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?

Kellycagedquoll

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.

Picture9

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.

DSC_0344

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.

IMG_2123

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).

MoreiraFig2

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.

IMG_2722

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.

MoreiraFif3a

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.

MoreiraFig4

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.

Tsujigazes

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.

TsujiFlowers

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.

Tsujiplates

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.

Tsuji2e

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.

Tsujifig2ab

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.

TsujiFig2cdnewnew

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.

Beautiful buds beset bumblebees with bad bugs

Sexual liaisons can be difficult to achieve without some type of purposeful motion.  Flowering plants, which are rooted to the ground, are particularly challenged to bring the male close enough to the female to have sex.  One awesome adaptation is pollen, technically the male gametophyte –  or gamete (sperm)-generating plant. These tiny males get to females either by floating through the air, or by being transferred by animal pollinators such as bees. Plants can lure bees to their flowers by producing nectar – a sugar rich fluid – which bees lap up and use as a carbohydrate source.  While nectaring, bees also collect pollen, either intentionally or inadvertently, which provides them with essential proteins. When bees travel to the next flower, they may inadvertently drop some of their pollen load near the female gametophyte – in this case a tiny egg-generating plant (though tiny, the female gametophyte is considerably larger than is the male gametophyte).  We call this process of “tiny boy meets tiny girl” pollination. Once the two gametophytes meet, the pollen produces one or more sperm, which it uses to fertilize an egg within the female gametophyte.  There is more to it, but this will hopefully clarify the difference between pollination and fertilization.

monardadidyma.jpg

Bumblebee forages on beebalm, Monarda didyma. Credit: Jonathan Giacomini.

All of this business takes place within the friendly confines of the flower.  The same flower may be visited by many different bees of many different species. While feeding, bees carry on other bodily functions, including defecation.  They are not careful about where they defecate; consequently a bee’s breakfast might also include feces from a previous bee visitor. Bumblebee (Bombus impatiens) feces carries many disease organisms, including the gut parasite Crithidia bombi, which can reduce learning, decrease colony reproduction and impair a queen’s ability to found new colonies. Because pollinators are so critical in ecosystems, Lynn Adler and her colleagues wondered whether certain types of flowers were better vectors for harboring and transmitting Crithidia bombi to other bumblebees.

Antirrhinummajus

Bumblebee forages on the snapdragon, Antirrhinum majus. Credit: Jonathan Giacomini.

The researchers chose 14 different flowering plant species, allowing uninfected bumblebees to forage on inflorescences (clusters of flowers) inoculated with a measured amount of Crithidia bombi parasites.  The bees were reared for seven days after exposure, and then were assessed for whether they had picked up the infection from their foraging experience, and if so, how intense the infection was. The researchers dissected each tested bee and counted the number of Crithidia cells within the gut.

researcher-photo.jpg

Researcher conducts foraging trial with Lobelia siphilitica inflorescence. Credit: Jonathan Giacomini.

Adler and her colleagues discovered that some plant species caused a much higher pathogen count (mean number of infected cells in the bee gut) than did other plant species.  For example bees that foraged on Asclepias incarnata (ASC) had four times as many pathogens, on average, than did bees that foraged on Digitalis purpurea (DIG) (top graph below). Bees foraging on Asclepias were much more likely to get infected (had greater susceptibility) than bees that foraged on several other species, most notably Linaria vulgaris (LIN) and Eupatorium perfoliatum (EUP) (middle graph). Lastly, if we limit our consideration to infected bees, the mean intensity of the infection was much greater for bees foraging on some species, such as Asclepias and Monarda didyma (MON) than on others, such as Digitalis and Antirrhinum majus (ANT) (bottom graph).

AdlerFig1

(Top graph) Mean number of Crithidia (2 microliter gut sample) hosted by bees after foraging on one of 14 different flowering plant species. This graph includes both infected and uninfected bees. (Middle graph) Susceptibility – the proportion of bees infected – after foraging trials on different plant species. (Bottom graph) Intensity of infection – Mean number of Crithidia for infected bees only. The capital letters below the graph are the first three letters of the plant genus. Numbers in bars are sample size.  Error bars indicate 1 standard error.

