microorganisms Article Influence of Lab Adapted Natural Diet and Microbiota on Life History and Metabolic Phenotype of Drosophila melanogaster Andrei Bombin * , Owen Cunneely, Kira Eickman, Sergei Bombin, Abigail Ruesy, Mengting Su, Abigail Myers, Rachael Cowan and Laura Reed * Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487, USA; [email protected] (O.C.); [email protected] (K.E.); [email protected] (S.B.); [email protected] (A.R.); [email protected] (M.S.); [email protected] (A.M.); [email protected] (R.C.) * Correspondence: [email protected] (A.B.); [email protected] (L.R.) Received: 9 November 2020; Accepted: 9 December 2020; Published: 11 December 2020 Abstract: Symbiotic microbiota can help its host to overcome nutritional challenges, which is consistent with a holobiont theory of evolution. Our project investigated the effects produced by the microbiota community, acquired from the environment and horizontal transfer, on metabolic traits related to obesity. The study applied a novel approach of raising Drosophila melanogaster, from ten wild-derived genetic lines on naturally fermented peaches, preserving genuine microbial conditions. Larvae raised on the natural and standard lab diets were significantly different in every tested phenotype. Frozen peach food provided nutritional conditions similar to the natural ones and preserved key microbial taxa necessary for survival and development. On the peach diet, the presence of parental microbiota increased the weight and development rate. Larvae raised on each tested diet formed microbial communities distinct from each other. The effect that individual microbial taxa produced on the host varied significantly with changing environmental and genetic conditions, occasionally to the degree of opposite correlations. Keywords: microbiota; 16S; metabolic phenotype; host microbiota interaction 1. Introduction The holobiont theory states that a host and its commensal microbiota possess a metagenome that expresses a synergistic phenotype, which is subjected to evolutionary forces as one complex organism [1]. A phenotype of this unit could be varied by genome modifications of the host, and its commensal bacteria. Metagenomic changes induced by bacteria have more potential for genetic variability and could arise by altering dominant species of bacteria, and acquisition of new strains of microorganisms from an environment [2]. Gut microbiota is one of the most important factors shaping metabolic phenotype and, as a consequence, is a key element in the development of metabolic and autoimmune diseases, cancer, and asthma [3,4]. Alterations in gut microbiota biodiversity and community structure are correlated with the development of the obese phenotype [5,6]. Transfer of the microbiota from an obese to a lean, axenic (microbiota free) individual significantly increases weight gain and adiposity, compared to axenic mice colonized with microbiota community from a lean organism [5,7,8]. These results suggest that obesity can be transferred from one individual to another; therefore, exhibiting some characteristics of an infectious disease [8]. Many species of Drosophila use rotting fruit as their primary host, including Drosophila melanogaster, and primarily consume the microorganisms growing in the rot. During colonization of a rotting fruit, Microorganisms 2020, 8, 1972; doi:10.3390/microorganisms8121972 www.mdpi.com/journal/microorganisms Microorganisms 2020, 8, 1972 2 of 27 Drosophila inoculates the substrate with their microbiota [9]. In addition, when females deposit embryos on a substrate, parental microbiota resides on the chorion of the egg [10]. The initial microbiota is gained by the first instar larvae through consumption of the chorion [10]. Later, during feeding, larvae acquire additional microbiota from the environment, representatives of which remain in the larval and pupal intestine until eclosion of the adult fly [11]. Wong et al. [12] showed that parental microbiota transferred with the chorion of the egg could modify the microbial community composition in a food substrate and in the offspring. In addition, the transfer of axenic Drosophila on food substrate would change the food microbial community to resemble the symbiotic microbiota composition that would develop in the host [12]. Symbiotic microbiota play an important role in D. melanogaster development and metabolic phenotype. Axenic flies have a longer development time, lower weight, protein, and glycogen content but higher free glucose and triglyceride levels [13–15]. There are similar negative phenotypic effects on axenic D. melanogaster and on those being raised on harmful diets such as high sugar and high fat [13,15–18]. Although axenic Drosophila consume less food, their energy