Facultative symbionts associated with aphid populations in citrus orchards in northern Tunisia

Like many insects, aphids (Hemiptera: Aphididae) can host a wide diversity of symbiotic bacteria that can be important drivers of their evolutionary ecology. In addition to the nutritional obligate symbiont Buchnera aphidicola, these phloem-sap feeding insects can host various facultative symbionts whose functional diversity depends on complex interactions with the host genotype and environmental factors. During sampling in citrus orchards in northern Tunisia, we collected aphids on citrus plants. The specimens belonged either to the cotton-melon aphid Aphis gossypii or the green citrus aphid Aphis spiraecola. We analysed the prevalence of Arsenophonus, Serratia symbiotica, Hamiltonella defensa and Regiella insecticola, four facultative symbionts frequently found in the genus Aphis and potentially associated with phenotypic effects related to nutrition, protection against parasites and tolerance of high temperatures. We found that the diversity and prevalence of facultative symbionts differed between these two aphid species that exploit similar ecological niches. In particular, we found a high prevalence of Arsenophonus in A. gossypii populations and that the defensive symbiont H. defensa was only present in A. spiraecola populations. These results are discussed in light of the ecology and life cycles of each of the species of aphid studied.


INTRODUCTION
Like most insects that feed on plant-phloem, aphids (Hemiptera: Aphididae) host symbiotic bacteria in their tissues that can have signifi cant consequences on their evolutionary ecology (Oliver et al., 2010;Ferrari & Vavre, 2011). The unbalanced diet of these sap-feeding insects renders them dependent on an obligate symbiont, B. aphidicola, which provides them with essential amino acids and vitamins (Douglas, 1998;Russell et al., 2017). Aphids can also carry an array of heritable facultative symbionts (e.g. H. defensa, S. symbiotica, R. insecticola, etc.) that occur more sporadically in host insect populations and are maintained in these by vertical transmission and intra-and interspecifi c horizontal transfers (Sandström et al., 2001;Caspi-Fluger et al., 2011;Gehrer & Vorburger, 2012). These facultative bacterial partners can impart dramatic phenotypic effects to their host, sometimes benefi cial and sometimes harmful depending on specifi c environmental conditions (Oliver et al., 2006(Oliver et al., , 2010. The benefi cial phenotypic effects include protection against parasitoid wasps and other parasites μM of each primer, 200 μM dNTP's, 1 × buffer and 0.625 unit of Taq DNA polymerase (Roche). The PCR reaction conditions consisted of 40 cycles of 95°C for 1 min, 55°C for 1 min 30 s and 72°C for 1 min 30 s. DNA samples found positive for the different symbiont species in the context of a previous deep 16S rRNA sequencing analysis were used as positive controls (Fakhour et al., 2018). Negative controls consisted of sterilized water instead of genomic DNA. The PCR products were stained with ethidium bromide and visualized on a 1% agarose gel. Half of the amplifi ed samples were sequenced and the resulting sequences were validated using BLAST on GenBank.

Statistical analyses
To evaluate potential factors contributing to the facultative symbiont infections, generalized linear models (GLMs) were examined on the basis of the presence/absence of the respective facultative symbionts in the two species of aphid (binomial error structure, logit-link functions). For each facultative symbiont, the model considered the species of aphid and site sampled as fi xed factors. We also carried out Fisher's exact tests to compare the global distribution of facultative symbionts between aphid species. To test the levels of co-occurrence of facultative symbionts, we performed a multiple regression analysis of the presence/absence of each symbiont against that of the other symbionts using generalized linear mixed models (binomial-error and logit-link functions). Finally, the graphical representation of facultative symbiont communities was done using the Mondrian function implemented in R (Gueguen et al., 2010). All statistical analyses were performed using the software R version 3.6.1 (R Development Core team, 2014).