It would be impossible to repeat this experiment on the 369,000 known species of flowering plants (with many more still to be identified).  So Adler and her colleagues really wanted to know whether there were some flower characteristics or traits associated with plant species that served as the best vectors of disease.  The researchers measured and counted variables associated with the flowers, such as the size and shape of the corolla, the number of open flowers and the number of reproductive structures (flowers, flower buds and fruits) per inflorescence.

bluelobelia.png

Flower traits measured by Adler and colleagues (example for blue lobelia, Lobelia siphilitica). CL is corolla length. CW is corolla width. PL is petal length. PW is petal width. Credit: Melissa Ha.

The researchers also wanted to know whether any variables associated with the bees, such as bee size and bee behavior, would predict how likely it was that a bee would get infected.  Surprisingly, the number of reproductive structures per inflorescence stood out as the most important variable. In addition, smaller bees were somewhat more likely to get infected than larger bees, and bees that foraged for a longer time period were more prone to infection.

AdlerFig2

Mean susceptibility of bees to Crithidia infection after foraging on 14 different flowering plant species, in relation to the number of reproductive structures (flowers, buds and fruits) per inflorescence.

These findings are both surprising and exciting. Adler and her colleagues were surprised to find such big differences in the ability of plant species to transmit disease.  In addition, they were puzzled about the importance of number of reproductive structures per inflorescence.  At this point, they don’t have a favorite hypothesis for its overriding importance, speculating that some unmeasured aspect of floral architecture influencing disease transmission might be related to the number of reproductive structures per inflorescence.

Penstemondigitalis

Bumblebee forages on Penstemon digitalis. In addition to the open flowers, note the large number of unopened buds.  Each of these counted as a reproductive structure for the graph above. Credit: Jonathan Giacomini.

The world is losing pollinators at a rapid rate, and there are concerns that if present trends continue, there may not be enough pollinators to pollinate flowers of some of our most important food crops. Disease is implicated in many of these declines, so it behooves us to understand how plants can serve as vectors of diseases that affect pollinators. Identifying floral traits that influence disease transmission could guide the creation of pollinator-friendly habitats within plant communities, and help to maintain diverse pollinator communities within the world’s ecosystems.

note: the paper that describes this research is from the journal Ecology. The reference is Adler, L. S., Michaud, K. M., Ellner, S. P., McArt, S. H., Stevenson, P. C. and Irwin, R. E. (2018), Disease where you dine: plant species and floral traits associated with pathogen transmission in bumble bees. Ecology, 99: 2535-2545. doi:10.1002/ecy.2503. 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.

Dinoflagellates deter copepod consumption

Those of us who enjoy eating seafood are dismayed by the dreaded red tide, which renders some of our favorite prey toxic to us.  A red tide occurs when dinoflagellates and other algae increase sharply in abundance, often in response to upwelling of nutrients from the ocean floor.  Many of these dinoflagellates are red or brownish-red in color, so large numbers of them floating on or near the surface give the ocean its characteristic red color. These dinoflagellates produce toxic compounds (in particular neurotoxins) that pass through the food web, ultimately contaminating fish, molluscs and many other groups of species.

redtideCreditMarufish:FlickrIsahayaBay

Red tide at Isahaya Bay, Japan.  Credit: Marufish/Flickr.

Did toxicity arise in dinoflagellates to protect them from being eaten by predators – in particular by voracious copepods?  The problem with this hypothesis is that copepods eat an entire dinoflagellate.  Let’s imagine a dinoflagellate with a mutation that produces a toxic substance. At some point the dinoflagellate gets eaten, and the poor copepod consumer is exposed to the toxin.  Maybe it dies and maybe it lives, but the important result is that the dinoflagellate dies, and its mutant genes are gone forever, along with the toxic trait. The only way toxicity will benefit the dinoflagellate individual, and thus spread throughout the dinoflagellate population, is if it increases the survival/reproductive success of individuals with the toxic trait. This can occur if copepods have some mechanism for detecting toxic dinoflagellates, and are therefore less likely to eat them.

Jiayi Xu and Thomas Kiørboe went looking for such a mechanism using 13 different species or strains of dinoflagellates that were presented to the copepod Temora longicornis. This copepod beats its legs to create an ocean current that moves water, and presumably dinoflagellates, in its direction, which it then eats.  For their experiment, the researchers glued a hair to the dorsal surface of an individual copepod (very carefully), and they then attached the other side of the hair to a capillary tube, which was controlled by a micromanipulator. They placed these copepods into small aquaria, where the copepods continued to beat their legs, eat and engage in other bodily functions.

照片 3

Aquarium with tethered copepod and recording equipment: Credit: J. Xu.