storage indices (triglyceride, glucose, glycogen, and trehalose levels) stay significantly higher than that of conventional flies [19]. In addition, the presence of the commensal microbiota allows Drosophila to maximize their lifespan and reproductive output [4]. Dobson et al. [14] showed that the abundance of specific bacterial taxa is associated with a change in metabolic phenotype: Acetobacter, Gluconobacter and Komagataeibacter are negatively correlated with a fly’s energy storage index. Lactobacillus is positively associated with triglyceride concentration and Achromobacter and Xanthomonadaceae have a positive correlation with glycogen levels [20,21]. In addition, Drosophila microbiota varies across genetic backgrounds, which makes it possible to establish associations between a host’s genes and microbiota dependent metabolic responses and particular symbiotic species [14,21,22]. Symbiotic microbiota allow Drosophila to overcome the nutritional limitation of their diet. Shin et al. [23] showed that axenic larvae raised on a casamino acid diet experienced a 90% body size reduction and were not able to survive to form pupae. However, the presence of only one bacterial species, Acetobacter pomorum, could restore survival and the normal rate of larval development via induction of the hosts’ insulin-like growth factor signaling [23]. Leitão-Gonçalves et al. [4] demonstrated that axenic flies express a strong preference for yeast-rich food due to their demand for essential amino acids. A. promorum and several Lactobacilli species are able to suppress the yeast appetite in Drosophila and shift the flies’ nutritional preference toward high-sugar concentration diets [4]. Their change in nutritional preference may be explained by competition between the host and its symbiotic microbiota for available sugars and through production of essential amino acids by the microbial community [4]. Microbiota composition of lab dwelling Drosophila primarily consists of Acetobacteraceae, Enterococcaceae, and Lactobacillaceae families [22,24]. Within these families, the influence of Acetobacter tropicalis, Enterococcus faecalis, Lactobacillus brevis, and Lactobacisul plantarum on a host’s metabolic phenotype has been studied more than others [15,22,23]. However, the microbiota of wild fly populations may differ in the diversity and the abundance of dominant species [24]. In the lab, Drosophila raised on fruits still exhibit a more complex and diverse community of symbiotic microbes compared to conventionally raised flies [25]. Lab food preservatives, especially methylparaben sodium salt (moldex), largely contribute to the difference between natural and laboratory associated microbiota communities [26]. Therefore, studying the evolutionary relationship of Drosophila and its microbiota, and the symbiont’s influence on fly’s metabolic phenotype only on standard lab microbiota, may be insufficient to understand the natural relationship and coevolution of the fly and its microbiota. With this work we wanted to address a series of specific questions and hypotheses: Hypothesis 1. How does a natural diet with a naturally occurring and/or maternally inherited community of microbes influence the life history and metabolic phenotypes of flies relative to a standard lab diet? Is there genetic variation in the phenotypic response to nutritional change and dietary and parental microbiota availability? We hypothesized that larvae raised on a natural diet will exhibit different life history traits and metabolic Microorganisms 2020, 8, 1972 3 of 27 phenotypes, comparing with larvae raised on a standard lab diet. We hypothesized that the presence of a maternally transmitted microbiota will significantly impact larvae phenotypes, and that this impact may vary across dietary treatments. Given prior findings on the roles of genetic variation on metabolic phenotypes, we hypothesized that there is genetic variation in phenotype that interacts with the dietary conditions and the availability of maternally transmitted microbiota. Hypothesis 2. Will the symbiotic microbiota community of the larvae raised on the natural diet be different from the lab food raised larvae? Will the presence of maternally inherited microbiota influence the formation of microbial communities? Is the microbiota community variable across host genotypes? We hypothesized that the gut microbial community composition and diversity will vary substantially across both dietary and parental microbiota conditions. We hypothesized that the maternally transmitted microbiota will have “founder effects” in the formation of the larval gut microbiome. We hypothesized that the composition of the microbial community will exhibit variation with host genotype. Hypothesis 3. Will specific