Low prevalence of secondary endosymbionts in aphids sampled from rapeseed crops in Germany

Abstract

Peach-potato aphids, Myzus persicae Sulzer (Hemiptera:Aphididae), and cabbage aphids, Brevicoryne brassicae Linnaeus (Hemiptera:Aphididae), are herbivorous insects of significant agricultural importance. Aphids can harbour a range of non-essential (facultative) endosymbiotic bacteria that confer multiple costs and benefits to the host aphid. A key endosymbiont-derived phenotype is protection against parasitoid wasps, and this protective phenotype has been associated with several defensive enodsymbionts. In recent years greater emphasis has been placed on developing alternative pest management strategies, including the increased use of natural enemies such as parasitoids wasps. For the success of aphid control strategies to be estimated the presence of defensive endosymbionts that can potentially disrupt the success of biocontrol agents needs to be determined in natural aphid populations. Here, we sampled aphids and mummies (parasitised aphids) from an important rapeseed production region in Germany and used multiplex PCR assays to characterise the endosymbiont communities. We found that aphids rarely harboured facultative endosymbionts, with 3.6% of M. persicae and 0% of B. brassicae populations forming facultative endosymbiont associations. This is comparable with endosymbiont prevalence described for M. persicae populations surveyed in Australia, Europe, Chile, and USA where endosymbiont infection frequencies range form 0–2%, but is in contrast with observations from China where M. persicae populations have more abundant and diverse endosymbiotic communities (endosymbionts present in over 50% of aphid populations).

Introduction

Aphids, such as the peach-potato aphid, Myzus persicae Sulzer (Hemiptera:Aphididae), and the cabbage aphid, Brevicoryne brassicae Linnaeus (Hemiptera:Aphididae), are important insect herbivores (Edde, 2021). Aphids are pests on many crops, including rapeseed (Zheng et al., 2020), where crop damage is caused through direct feeding (Dedryver et al., 2010) and the transmission of Turnip Yellows Virus, TuYV, (Asare-Bediako et al., 2020). Aphids reduce rapeseed yields directly through feeding activities and indirectly via the transmission of plant viruses: Depending on the rapeseed variety and crop growth stage at the time of inoculation, TuYV infection can reduce yields by 26–40% (Congdon et al., 2020).

Currently, aphid populations are primarily controlled using insecticides. However, due to more stringent regulations (EU 2009), greater environmental concerns around insecticide use (Goulson, 2013), and the emergence of aphid populations with insecticide resistance or reduced insecticide sensitivity (Bass et al., 2015; Bass and Nauen, 2023), there is a growing need for more sustainable pest management practices (Ali et al., 2023). One avenue that is being explored is the promotion of natural enemy populations, such as parsitoid wasps, that can provide natural pest regulation services (Ali et al., 2023; Elliott et al., 2023). The main parasitoids of M. persicae and B. brassicae include Aphidius ervi Haliday (Hymenoptera:Braconidae), A. colemani Viereck (Hymenoptera:Braconidae), and Diaeretiella rapae McIntosh (Hymenoptera:Braconidae) (Mehrparvar et al., 2019; Ward et al., 2022). By promoting the abundance and activities of these parasitoids farmers could increase the provision of natural pest regulation services and reduce reliance on chemical-based pest control methods. However, aphid-encoded factors can influence the success of these parasitoids.

The majority of aphid species form an essential (obligate) relationship with the endosymbiont Buchnera aphidicola. B. aphidicola supplements the aphid diet by providing access to essential amino acids (Douglas and Prosser, 1992). Aphids can also form a diverse range of non-essential (facultative) endosymbiotic relationships. These facultative endosymbionts are key drivers of phenotypic diversity in aphids (Zytynska et al., 2021) and around nine facultative endosymbionts have been described (Zytynska and Weisser, 2016). The most common facultative endosymbionts that have been described to associate with aphids are Spiroplasma spp., Regiella insecticola, Hamiltonella defensa, Rickettsiella sp., Fukatsuia symbiotica (previously pea aphid x-type symbiont, PAXS; Patel et al., 2019), Serratia symbiotica, Rickettsia spp., Arsenophonus spp., and Wolbachia spp. (Zytynska and Weisser, 2016). Associations with endosymbionts can occur in single infections (i.e., one facultative endosymbiont), co-infections (two endosymbionts), or multiple co-occurring infections (Zytynska et al., 2023).