The researchers then added a measured amount of one type of dinoflagellate into the aquarium, and using high resolution videography, watched the copepods feed over the next 24 hours.

Picture1

Tethered copepod beats its legs to attract a dinoflagellate (round blue circular cell). Credit: J. Xu.

Twelve of the dinoflegellate strains were known to be toxic, though they had several different types of poison. Protoceratium reticulatum was a nontoxic control species of dinoflagellate.  As you can see below, on average, copepods ate more of the nontoxic P. reticulatum than they did of any of the toxic species.

XuFig1

Average dinoflagellate biomass ingested by the tethered copepods.  P. reticulatum  is the nontoxic control.  Error bars are 1 SE.

Xu and Kiørboe identified two major mechanisms that underlie selectivity by the copepod predator.  In many cases, the copepod successfully captured the prey, but then rejected it (top graph below). For one strain of A. tamarense prey, and a lesser extent for K. brevis prey, the predator simply fed less as a consequence of reducing the proportion of time that it beat its feeding legs (bottom graph below).

XuFig3bd

Copepod feeding behavior on 13 dinoflagellate prey species.  Top graph is fraction of dinoflagellates rejected, while bottom graph is the proportion of time the copepods beats its feeding legs in the presence of a particular species/strain of dinoflagellate.  

If you look at the very first graph in this post, which shows the average dinoflagellate biomass consumed, you will note that both strains of K. brevis (K8 and K9) are eaten very sparingly.  The graphs just above show that the copepod rejects some K. brevis that it captures, and beats its legs a bit less often when presented with K. brevis. However, the rejection increase and leg beating decreases are not sufficient to account for the tremendous reduction in consumption. So something else must be going on.  The researchers suspect that the copepod can identify K. breviscells from a distance, presumably through olfaction, and decide not to capture them. This mechanism warrants further exploration.

One surprising finding of this study is that the copepod responds differently to one strain of the same species (A. tamarense) than it does to the other strains.  Xu and Kiorbe point out that previous studies of copepod/dinoflagellate interactions have identified other surprises.  For example, there are cases where a dinoflagellate strain is toxic to one strain of copepod, but harmless to another copepod strain of the same species. Also, within a dinoflagellate species, one strain may have a very different distribution of toxins than does a second strain.  So why does this degree of variation exist in this system?

The researchers argue that there may be an evolutionary arms race between copepods and dinoflagellates.  The copepod adapts to the toxin of co-occurring dinoflagellates, becoming resistant to the toxin. This selects for dinoflagellates that produce a novel toxin that the copepod is sensitive to. Over time, the copepod evolves resistance to the second toxin as well, and so on… Because masses of ocean water and populations of both groups are constantly mixing, different species and strains are exposed to novel environments with high frequency. Evolution happens.

note: the paper that describes this research is from the journal Ecology. The reference is Xu, J. and Kiørboe, T. (2018), Toxic dinoflagellates produce true grazer deterrents. Ecology, 99: 2240-2249. doi:10.1002/ecy.2479. 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.

Savanna plant survival: hanging out in the right crowd

Tyler Coverdale first visited the Mpala Research Centre in Laikipia, Kenya in 2013, and immediately became painfully aware of the abundant spiny and thorny plants that cover the savanna.  Spines help defend the plants from voracious elephants, giraffes and numerous other herbivores that depend on vegetation for their sustenance.

Camels

Camels browsing on  Barleria trispinosa at Mpala Research Centre, Kenya. Credit Tyler Coverdale.

Acacia trees such as Acacia etbaica (left foreground below) dominate the landscape, and may be associated with smaller shrubs, such as Barleria trispinosa. In the photo below, there is one B. trispinosa plant immediately below (on the right side) the acacia tree, and a second B. trispinosa plant to its right, more out in the open.  Coverdale realized that being situated immediately below a spiny acacia tree might be advantageous to B. trispinosa, which could be protected from the ravages of elephants and giraffes by the acacia thorns .

MRC landscape

Credit: Tyler Coverdale.

As you might guess by its name, B. trispinosa is itself a very spiny plant, which should help protect it from browsers.  Nonetheless, it still gets eaten, so Coverdale and his colleagues explored whether being under acacias would reduce how much it, and two other related species, got browsed.

Barleria trispinosa

Barleria trispinosa out in the open. Credit: Tyler Coverdale.

