Research Overview

Honeypot ants are distributed globally, and have evolved repletism, a characteristic where certain ant workers serve as a food storage vessel for the rest of the colony in times of scarcity. These ants belong to at least six different genera that have evolved independently in semi-arid habitats, and are a classic example of convergent evolution. Members of The Khadempour Lab are interested in learning whether these ants have a resident microbiome, and if so, what its functions might be, and whether it will have also converged across different ant species. Dr. Khadempour has also established the International Consortium of Honeypot Ant Researchers, which is a collaborative network of cool people that are interested in these cool ants. If you think you can contribute to the consortium, send Dr. Khadempour an email.

Like other pitcher plant species, Sarracenia purpurea live in soils that are relatively poor in nutrients. To compensate for low nutrients they are carnivorousCarnivorous plants are plants that derive some or most of their nutrients (but not energy) from trapping and consuming animals or protozoans, typically insects and other arthropods. The insects crawl into the pitchers and become trapped in a pool of rainwater and then are broken down by a community of bacteria. These bacteria are consumed by protozoa in the genus Tetrahymena. The protists are then consumed by mosquito larvae.

My first experiment with this system was designed to test the bottom-up effects of bacteria on the protists. I wanted to determine if all bacteria are the same for the protists or if some bacteria are better for protist growth. We found that it does make a difference. Some strains help the protists to multiply quickly, while others kill all the protists in the sample. We also combined different bacterial strains together to create "good communities" and "bad communities" and found that the effect of bacteria on protists fitness persists when the bacteria are in a community.

Leaf-cutter ants grow a fungal cultivar that serves as their primary food source. The fungus is responsible for breaking down plant biomass that the ants cut and provide. It produces gongylidiaHyphal swellings (hypha are the fibers that make the "body" of a fungus) produced by higher attine cultivars. They contain an abundance of sugars and lipids and serve as the ant's primary food source. They also contain biomass-degrading enzymes that ants ingest then deposit on the top of the fungus garden when they defecate. that the ants eat. A monophyletic groupA monophyletic group, or clade, is a group of organisms that consists of all the descendants of a common ancestor. of ants within the genus AttaAtta are one genus of leaf-cutter ants. The other genus of leaf-cutters are Acromyrmex. Atta have larger colonies than Acromyrmex with more size polymorphism. have transitioned to cutting grasses instead of the usual dicots. I am interested in whether the fungus associated with these ants has also undergone an evolutionary transition.

To examine this, we are sequencing the genomes of eight fungal strains from four species of ants (two strict grass-cutters, one dicot-cutter and one species that cuts both grasses and dicots). We are examining these genomes for adaptations to their particular substrate. We are also looking into the genomes and comparing them to close relatives that are not symbiontsAn organism that lives in close association with another.. Doing so will allow us to test if there are genomic adaptations for symbiosisThe living-together of unlike organisms. Can be mutualism, commensalism or parasitism..

Leaf-cutter ant fungus gardens house a communityInteracting group of various species in a common location. of bacteria. We know very little about what these bacteria may or may not be doing in the fungus garden. Previous research indicates that bacteria fix nitrogenA process by which an organism takes N₂, which is an inorganic form of nitrogen that many organisms cannot use, and changes it into an organic, usable form., making it available for the ants (the plants they indirectly consume do not have enough nitrogen). Through previous metagenomic sequencingSequencing all of the DNA in a sample. We can infer both the identities of the organisms in a sample and their functional capacities., there was also an indication that the bacteria could be providing the system with vitamins and nutritional supplements.

My work takes our knowledge of these bacterial communities further. I used metagenomic sequencingSequencing all of the DNA in a sample. We can infer both the identities of the organisms in a sample and their functional capacities. to compare fungus gardens from grass-cutter and dicot-cutter ants. I found that the bacterial communities significantly differed between the two systems, both in the communityInteracting group of various species in a common location. composition (who’s there) and functional capacity (what they’re capable of doing).