RESULTS
Of the 68 citrus aphid colonies sampled at 9 locations in northern Tunisia, we identifi ed 19 colonies of the cotton-melon aphid, A. gossypii, and 49 colonies of the green citrus aphid, A. spiraecola. For A. gossypii,89% (17/19) of the colonies were infected, whereas only 45% (22/49) of the A. spiraecola colonies were positive for facultative symbionts (Fig. 1). In general, these two aphids differed in terms of the species composition of their symbionts (Fisher's exact test, p < 0.0001). More precisely, for Arsenophonus, its prevalence was signifi cantly higher in A. gossypii than in A. spiraecola (χ 2 = 18.38, df = 1, p < 0.001, Fig. 2A). Arsenophonus was clearly the facultative symbiont that was most frequently recorded in the citrus aphids studied with a prevalence reaching 73% (14/19) in A. gossypii, either as a single infection (47%) or in combination with other symbionts (26%) (Fig. 1). In A. spiraecola colonies, the prevalence of this symbiont was 18% (9/49). In terms of infection with the symbionts S. symbiotica and in the Mediterranean basin (Halima-Kamel & Hamouda, 2004;Jacas et al., 2010). The four targeted symbionts are frequently found in aphids of the genus Aphis (Najar-Rodríguez et al., 2009;Brady & White 2013;Brady et al., 2014;Wulff & White, 2015) and are known to be involved in a variety of effects including resistance to parasitoids, tolerance of high temperatures and host nutrition (Oliver et al., 2003(Oliver et al., , 2006Tsuchida et al., 2004;Burke et al., 2010;Duron, 2014;Wulff & White, 2015). It is hypothesized that aphids exploiting similar ecological niches, although belonging to different species or to geographically distant populations, tend to exhibit similar combinations of symbionts (McLean et al., 2011;Henry et al., 2013Henry et al., , 2015Brady et al., 2014). In citrus orchards where the sampling was carried out, A. gossypii and A. spiraecola populations exploit similar niches (e.g. they feed on the same host-plant species, are attacked by the same natural enemies and are subject to similar climactic conditions) (Boukhris-Bouhachem, 2011; Limem Sellami et al., 2013;Elhaddad et al., 2016;Boukhris-Bouhachem et al., 2017). Thus, determining the presence of these facultative symbionts in A. gossypii and A. spiraecola also offer the possibility of testing the hypothesis that populations of these two aphid species are likely to host similar combinations of symbionts.

Aphid collection
During sampling in the North of Tunisia [on the Cap Bon peninsula and around the capital Tunis where citrus crops are concentrated (Metoui et al., 2014)], we collected specimens of A. gossypii and A. spiraecola (Table S1). The samples consisted of three wingless parthenogenetic adult females from the same colony (i.e. from the same leaf). A total of 68 colonies at 9 locations were sampled. Samples collected at the same location were at least 500 m apart and came from different orchards. Aphids were stored in 95% ethanol at 4°C until used and then identifi ed based on morphological criteria (Blackman & Eastop, 2000).

Species-specifi c screening for facultative symbionts
DNA of the collected aphids was extracted using the DNeasy Blood & Tissue Kit (QIAGEN) following the instructions of the manufacturer. Each DNA extraction was performed on a pool of three individuals from the same colony to reduce the risk of missing infection when facultative symbionts are present. We screened each specimen for the facultative symbiont species Arsenophonus, S. symbiotica, R. insecticola and H. defensa. We amplifi ed a partial region of the 16S rRNA gene using specifi c primers (Table 1). The PCR assays were performed in a fi nal volume of 15 μl containing 1 μl of the template DNA lysate, 0.5 We also determined the prevalence of multiple infections, that is, the presence of more than one facultative symbiont species in the same sample. Twenty-one percent (14/68) of the samples were infected by at least two facultative symbionts. For A. gossypii, all possible combinations of Arsenophonus, S. symbiotica and R. insecticola were recorded other than a dual infection with S. symbiotica and R. insecticola (Fig. 1A). For A. spiraecola, we recorded single infections with each symbiont, but samples with two or three symbionts were uncommon and followed no discernible pattern (Fig. 1B). If we only consider those samples infected with at least one facultative symbiont, the prevalence of multiple infections is 29% (5/17) in A. gossypii and 41% (9/22) in A. spiraecola with no signifi cant difference between the two aphids (Fisher's exact test, p = 0.52). However, none of the combinations were recorded signifi cantly more or less frequently than expected by chance. Even the combination Arsenophonus-S. symbiotica of 16% (3/19) for the A. gossypii samples did not deviate signifi cantly from a random association (χ 2 = 0.14512, df = 1, p = 0.70).

