) Baccharis salicifolia. The induction effect is the log response ration (LRR) which is the natural log of (emission by the herbivore induced plants divided by the emission by the control plants).  Error bars are 95% confidence intervals.

Having discovered that females emit much more pinocarvone than males, the next question was whether females are more sensitive to pinocarvone, or in fact to any of the other VOCs.  So Moreira and his colleagues exposed plants to one of three treatments: 100 ul of pure pinocarvone, 100 ul of six VOCs including pinocarvone, and a control (no VOCs).  They discovered that all experimental treatments reduced herbivory in comparison to the controls, but that there was no difference between males and females in how they responded.

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Reproductive performance of aphids raised on female plants (left graph) or male plants (right graph) subjected to pinocarvone or a blend of six VOCs (including pinocarvone) in comparison to reproductive performance on untreated control plants (dashed line on top of each graph).  Shading surrounding dashed line indicates 1 SE.  Error bars are 1 SE.

This lack of different response between male and female plants to pinocarvone was a bit surprising; the researchers speculate that both males and females have pinocarvone receptors, but that female receptors are more sensitive (or numerous). If true, natural emissions of pinocarvone may suffice to induce a response in female but not male plants. But the artificial emitters may have released enough pinocarvone to stimulate male plants to respond as well. Clearly there is much more work to do here.

The researchers also wanted to know whether plants were more sensitive to VOCs produced by genetically identical plants (clones) in comparison to genetically-distant plants.  They discovered no influence of genetic relatedness on plant response to herbivory.  This is important, because from an evolutionary standpoint, there is no obvious reason why a plant would want to warn an unrelated plant that it was about to get eaten. An adaptive explanation is that relatives may tend to live near each other, so an emitter plant still benefits indirectly by promoting the survival of relatives who carry a proportion of genes identical to its own genetic constitution. One possible non-adaptive explanation is that a plant may use VOCs as a way of quickly communicating with itself, informing distant tissues that they need to produce defense compounds.  Nearby plants may simply be eavesdropping on this conversation, and using it to their advantage.

note: the paper that describes this research is from the journal Ecology. The reference is Moreira, X., Nell, C. S., Meza‐Lopez, M. M., Rasmann, S. and Mooney, K. A. (2018), Specificity of plant–plant communication for Baccharis salicifolia sexes but not genotypes. Ecology, 99: 2731-2739. doi:10.1002/ecy.2534. Thanks to the Ecological Society of America for allowing me to use figures from the paper. Copyright © 2018 by the Ecological Society of America. All rights reserved.

 

Girl and boy flowers support different microbe communities.

Most of us are accustomed to thinking about sexual dimorphism in animals.  Male lions have manes, and male deer have antlers and generally larger bodies than female deer.  In many species, male birds have more complex sings and more colorful plumage. Perhaps less familiar is that female insects are generally larger than males of the same species.  But many of us are unaware that sexual dimorphism exists in some plant species as well.

As a child, Kaoru Tsuji spent considerable time watching insects on plants.  Later, as an undergraduate at Kyoto University in Japan, she noticed that larvae of a particular geometrid moth only visited male Eurya japonica plants, but not females. This led to her graduate work on how plant sexes affect herbivorous insects, and later, more broadly, on how plant sexual dimorphism affects other species in the community.

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Kaoru Tsuji gazes at female Eurya emarginata plant. Credit: Noriyo Tsuji.

At the 2014 Ecological Society of America meetings, Tsuji heard Tadashi Fukami talk about microbial communities in flower nectar, and realized that she could learn to apply Fukami’s techniques to the microbial communities living within Eurya flowers.  After working three months in Tadashi’s lab, Tsuji was now ready to explore whether two plant species, Eurya japonica and Eurya emarginata, host different communities of bacteria and fungi in the flowers of male and female plants.

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Male and female flowers of the two study species visited by pollinators.  These photos are not to scale; in actuality the male flower is substantially larger.  You can get a sense of this by noting that the same insect pollinator, the fly Stomorhina obsoleta, is pictured in figure a and near the top left of figure b.

For both species, male flowers tend to be larger, while female flowers tend to have sweeter nectar. Higher sugar levels will increase the chemical stress experienced by microbial organisms living in the nectar. Because the inside of a microbial cell has a lower sugar concentration (and thus a higher water concentration) than the sugar rich nectar environment, water tends to leave the microbial cell, leading to severe dehydration. Thus Tsuji and Fukami expected to find lower microbial abundance in female flower nectar.

