Accessing the Hidden Microbial Diversity of Aphids: an Illustration of How Culture-Dependent Methods Can Be Used to Decipher the Insect Microbiota

Abstract

Microorganism communities that live inside insects can play critical roles in host development, nutrition, immunity, physiology, and behavior. Over the past decade, high-throughput sequencing reveals the extraordinary microbial diversity associated with various insect species and provides information independent of our ability to culture these microbes. However, their cultivation in the laboratory remains crucial for a deep understanding of their physiology and the roles they play in host insects. Aphids are insects that received specific attention because of their ability to form symbiotic associations with a wide range of endosymbionts that are considered as the core microbiome of these sap-feeding insects. But, if the functional diversity of obligate and facultative endosymbionts has been extensively studied in aphids, the diversity of gut symbionts and other associated microorganisms received limited consideration. Herein, we present a culture-dependent method that allowed us to successfully isolate microorganisms from several aphid species. The isolated microorganisms were assigned to 24 bacterial genera from the Actinobacteria, Firmicutes, and Proteobacteria phyla and three fungal genera from the Ascomycota and Basidiomycota phyla. In our study, we succeeded in isolating already described bacteria found associated to aphids (e.g., the facultative symbiont Serratia symbiotica), as well as microorganisms that have never been described in aphids before. By unraveling a microbial community that so far has been ignored, our study expands our current knowledge on the microbial diversity associated with aphids and illustrates how fast and simple culture-dependent approaches can be applied to insects in order to capture their diverse microbiota members.

New Insights into the Nature of Symbiotic Associations in Aphids: Infection Process, Biological Effects, and Transmission Mode of Cultivable Serratia symbiotica Bacteria

“…The aphid-associated bacterium S. symbiotica has typically been described to be a strict endosymbiont, but the discovery of cultivable strains that are potentially capable of growing outside aphid hosts shows a different aspect of this symbiont species and raises questions about its associated biological effects, as well as the nature and the durability of the associations in which it is involved. These strains have been isolated from Aphis species but have not been clearly localized in their original hosts (36,37). Nevertheless, a new field study revealed that these cultivable strains belong to the same clade as other S. symbiotica strains that reside in the aphid gut harvested in the field (38), suggesting that they would derive , and weight of emerged parasitoids (C).…”

“…Cultivable S. symbiotica strain CWBI-2.3 T (S1ϩ), isolated from a field-collected A. fabae aphid (36,39), was used in this study. In addition, we isolated two novel strains of S. symbiotica as described previously (36,37): strain 24.1 (S2ϩ) from a field-collected A. fabae aphid and strain Apa8 A1 (S3ϩ) from an Aphis passeriniana aphid collected in Tunisia. These strains, which had a free-living capacity, were preserved in frozen stocks at Ϫ80°C and cultured at 20°C in 863 medium (1% yeast extract, 1% casein peptone, 1% glucose) as described previously (36).…”

“…Recently, several strains of S. symbiotica have been successfully isolated from aphids in the Aphis genus and cultivated freely in an axenic pure medium without insect cell lines and fetal bovine serum (FBS) (36,37). A new field study just revealed that these cultivable strains belong to the same clade as the S. symbiotica strains residing in the gut of field-collected aphids, suggesting that they are most likely gut-associated symbionts (38).…”

Draft Genome Sequences of Two Cultivable Strains of the Bacterial Symbiont Serratia symbiotica

“…We report here the draft genome sequences of two S. symbiotica strains displaying free-living capacities, namely, strain 24.1, previously isolated from the black bean aphid Aphis fabae, and strain Apa8A1, previously isolated from the sage aphid Aphis passeriniana (7). Bacterial cultures were started from single colonies and grown in 863 medium at 20°C (6).…”

“…In the pea aphid Acyrthosiphon pisum (subfamily Aphidinae), S. symbiotica includes intracellular strains of a facultative nature showing moderately reduced genome sizes (3,(20)(21)(22). In addition to co-obligate and facultative strains, S. symbiotica also includes strains capable of growing independently from their host aphid on an artificial rich medium (6,7). The genomic features of the strain CWBI-2.3 T , isolated from A. fabae, suggest that it may represent a missing link in the evolution of a free-living lifestyle toward a host-dependent lifestyle (1, 2, 6, 7).…”