DISCUSSION
In this study, we collected citrus aphids from orchards in northern Tunisia. We identifi ed the individuals collected and found that they all belonged to either the species A.  gossypii or A. spiraecola. We determined whether these two species were infected with facultative symbionts. Four symbionts, namely Arsenophonus, S. symbiotica, R. insecticola and H. defensa, were recorded either in A. gossypii or A. spiraecola. These symbionts are widely recorded occurring in the genus Aphis (Carletto et al., 2008;Jones et al., 2011;Brady & White, 2013;Jousselin et al., 2013;Arneodo & Ortego, 2014;Brady et al., 2014;Wulff & White, 2015;Zhao et al., 2016;Zytynska & Weisser, 2016;Desneux et al., 2018) and are known to have associated effects that may contribute to the ecological success of their host. The majority of the A. spiraecola samples were not infected with facultative symbionts, although all the targeted symbionts were detected in this species. However, in A. gossypii, the presence of at least one facultative symbiont is almost systematic. H. defensa was recorded in A. spiraecola but not in A. gossypii even though previous studies have reported the presence of H. defensa in this aphid (Zhao et al., 2016;Ayoubi et al., 2020). We recorded similar percentage infections of A. gossypii and A. spiraecola with S. symbiotica and R. insecticola, but the percentage infection of these aphids with Arsenophonus differed. In A. gossypii populations, the prevalence of Arsenophonus was around 75%, which is similar to that recorded in other studies (Jones et al., 2011;Zhao et al., 2016;Ayoubi et al., 2018;Zhang et al., 2018).
Although the effects associated with the symbionts identifi ed in A. gossypii and A. spiraecola are unknown, inferences can be drawn based on their functions in other aphid species. H. defensa is well known for protecting the pea aphid A. pisum (Oliver et al., 2003(Oliver et al., , 2009 and black bean aphid Aphis fabae (Schmid et al., 2012;Rouchet & Vorburger, 2014) against parasitoids. In A. pisum, S. symbiotica has some ability to protect its host against parasitoids (Oliver et al., 2006;Pons et al., 2019) and high temperatures (Montllor et al., 2002;Burke et al., 2010). R. insecticola is reported to affect the performance of A. pisum on host plants (Tsuchida et al., 2004) and protect A. fabae against parasitoids (Vorburger et al., 2010). The effects associated with Arsenophonus are unclear. Bacteriophages required for protective symbiosis are recorded in various strains of this symbiont (Duron, 2014), but no defensive properties are reported for Aphis glycines infected with Arsenophonus (Wulff et al., 2013). Due to the high prevalence of Arsenophonus in populations of A. gossypii it is suggested that this facultative symbiont may be involved in host nutrition by mediating host plant range (Wagner et al., 2015;Zhao et al., 2016;Tian et al., 2019;Ayoubi et al., 2020).
Previous large-scale surveys indicate that aphids exploiting similar ecological niches, although belonging to different species or geographically distant populations, tend to have similar combinations of facultative symbionts (Ferrari et al., 2012;Henry et al., 2013Henry et al., , 2015Brady et al., 2014;Wagner et al., 2015). In our study, A. gossypii and A. spiraecola were collected from similar food resources (i.e. citrus plants) distributed over a small geographical area and therefore subject to similar climatic conditions. In ad-dition, the two species share many natural enemies (Boukhris-Bouhachem, 2011;Limem Sellami et al., 2013), are frequently tended by ants (Kaneko, 2018;Karami-Jamour et al., 2018) and transmit the same viruses, such as citrus tristeza virus (CTV) (Elhaddad et al., 2016;Boukhris-Bouhachem et al., 2017). In our study, the two aphids differ in terms of symbiont composition, while they occupy very similar ecological niches. The factors that may determine the presence of facultative symbionts in aphid populations remain poorly understood. A recent study indicates that the bacterial fl ora associated with A. gossypii is strongly affected by its host plants (Xu et al., 2019). However, other studies indicate that the composition of microbial communities hosted by aphids may largely depend on host genotype (Fakhour et al., 2018;McLean et al., 2019). In addition, it is possible that microclimatic conditions and other local variables (natural enemies, surrounding vegetation, etc.) have a major infl uence on the structure of symbiotic communities (Zytynska et al., 2019). A. gossypii and A. spiraecola are both highly polyphagous species and can readily switch from one host plant species to another. Therefore, it is also possible that their feeding behaviour and the food environment they encountered previously determines the composition of the symbiont community associated with them.
