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The interaction between a butterfly, its microbiome and the environment

Final Report Summary - BXMXE (The interaction between a butterfly, its microbiome and the environment)

It is now recognized that an organism does not function alone, with individuals being in intimate association with a resident microbial community. This microbiome can profoundly affect the biology and ecology of an organism, and as such may influence the direction or strength of evolution. Given that global environmental change is having major consequences for biodiversity and the integrity of natural ecosystems, how organisms respond and adapt to changing conditions, and how the bacterial microbiome affects the ability of hosts to adapt, is a timely and important question in biology.
The overall aim of this project is to investigate the impact of the bacterial microbiome on host fitness and local adaptation in the context of the changing environment by focusing on the interaction between the butterfly Colias eurytheme, its microbiome and temperature. The specific objectives include: a) characterising the bacterial microbiome of C. eurytheme, b) mapping the geographical pattern of microbiome diversity in butterflies collected across a latitudinal (temperature) cline; and c) experimentally investigating the role of temperature in the host/microbiome interaction.

Characterising the bacterial microbiome of C. eurytheme
To characterise the bacterial microbiome of C. eurytheme butterflies, 35 wild adults were collected from Pennsylvania, USA. The bacterial community of the thorax and abdomen of each sample was identified through sequencing of a hypervariable region of the bacterial 16S rRNA gene using Illumina MiSeq technology. Alongside, 6 adults of the closely related Colias philodice, collected at the same time, were similarly sequenced to ascertain whether the bacteria they harbour differ. Several fitness parameters were measured including body mass, thorax mass, abdomen mass, age, and peak metabolic rate (PMR; an indicator of flight performance). These were taken to provide insight into any relationship between the microbiome and host fitness.
Body mass was found to significantly vary with PMR in that heavier individuals have a higher PMR. This relationship was true for both males and females, and gender was also found to vary significantly with PMR, with females generally being heavier than males (Figure 1). Colias eurytheme males were also significantly heavier than C. philodice males (p=0.0213). The data also suggest a relationship between microbial community composition and host body mass, thus indirectly the constituents of the microbiome may affect the flight performance of the butterfly. Additionally there was an effect of tissue upon microbiome composition (Figure 2). There was an overall general trend of bacteria being at a higher density in the abdomen than in the thorax. Certain bacteria were almost exclusively found in the abdomen including an Acinetobacter. This may be expected as many bacteria are found in the gut within the abdomen as a result of being ingested. There was no direct evidence of a difference in the microbiome between males and females, or between the two species. Both species occur in the same habitat and share host plants (through which they can acquire bacteria), which may explain the observed overlap in bacterial community composition.
High-throughput sequencing data revealed the microbiome of Colias butterflies to be dominated by five bacteria from three diverse phyla: the Proteobacteria, Bacteroidetes and Firmicutes. The most abundant genus was Asaia, a Proteobacteria that has been identified in many insects including butterflies. Two other almost equally abundant taxa were an unclassified bacterium from the family Flavobacteriaceae, and a bacterium from the genus Acetobacter. Acetobacter bacteria have been found in many arthropods that live on sugar-based diets such as nectar, suggesting that the presence of this acetic acid bacterium could be linked to sugar metabolism in the butterfly. The remaining two bacteria found in the core Colias microbiome were an unclassified bacterium from the family Enterobacteriaceae, and a member of the genus Fructobacillus. Fructobacillus is found in flowers and so may be acquired through the host plant. This bacterium, which has been found in honeybees, may also aid digestion of the sugar compounds found in nectar.

Mapping microbiome diversity across a latitudinal (temperature) cline
Temperature is a key environmental factor implicated in global environmental change, and is also integral to host biology, particularly in thermally sensitive ectotherms such as butterflies. Thus butterflies are prone to constraint in which only a narrow range of thermal conditions are optimal for flight activity. As flight underpins most butterfly fitness traits, the host is expected to evolve to maximize the thermal range in which it can fly. Due to the intimacy of the relationship between host and microbiome there is also the expectation that 1) changes in temperature will affect the host/bacteria interaction with subsequent consequences on host fitness, and/or 2) heritable bacteria with a vested interest in maximising host reproductive fitness will influence the thermal tolerance and adaptation of the host.

To identify bacteria that may influence the thermal tolerance of C. eurytheme, 338 adults were collected from 14 sites at 11 points along a latitudinal cline in Eastern U.S.A.; between Georgia in the south and New York State in the north (Figure 3. Map showing location of sample sites across east coast USA). This represents a temperature cline with southernmost populations experiencing warmer conditions than that of the north. Both males and females were collected, as was both C. eurytheme and C. philodice, in order to investigate any gender or species-specific variation. At each collection site, the latitude, major host plant present, and humidity were recorded. Body size was estimated from the wings, as was the age of the butterfly. Both size and age of butterfly may impact microbiome composition and so should if possible be incorporated into the analysis. Of the individuals collected, the bacterial microbiome was sequenced from 253 abdominal samples (Table 1). This dataset has recently been received and is currently being analysed.

Investigating the role of temperature in the host/microbiome interaction
To further investigate the relationship between temperature, host butterfly and the microbiome I reared offspring from wild-caught C. eurytheme (from Georgia (GA) in the south, and Pennsylvania (PA) in the north) in the laboratory, manipulated the associated microbiome and then measured host fitness-related traits. Both GA and PA larvae were reared concurrently at three temperature regimes: cool, optimal and hot. To reflect the natural scenario I used temperature-controlled cabinets that allowed temperature to cycle over the course of the day. Within this experiment, I also treated a subset of the butterflies with antibiotics to perturb the microbiome, with a further subset of these butterflies being re-infected with bacterial samples (feces) collected from similar age caterpillars of control butterflies. From each experimental group larvae and adults were sampled at different time points. I measured fitness indicators including development time, survival, body mass and growth rate for all individuals, and resting (larvae) and peak (adults) metabolic rate for a subset. I will sequence the microbiome of individuals from each temperature condition and manipulation group to determine whether particular bacteria or combinations of bacteria are more likely to infect butterflies with higher fitness under different temperature regimes. This work will also elucidate the effect of temperature upon the composition and bacterial abundance within the microbiome.
Socio-economic impacts of the project
This research investigates whether bacteria influence host fitness in differing environments. The results may allow insight into potential mechanisms for pest control: C. eurytheme can be a pest of Alfalfa crops in California, therefore specific alteration of the microbiome could be used to reduce the impact of their behaviour. This knowledge can also be extended to other insect pests. More fundamentally, in order to understand how to preserve biodiversity it is important to understand basic organismal biology; these results will be important for the understanding of how animals respond and adapt to thermal conditions. Knowledge of the influence of the bacterial microbiome upon host biology is also extremely important for conservation biology. Movement of organisms via range shifts precipitated by changing habitat or thermal conditions is likely to cause microbiome dysbiosis. The implications of this for host fitness may be large, and thus comprehension of these interactions is important. In a more practical vein, when considering re-introducing endangered organisms, the results of this project will be important as source populations may have a vastly different microbiome to that of the sink population. The consequences of this may be devastating if there is a specific interaction between the microbiome, host fitness and environment.
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