Microb Ecol https://doi.org/10.1007/s00248-017-1092-x INVERTEBRATE MICROBIOLOGY Accessing the Hidden Microbial Diversity of Aphids: an Illustration of How Culture-Dependent Methods Can Be Used to Decipher the Insect Microbiota Alina S. Grigorescu1 & François Renoz2 & Ahmed Sabri3 & Vincent Foray 4 & Thierry Hance2 & Philippe Thonart1 Received: 6 June 2017 /Accepted: 10 October 2017 # Springer Science+Business Media, LLC 2017 Abstract Microorganism communities that live inside insects symbionts and other associated microorganisms received can play critical roles in host development, nutrition, im- limited consideration. Herein, we present a culture- munity, physiology, and behavior. Over the past decade, dependent method that allowed us to successfully isolate high-throughput sequencing reveals the extraordinary mi- microorganisms from several aphid species. The isolated crobial diversity associated with various insect species microorganisms were assigned to 24 bacterial genera from and provides information independent of our ability to the Actinobacteria, Firmicutes, and Proteobacteria phyla culture these microbes. However, their cultivation in the and three fungal genera from the Ascomycota and laboratory remains crucial for a deep understanding of Basidiomycota phyla. In our study, we succeeded in isolat- their physiology and the roles they play in host insects. ing already described bacteria found associated to aphids Aphids are insects that received specific attention because (e.g., the facultative symbiont Serratia symbiotica), as well of their ability to form symbiotic associations with a wide as microorganisms that have never been described in aphids range of endosymbionts that are considered as the core before. By unraveling a microbial community that so far has microbiome of these sap-feeding insects. But, if the func- been ignored, our study expands our current knowledge on tional diversity of obligate and facultative endosymbionts the microbial diversity associated with aphids and illustrates has been extensively studied in aphids, the diversity of gut how fast and simple culture-dependent approaches can be applied to insects in order to capture their diverse microbiota Alina S. Grigorescu and François Renoz contributed equally to this work. members. Electronic supplementary material The online version of this article . (https://doi.org/10.1007/s00248-017-1092-x) contains supplementary Keywords Insect Aphid microbiota Culture-dependent material, which is available to authorized users. method . Molecular phylogeny . Symbiotic bacteria * Alina S. Grigorescu [email protected] Introduction * François Renoz [email protected] Insects owe to a great extent their adaptation to different en- vironments to the relationships they developed with various – 1 Walloon Center of Industrial Biology, Université de Liège, microorganisms [1 4]. Aphids (Hemiptera: Aphididae) are Sart-Tilman, B40, 4000 Liège, Belgium phloem sap-feeding insects that represent a relevant model 2 Earth and Life Institute, Biodiversity Research Center, Université to study such insect-bacteria relationships [5]. Indeed, they Catholique de Louvain, Croix de Sud 4-5, bte L7.07.04, have established associations with (i) obligate (or primary) 1348 Louvain-la-Neuve, Belgium endosymbionts, which are harbored in special host cells called 3 Artechno SA, Rue Herman Meganck 21, 5032 Isnes, Belgium bacteriocytes and fundamentally supply nutritional roles – 4 Present address: Centre de Recherches de Biochimie [6 8], and (ii) facultative (or secondary) symbionts, which Macromoléculaire (UMR-CNRS 5237), 1919, Route de Mende, can be localized in a variety of host tissues and can enhance 34293 Montpellier Cedex 05, France the adaptation of aphids to specific environmental conditions. Grigorescu A. S. et al. For example, facultative symbionts have shown their ability to complementing metagenomic studies [29, 33]andthefact improve host heat tolerance [9, 10], host resistance to natural that no recent culture-dependent studies were done on enemies [11–14], host plant specialization [15, 16], and host aphids, we chose this old-fashioned approach in order to survival [17] or to interfere with predation by inducing aphid assess the diversity of microorganisms that can be found body color modification [18]. Yet, culture-dependent studies associated with different aphid species and to try to define performed about 20 years ago showed that the aphid microbi- the core bacterial taxa that can be found in aphids beyond ota is not only limited to the obligate and facultative symbi- their obligate and facultative symbiotic partners. In our onts but also included microorganisms isolated from the gut study, we considered both aphid species that were labora- lumen [19–21]. These studies, however, remained obscured tory reared as well as specimens found in wild popula- by the importance given to the known endosymbionts and by tions. The culturing method previously developed by the fact that even after those initial findings on the gut micro- Sabri et al. [34] was chosen because it is the only one biota of aphids, these insects were still thought to have an that proved successful for isolating a free-living strain of almost-sterile digestive tube [19]. Thus, no major efforts were S. symbiotica, a facultative symbiont which was otherwise put afterwards to unravel the rare members of the aphid mi- considered non-culturable, as well as other bacterial iso- crobiota, leaving this category of microorganisms associated lates from the aphid honeydew [30, 35]. In the present with aphids largely unknown. study, our culture-dependent approach succeeded to iso- Culture-dependent techniques were the first approach that late more than 20 bacterial genera and three fungal gen- scientists used for studying microbial diversity but lost power era. The results obtained here illustrate the power that with the rise of metagenomic strategies. These new powerful culture-dependent methods can have to reveal the hidden approaches provided additional exploration venues for aphid part of the microbiota of an insect. microbiota, given that most of the known associated endo- symbionts are non-culturable due to their adaptation to a host-dependent lifestyle [22, 23]. Thus, some culture- Materials and Methods independent studies concluded that the known obligate and facultative symbionts represent the major flora associated with Aphid Strains aphids [24–27]. Nevertheless, a few additional bacterial strains, similar to Pseudomonas sp., Acinetobacter sp., Fifteen aphid species and strains (seven laboratory reared and Pelomonas sp., and Burkholderia sp. [28]; Staphylococcus eight from wild populations) were used in this study as listed sp. [26]; and Erwinia sp. and Pantoea sp. [27], were also in Table S1. The laboratory-reared aphid clones were main- detected, suggesting that the aphid microbiota is more diverse tained in continuous parthenogenetic culture on Vicia faba or than previously thought. At the same time, several microbial Cucumis sativus plants at 20 ± 2 °C and long-day conditions culture-dependent approaches have succeeded to isolate some (16 h of light and 8 h of darkness). From time to time, several aphid microbial partners, suggesting that these approaches are adult individuals of different ages were removed from the host able to detect microorganisms that may be present in such low plants for dissections, as described below. Most of the aphids concentrations that are not detected by metagenomic ap- from wild populations used here came from a sampling cam- proaches [29]. If metagenomic sequencing provides some in- paign carried on in May 2013 in Tunisia. The aphid species formation independent of our ability to culture bacteria asso- were identified according to the morphological criteria and the ciated to insects, their cultivation in the laboratory is required host plant [36]. Aphis fabae came from a previous collection for better valuing their physiology and the role they play in campaign carried on in Belgium in May 2011. After collec- host insects. For example, Leroy et al. [30] showed that tion, the aphids were placed in small humid plastic containers Staphylococcus sciuri, a bacterium isolated from with few leaves from the host plants to keep them alive. For Acyrthosiphon pisum honeydew, is responsible for the attrac- each collected aphid sample, adult individuals were dissected tion of the aphid natural enemies, and Fischer et al. [31]dem- within 10 days after their collection. onstrated the existence of Staphylococcus sciuri strains which release semiochemical factors promoting the attraction of ants Isolation of Microorganisms from Wild and Laboratory in myrmecophilous relationships. The isolation and culturing Aphids of different members of the aphid microbiota also represents a great advantage for performing high-throughput analyses, In order to isolate microorganisms from the body cavity of such as the genome sequencing of Serratia symbiotica strain aphids, individuals (i.e., between 10 and 90 as listed in CWBI-2.3T, a facultative symbiont that was isolated from the Table S1) of each aphid strain were first surface sterilized by black bean aphid Aphis fabae [32]. sequentially dipping them into 96% denatured ethanol for 3– Given the many advantages that a culture-dependent 4 min and into 4% bleach for about 1 min and then washed approach has in terms of future possible studies and with sterile water. Following this disinfection step, two Accessing the Hidden Microbial Diversity of