The bacterial genus Pantoea was the most abundant bacteria in the system, and is one of the bacteria that are known to fix nitrogenA process by which an organism takes N₂, which is an inorganic form of nitrogen that many organisms cannot use, and changes it into an organic, usable form. in the system. The diversity of bacteria was higher in the dicot-cutter ant fungus gardens, reflecting the higher diversity of plants that are incorporated by dicot-cutter ants.

The grass-cutter ant fungus gardens were enriched for genes related to nitrogen fixation, amino acid metabolism, siderophoreProteins that are responsible for iron acquisition in low-iron environments. production and terpenoid biosynthesis. These are correlated with lower amounts of nitrogen, amino acids, iron and terpenoids in grasses compared to dicots and we believe that this suggests that the bacteria are compensating for a lack of these materials and could facilitate the ants’ transition from dicots to the less nutritious grasses.

Lipids are an important class of a molecules that animals use as long term energy storage and also for signaling. In collaboration with Pacific Northwest National Labs (PNNL) we are tracking the identity of lipids from leaf material as they works their way through the fungus garden and into gongylidia. We show that the lipid identities and abundances change significantly through the fungus gardens and that the gongylidia (which the ants eat) are distinct from the rest of the garden. We also performed a behavioral experiment and showed that Atta leaf-cutter ants are attracted to lipids that are found in the gongylidia.

Most leaf-cutter ants act as extreme generalist herbivores. This is very rare in the animal kingdom. I wanted to determine if the fungus in the leaf-cutter ant fungus gardens, which is responsible for breaking down plant biomass in the system, always produces the same enzymes no matter what the ants feed it, or if it responds differently to different substrates.

I set up an experiment with subcolonies of ants and their fungus, and fed them different substrates: leaves, flowers, Quaker Instant Oatmeal, and a mixture of all three. After two weeks of feeding, I sent the fungus garden material for metaproteomic analysisA method to identify and quantify all of the proteins in a sample and determine what organism produced them. Genomes for the target organisms are necessary to be able to identify the proteins with spectral data. at PNNL. We determined that the fungus responds in a flexible, substrate-specific manner to the substrates that the ants incorporate. One important finding was that the fungus only produces enzymes to degrade recalcitrantBiomass that is difficult to break down, requiring specialized enzymes. biomass when in needs to: it does not produce them when it is fed oats, either on their own or in a mixture with leaves and flowers. We believe that the flexible, substrate-specific nature of the fungus helps the ants to function as generalist herbivores.

Mountain pine beetles are major native forest pests in western North America. The beetles are a type of bark beetles - they attack trees and burrow under the bark in the phloem where they lay eggs and where their young develop. The beetles are associated several species of fungi. Some of these species have been closely studied while others have not.

Being able to easily identify and detect these fungi is important for the wood products industry, so I designed target-specific PCR primersOligonucleotide segments that align with a section of DNA that is unique to a species or group. When running a PCR with DNA extracted from either the target or a mixed sample containing the target, you should have successful DNA amplification. for this task. Once I had the primers, I was able to use them in a study where I could identify and quantify the fungi over the lifecycle of the beetle in pine trees using qPCRQuantitative PCR - a method to quantify the abundance of an organism or gene in a sample with primers that are targeted to the gene of interest, and a fluorescent dye that binds to the amplified DNA.. I found that three of the species of fungi (that are most often studied because they grow quickly in culture) are abundant in the early stages of the beetle lifecycle. These fungi have the ability to use pine terpenes as a carbon source, and likely serve to detoxify the phloem environment for developing larvae. The fourth fungus grows much more slowly and likely serves as a nutritional source. It was significantly more abundant toward the end of the beetle lifecycle in the tree, in the beetle’s meconiumBefore a larval mountain pine beetle pupates, it voids its gut and lines the walls of its pupal chamber with that material, this material is called the meconium. When it completes its pupation and becomes a teneral adult, it eats through this nutrient rich meconium before leaving the tree., which the beetle consumes before leaving the tree.