The first study was observational – a survey of the damage three species of Barleria suffered when they were under (associated with) acacia trees vs. unassociated with acacia trees. For each Barleria species, the researchers haphazardly chose 10 stems from eight associated and eight unassociated plants, and measured the proportion of these stems that showed physical evidence of being browsed.  As the figure below shows, browsing was sharply lower for each species when it was associated with an acacia plant.

CoverdaleFig1A

Percentage of stems damaged by browsers for three Barleria species in relation to whether they were associated or unassociated with an acacia tree.* indicates significant differences between means in all figures.

The understory plant community associated with acacias is much denser than the plant community out in the open, so the researchers wondered whether it was the acacia itself, or the other plants associated with it, that were providing protection. They set up an experiment using focal B. trispinosa plants with four treatments (A) unmanipulated control, (B) overstory removal, (C) overstory + understory removal, (D) a procedural control with overstory + understory removal, with the focal plant enclosed in a metal cage to protect it from predators (see Figure below).

CoverdaleS1

Coverdale and his colleagues ran the experiment for one month.  They discovered that removing overhanging acacia branches sharply increased herbivory, but the additional removal of understory neighbors had little additional effect.  Both the unmanipulated controls and procedural controls were unaffected.

CoverdaleFig1B

Change in % of stems browsed for (A) unmanipulated control (left bar), (B) overstory removal (second from left bar), (C) overstory + understory removal (second from right bar), (D) a procedural control (right bar).  Different letters above bars indicate significant differences between the mean values.

The researchers then investigated how useful these spines are to unassociated B. trispinosa plants. They set up another experiment with four types of spine treatments: (A) unmanipulated controls, (B) 50% spine removal, (C) 100% spine removal, (D) procedural control with 100% spine removal + enclosure within a predator-proof cage. These cages were vandalized shortly after the experiment was set up, so the researchers chose eight plants from a nearby plot (that had all predators excluded for a different experiment) as their procedural control. They discovered that spines are very useful to protect against predators in unassociated B. trispinosa.

CoverdaleFig1C

Change in % of stems browsed for (A) unmanipulated control (left bar), (B) 50% spine removal (second from left bar), (C) all spines removed (second from right bar), (D) procedural control (right bar).

If you were a plant living under the protection of an acacia tree, it would make sense for you to reduce your investment in thorns, so you could allocate more resources to growth and reproduction.  Does Barleria do this?

CoverdaleFig2

Several lines of evidence indicate that all three Barleria species reduce their investment in spines when associated with an acacia. First, a survey of spine density shows a reduced number of spines for all three species when they were associated with acacia trees (top graph).  Second, the spines that are present are significantly shorter in Barleria species associated with acacia trees (middle graph).  In a final survey, Coverdale and his colleagues cut all of the spines off of associated and unassociated Barleria.  For each plant, the researchers calculated the dry weight of spines and of all the other plant tissue.  For each Barleria species, the defensive investment – the ratio of spines to total mass, was substantially reduced in acacia-associated plants in comparison to unassociated plants (bottom graph).

Lastly, can plants react adaptively to browsing?  In other words, will understory plants produce more thorns if they are browsed?  To explore this question, the researchers used scissors to simulate moderate (25%) or heavy (50%) browsing.  They discovered a significant increase in spines produced by unassociated plants one month after clipping. Ecologists call this an induced defense. This induced defense is strongly suppressed in plants that have lived under the protection of acacia trees – in fact there was no significant response to experimental browsing in acacia-associated B. trispinosa plants. The researchers don’t know how long this suppression of induced responses persists. Would browsing induce increased spine growth in B. trispinosa six months, a year or two years after its protective acacia tree died?

Coverdale and his colleagues conclude that the overall benefit of association is positive to the plant populations.  Their studies show better survival and higher reproductive rates of acacia-associated understory plants. There is probably a cost associated with too many offspring competing for resources within a small area, as seedlings tend to grow within 1 meter of their parents.  However the reduction in defense costs probably overrides this cost of competition, leading to increased population size.  The researchers suggest a long-term study of population growth rates for acacia-associated and unassociated plants for several different species to see how general these effects are, and to explore whether other factors, such as soil moisture and nutrient levels influence the allocation and induction of defensive structures such as spines and thorns.

note: the paper that describes this research is from the journal Ecology. The reference is Coverdale, T. C., Goheen, J. R., Palmer, T. M. and Pringle, R. M. (2018), Good neighbors make good defenses: associational refuges reduce defense investment in African savanna plants. Ecology, 99: 1724-1736. doi:10.1002/ecy.2397. 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.