Introduction
Insects are commonly associated with symbiotic microbes which could bring some advantages to themselves. The pea aphid Acyrthosiphon pisum , a model insect for endosymbiont studies, usually hosts one obligate symbiont and several facultative symbionts (Douglas & Prosser, 1992; La Pena, Vandomme, & Frago, 2014). Since phloem sap is a suboptimal diet for aphids due to imbalanced amino acids: carbohydrates proportions, the obligate symbiont, Buchnera aphidicola that resides in aphid bacteriocytes offers essential amino acids to aphids (Douglas, 1998). By contrast, facultative symbionts are usually not required for survival and reproduction of aphids but are able to improve their fitness particularly when they are confronted by various biotic and abiotic challenges. Studies indicate that some facultative symbionts increase heat tolerance (Doremus & Olive, 2017), defense against the natural enemies and fungi (Łukasik, Van Asch, Guo, Ferrari, & Godfray, 2013; Oliver, Russell, Moran, & Hunter, 2003), and host plant adaptation of aphids (Leonardo & Muiru, 2003). Previous research shows that an elicitor protein GroEL from B. aphidicolatriggers plant resistance against aphids (Chaudhary, Atamian, Shen, Briggs, & Kaloshian, 2014). Hence, endosymbionts may modify the components of salivary proteins that modulate aphid feeding on plants. Very little is known, however, how facultative endosymbionts affect aphid feeding by altering aphid salivary proteins.
Aphids use their stylets to obtain nutrients from phloem sieve elements of the host plants (Tjallingii & Esch, 1993). While probing, aphids secrete saliva to inhibit plant defense response caused by wounding due to aphid feeding as well as clogging of the fluid in the phloem tissue usually induced by Ca2+ influx (Aidemark, Andersson, Rasmusson, & Widell, 2009). Plants typically initiate rapid cytosolic Ca2+ sparks upon aphid probing (Vincent et al., 2017), resulting in the activation of local defenses including reactive oxygen species (ROS) and phytohormone signaling (Tian et al., 2019). ROS-induced cell death prevents the aphid stylet from reaching the phloem (Laitinen et al., 2017) and triggers the salicylic acid (SA) signaling pathway to confer the local plant resistance to aphids (Hogenhout & Bos, 2011; Jaouannet et al., 2014). In addition, Ca2+ is also a long-distance signal that can be transmitted from injured cells to uninfected tissues, activating the wound-related systemic jasmonic acid (JA) signaling pathway (Farmer, Gasperini, & Acosta, 2014). Both synergistic and antagonistic JA- and SA-regulated defense have been reported in plant responses to aphids (Moran and Thompson,2001; Moran et al., 2002; De Vos et al., 2005; Mewis et al., 2006; Pegadaraju, 2005). Conversely, Ca2+-binding proteins secreted by aphids into plant tissues have been shown to facilitate aphids feeding. For example, Armet, a salivary protein from the pea aphid sequesters Ca2+ to counteract the Ca2+-triggered occlusion in Vicia fabae , which prolongs aphid phloem feeding time (Wang et al., 2015). Effectors containing Ca2+-binding domains presumably could efficiently quench the cytosolic Ca2+ elevation of host plants, which consequently improves aphid feeding.
The facultative symbiont Serratia symbiotica increases heat tolerance of A. pisum , and enhances resistance to parasitoids and predators (Costopoulos, Kovacs, Kamins, & Gerardo, 2014; Hopper et al., 2018; Montllor, Maxmen, & Purcell, 2002), but little in known of whether and how S. symbiotica benefits aphid feeding. Previous studies showed that pea aphid clones collected from 11 legume plants differed in their facultative symbionts and infectious rate (Frantz, Calcagno, Mieuzet, Plantegenest, & Simon, 2009; Henry et al., 2013; Simon et al., 2003), suggesting that host plant species impact the facultative symbionts associated with aphids. During July-August of 2015-2019, we intended to determine the prevalence of endosymbionts of pea aphids in Medicago sativa fields in Yinchuan city, Ningxia province, China, and identified 5 endosymbionts within 154 sampled pea aphids (Fig. S1). S. symbiotica had a relatively high infectious rate (85%) in all sampled aphids, we hypothesized that infection ofS. symbiotica may benefit the aphid growth on Medicagoplants by facilitating aphid feeding. To test the hypothesis, we monitored aphid feeding behavior, performed aphid salivary gland transcriptomic analysis in the presence and absence of S. symbiotica . Histidine-rich calcium-binding protein-like (GeneID: 103308203) (A. pisum HRC , ApHRC ) that encodes a salivary protein was up-regulated by S. symbiotica . Further investigation indicated that ApHRC suppressed Ca2+ elevation, which otherwise would have induced the plant defense response. Our study has demonstrated that S. symbiotica infection can modify aphid salivary composition by modulating salivary gland gene expression, which suppressed plant defense and benefited aphid feeding.