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Objectives
In this simple study, I aim to compare the genome of the novel Orbus bacterium Schmidhempelia to known strains of Bifidobacterium, both of which were isolated from the guts of Honey Wasps. To do this, I will use the Rapid Annotation using Subsystem Technology (RAST) program to annotate the genomes of each to be used in analysis.
Introduction
Honey bees and other insect pollinators have been studied extensively on their environmental and economic impact to countries across the globe (Corbet et al., 2015). In many of these species, such as the honey bee (Apis mellifera), microbial symbionts are harbored within the gut that can perform a variety of functions (Kwong and Moran, 2016). These bacteria may aid in pathogen defense and biofilm formation (Cox-Foster, et al., 2007), eusocial behaviors and queen rearing (Tarpy et al., 2015), honey production (Engel, 2012), and in this study’s case, digestion and metabolism, which has been studied in other contexts (Zheng, et al., 2016).
Lactobacillus (Class: Bacilli) and Bifidobacterium (Class: Actinobacteria) are anaerobic bacteria genera that are often studied in the context of human probiotic function (Jaskari, et al., 1998). Both classes, Bacilli and Actinobacteria, have been proven to aid in carbohydrate digestion and enzymatic function, in the guts of bees and other insects. This includes the honey bee, bumble bee (bombus), and aphids (Aphididae) (Lee et al., 2014). By understanding how the microbiome works, certain steps could be taken to understand how these microorganisms interact with their hosts to aid them in digestion and other functions. This information could be used to help understand how to preserve Hymenoptera diversity, including bees (Kwong & Moran 2016).
Nine bacterial species clusters make up the core microbiota of the A. mellifera gut microbiome in 95% of individuals (Kwong & Moran 2016). All of the bacteria are known to be primarily obligate and unable to live outside of the bee’s gut, however some are found environmentally and in other insects (Kwong & Moran 2016). Some of the symbiont bacteria such as Lactobacillus, that tightly coevolved with the host tend to have smaller genomes, lacking essential genes that the host substitutes. However, they provide essential genes for the host, such as ones aiding in digestion (McCutcheon & Moran 2012). These bacteria are often found in eusocial insects, such as many bees, wasps, and ants (Holm 2017).
The newly discovered gammaproteobacterium from the Orbales order, Gilliamella apicola and has been found to be in both in the honey bee (A. mellifera) and the bumble bee (Bombus) gut. It has been shown to be closely tied to the gut of the worker honey bee, but some members do exist environmentally (Martinson et al., 2014). Gilliamella apicola has been found to possess carbohydrate digestion abilities that assist bees (Zheng et al. 2016). Specifically, they can break down the sugar mannose which is toxic to the honey bee (Sols, Cadenas, & Alvarado 1960; Zheng et al. 2016). In bees, mannose causes the metabolic enzyme mannosephosphate isomerase to become saturated as it tries to break down the substance. This causes a shortage in the enzyme, and an accumulation of mannose-6-P, which leads to a major depletion in ATP (Fuente et al., 1986).
The honey wasp is still relatively unstudied and holds the potential to reveal more about Orbales and the evolution of Hymenopteran digestion. Other insects have been found to have closely associated microbiota, but the honey wasp, or Brachygastra melifica, produces and stores honey in a similar way to A. mellifera, making it a good model organism to contrast the honey bee (Hunt et al. 1998).
In this study, Bifidobacterium is analyzed and compared along with novel species Schmidhempelia that has yet to be classified (Koch & Schmid-Hempel, 2011).
Methods
Full genome samples were obtained from Jo-Anne Holley of the Moran Lab to be analyzed an annotated. These genomes represent bacteria harvested from the gut microbiome of the Mexican Honey Wasp (Bracygastra mellifica). The 16S sequences of the bacteria were run through BLASTn to verify their identity (Morgulis et. al, 2008 & Zhang et. al, 2000).
To determine genome functionality, the full genome sequences were run through RAST (Aziz et. al, 2008). In both samples, the RASTtk annotation scheme was used. Gene calls were not preserved, nor were frameshifts fixed. Errors were automatically fixed, and gaps were backfilled. Surface-level information gathered from the computations and subsystem statistics were used in analysis.
Results and Discussion
The BLASTn search confirmed the identities of the two samples via the 16s ribosomal subunit sequence. Sample wjB1 was confirmed as Bifidobacterium commune (FIGURE 1), and sample wjB12 was traced to a novel bacterium identified by Koch and namesake Schmid-Hempel. This is expected, as Schmidhempelia has yet to be formally classified (FIGURE 2).
FIGURE 1: BLASTn top-hit for sample wjB1 identifies it as Bifidobacterium commune.
FIGURE 2: BLASTn top-hit for sample wjB12 identifies it as the novel bacterium identified in Koch & Schmid-Hempel, 2011.
From the surface-level analysis, it was found that the Bifidobacterium genome (FIGURE 3a) was 1.32 as large as the Schmidhempelia genome (FIGURE 4a), and had a much higher GC content. Bifidobacterium has a higher devotion to carbohydrate, protein, and amino acid metabolism as compared to Schmidhempelia (FIGURE 3b). Schmidhempelia has a higher devotion to nucleic acid metabolism and vitamin/cofactor synthesis as compared to Bifidobacterium (FIGURE 4b).
FIGURE 3: Graphical depiction of genome functionality in Bifidobacterium (wjB1), isolated from the guts of a Honey Wasp.
FIGURE 4: Graphical depiction of genome functionality in Schmidehmpeila (wjB12), isolated from the guts of a Honey Wasp.
The gene density of the two species can be calculated by dividing the number of coding sequences by the size of the genome. For the two samples:
Bifidobacterium = (2192)/(2,551,425) = 0.00086 coding regions/nucleotide
Schmidhempelia = (4752)/(1,933,285) = 0.0025 coding regions/nucleotide
Conclusions
Given that bacteria tend to lose genome size and become more dependent on the host genome the more tightly a symbiosis ingrains itself, I can support the hypothesis that Schmidhempelia carries a more tightly regulated symbiosis with the Honey Wasp due to it's more compact genome structure. The GC content may play a role in this as well, as it has been reported that strong symbioses show higher biases in this metric (McCutcheon & Moran, 2012).
This analysis could be improved by looking further into the specific proteins found in the metabolic pathways of each bacteria, as Honey Wasps carry a unique dietary pattern that is different than that of the Honey Bee. If these metabolic pathways are found to be significantly different, it could indicate the beginning of an early divergence of the bacteria found in the guts of Honey Bees from the bacteria found in the guts of Honey Wasps.
References
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Acknowledgments
I would like to thank Jo-Anne Holley of the Moran lab and Bugs in Bugs FRI stream for graciously allowing me to use these genomes to complete this class project, even after 4 years of me being a mediocre mentor. She has helped guide me through the jungle of academia, and has given me so much opportunity throughout my undergraduate career.
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