Complicating this situation, animal visitors, such as bees and flies, also influence the microbial community in at least two ways.  First, many nectar-colonizing microbes depend on animals to disperse them to new flowers. Second, the interaction of nectar production, water evaporation and consumption by bees and flies can change the concentration of sugar in the nectar.  If there are few (or no) animals drinking the nectar, water will evaporate, sugar will remain, and the nectar will become more and more concentrated (sweeter) as more nectar is secreted over time.  But if nectar gets consumed, the new secretions will simply replace the old nectar, and sugar levels should be relatively constant. Thus flowers without animal visitors should impose more chemical stress on microorganisms by virtue of being sweeter.

The researchers sampled nectar from 1736 flowers, and grew the nectar microbes on agar plates supplied with nutrients that would support either bacterial or fungal growth.  In addition, the researchers also placed small-mesh bags over a subset of these flowers (before they opened), to reduce animal visitation.  After five days they counted the number of colonies formed, to estimate microbial abundance. Unfortunately, microbes were rarely found in E. japonica, so most of the data are for E. emarginata flowers only.

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Female flowers of Eurya emarginata visited by a fly, Stomorhina obsoleta. Agar plates showing isolated colonies of nectar-colonizing microbes are superimposed. Left and right plates have yeast and bacterial colonies, respecitevly, both isolated from E. emarginata nectar. Credit: Kaoru Tsuji and Yuichiro Kanzaki.

First, as expected, female flowers had higher nectar sugar levels than did male flowers (the Brix value measures sucrose concentration).  In addition, putting a fine mesh bag over the buds substantially increased sugar levels in nectar from flowers of both sexes.

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Sucrose (Brix) concentration of exposed and bagged E. emarginata flowers of both sexes.  For box plots, the dark horizontal bar is the median value, while the box encloses the 25th and 75th percentile.

The proportion of flowers in which fungi and bacteria were detected was much greater in male flowers than in female flowers.  In male flowers only, bagging the flowers decreased fungal frequency but not bacteria frequency.

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The proportion of exposed and bagged E. emarginata flowers whose nectar, when cultured in the appropriate medium, generated fungal colonies (top graph) and bacterial colonies (bottom graph).

The researchers used colony forming units (CFUs) – the number of viable colonies on the agar plate – as their measure of bacterial abundance.

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Abundance of fungi (top) and bacteria (bottom) cultured in agar plates, that were swabbed with nectar derived from exposed and bagged E. emarginata flowers of both sexes. Note that the y-axis is log10 CFUs, so an increase from 3 to 4 (for example) is actually a tenfold increase in number of CFUs.

As expected, fungi were less abundant in female flower nectar than in male flower nectar.  In addition, bagging the flowers substantially reduced fungal abundance. Bacteria were also less abundant in female flower nectar than in male flower nectar.  Surprisingly, bagging the flowers substantially increased bacterial abundance, despite the increased chemical stress and decreased visitation by animal visitors.

Why did bacterial abundance increase when flowers were bagged?  The researchers hypothesize that reduced fungal dispersal from bagging caused competitive release of bacteria from the fungi.  Presumably the fungi and bacteria compete for essential resources (such as amino acids) in the nectar.  Because the bags reduce fungal abundance, there are fewer fungi to out-compete the bacteria, leading to an increase in bacterial abundance.

The researchers used DNA analysis to characterize which microbial species were found in female vs. male flowers.  They discovered major differences in species composition between the sexes.  Taken together with the data on frequency and abundance, it is clear that sexual dimorphism in these plants influences microbial communities in significant ways.  Tsuji and Fukami suggest that sexual dimorphism in many species may have profound community-wide consequences that researchers are only beginning to understand and uncover.

note: the paper that describes this research is from the journal Ecology. The reference is Tsuji, K. and Fukami, T. (2018), Community‐wide consequences of sexual dimorphism: evidence from nectar microbes in dioecious plants. Ecology, 99: 2476-2484. doi:10.1002/ecy.2494. Thanks to the Ecological Society of America for allowing me to use figures from the paper. Copyright © 2018 by the Ecological Society of America. All rights reserved.