There was a high incidence of multiple infections, of up to 42%, in the A. spiraecola samples, which did not differ from that recorded for A. gossypii. Further, there was no evidence for preferential associations or the exclusion of particular symbionts. Multiple infections by facultative symbionts are mostly reported for the pea aphid A. pisum (Leonardo & Muiru, 2003;Oliver et al., 2006;Nyabuga et al., 2010;Ferrari et al., 2012;Henry et al., 2013;Russell et al., 2013). The reports for the genus Aphis are confl icting as A. fabae, A. craccivora and A. gossypii are rarely reported hosting more than one facultative symbiont (Chandler et al., 2008;Najar-Rodríguez et al., 2009;Brady et al., 2014;Henry et al., 2015;Zhao et al., 2016;Zhang et al., 2018) whereas one individual of the black bean aphid A. fabae can harbour up to four different symbionts (Zytynska et al., 2015). Our results are consistent with the latter study and indicate that multiple infections by different symbionts are probably less rare in the genus Aphis than previously thought. However, in the context of this study, artefacts due to the methodology cannot be excluded. Indeed, we assessed the presence of facultative symbionts using three individuals per colony and contamination from other colonies cannot be completely excluded. Host infection by several facultative symbionts is known to affect host fi tness in different ways dependent on the symbionts involved (Oliver et al., 2006;Łukasik et al., 2013;Tsuchida et al., 2014;Leclair et al., 2016). Specifi c multiple infection patterns could either result in an amplifi cation of the benefi cial effects associated with each symbiont (Oliver et al., 2006) or in severe costs for host aphids (Oliver et al., 2006). However, apparent preferential associations or exclusion of symbionts might be explained by drift in certain cases (Mathé-Hubert et al., 2019). Patterns of multiple infections in species of aphids may also depend on specifi c features related to their respective lifecycle, such as feeding behaviour (i.e. the degree of polyphagy), alternation between a primary and a secondary host plants (i.e. heteroecious cycle) or a sexual reproductive phase that promotes the exchange of facultative symbionts between males and females (Moran & Dunbar, 2006).
In conclusion, the prevalence of several facultative symbionts in populations of two species of aphids was quantifi ed. In the citrus orchards in northern Tunisia, A. gossypii and A. spiraecola exploit similar niches and are major pests. It is important to keep in mind that, despite the fact that the four targeted symbiont species are frequently found in aphids of the genus Aphis, other facultative symbionts can infect these aphids. For example, several studies report the presence of Wolbachia in A. gossypii (Jones et al., 2011;Zhao et al., 2016). In addition, like other fi eld studies, this study is a snapshot as the prevalence of symbionts in insect populations can change depending on environmental pressures. However, it is relevant to evaluate the presence of facultative bacteria in insect pests, as in addition to being benefi cial for their hosts they may make them more vulnerable to certain stresses. Due to their associated effects, bacterial symbionts can interfere with control programs, be it biological control or chemical control.

DECLARATION OF INTEREST. None.
AUTHOR CONTRIBUTIONS. FR conceived and designed the study; VF and KLG sampled the aphids; FR, IP and CN did the research; FR, VF and IP analysed the data; FR wrote the fi rst draft of the manuscript; IP, VF and TH revised the manuscript. All authors approved the manuscript for publication.
ACKNOWLEDGEMENTS. This work was supported by the Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture (FRIA grant no. 1.E074.14) and the FRS-FNRS in Belgium (FRFC 6886819) (http://www.fnrs.be). Field sampling was possible due to a grant from Wallonie-Bruxelles-International (WBI) in the context of bilateral cooperation between Tunisia and Belgium (Axe 2 -Lutte biologique contre les ravageurs en cultures d'agrumes 2012-2014). The funding agencies had no role in the design of the study, data collection and analysis, decision to publish or preparation of the manuscript. This paper is publication BRC 342 of the Biodiversity Research centre (UCL/ ELI/BDIV).