Facultative endosymbionts influence the phenotype of many aphid species (Zytynska and Weisser, 2016; Zytynska et al., 2021) and a key endosymbiont-derived phenotype is protection against parasitoid wasps (Oliver and Higashi, 2019; Zytynska et al., 2021). In most aphid species protection against parasitism is conferred by the defensive endosymbiont, H. defensa (Leybourne et al., 2020; Zytynska et al., 2021). Other endosymbionts can also provide protective services, for example in M. persicae protection against parasitism is associated with R. insecticola (von Burg et al., 2008; Vorburger et al., 2009). It has also been suggested that facultative endosymbionts, including these defensive endosymbionts, can influence aphid susceptibility to insecticides (Li et al., 2021). However, these observations were made by manipulating aphid endosymbiont communities in controlled environments (Li et al., 2021) and were not observed for aphid populations with naturally occurring endosymbiont associations (Leybourne et al., 2023). For the potential success of aphid control strategies to be better estimated the natural prevalence of potentially defensive endosymbionts in aphid populations needs to be determined, particularly for endosymbionts that can potentially disrupt the success of biocontrol agents such as parasitoid wasps.

The endosymbionts of M. persicae have been surveyed for several geographic regions, including Australia (Yang et al., 2023a; Yang et al., 2023b), China (Xu et al., 2021; Yang et al., 2023b), Colombia (Gallo-Franco et al., 2019), and The Netherlands (Beekman et al., 2022). Endosymbionts in these M. persicae populations include R. insecticola (potentially defensive), Wolbachia spp., Rickettsia spp., Arsenophonus spp., S. symbiotica, H. defensa (potentially defensive), and Spiroplasma spp. (von Burg et al., 2008; Vorburger et al., 2009; Henry et al., 2015; Xu et al., 2021; Yang et al., 2023b). The prevalence of endosymbionts in M. persicae is highly variable, with some populations forming no endosymbiotic associations (Yang et al., 2023a) and others showing high levels (>40%) of endosymbiont infection (Xu et al., 2021). For B. brassicae, several studies have screened for endosymbionts but no associations have been detected for this aphid (Clark et al., 2012; Yang et al., 2023a).

Here, we sampled M. persicae and B. brassicae populations, alongside mummified aphids, from 14 winter rapeseed fields. We sampled aphids along two transects, one next to the field edge and the second within the crop, and used a multiplex diagnostic PCR method to characterise the facultative endosymbiont community for each aphid population. Aphids were sampled from a key rapeseed production region in Lower Saxony, Germany (fig. 1A). Sampling occurred between 26–28th October 2021, a time-period that reflects peak autumn aphid activity and virus risk (Ellis et al., 2014). The aim of our research was to gain insights into the prevalence of potentially defensive endosymbionts in naturally-occurring aphid populations for these two agriculturally important aphid species.

Figure 1. (A) Study region (shaded grey box) in Lower Saxony, North Germany and location of the 14 study sites (white circles). (B) Graphical representation of the trial design followed at each field site: Each field site was adjacent to another agricultural field with the first transect 5 m away from the field edge and the second transect 20 m into the crop; each transect contained five 2 m² quadrats. (C) Overview of the DNA extraction process. Maps were created in ggmap (v.3.0.2) with the base map obtained from Google Map Services. This graphic was prepared in bio-render.

Methods

Sampling procedure

We sampled 14 winter rapeseed fields in an agriculturaly important region of Lower Saxony, Germany (fig. 1A). In each field, sampling occurred along two 100 m transects (fig. 1B). One was 5 m from the field edge and the second 20 m into the crop. We sampled at two distances into the field as recent results in cereal aphids indicate that this can influence the prevalence and diversity of aphid endosymbionts (Zytynska et al., 2023); as agricultural systems are not homogenous we did not want to exclusively sample from the centre of the crop as this would not provide a realistic representation of the aphid and endosymbiont communities. Transects were divided into five equidistant sampling regions and a 2 m² quadrat was surveyed at each sampling region. One aphid colony (hereafter population) was sampled per quadrat. The number of individual aphids per population ranged from one to 20 and all aphids were pooled into one sample. Where present, an aphid mummy was also collected from each quadrat. Mummies were identified morphologically. Aphid and mummy samples were stored at −20 °C until DNA extraction.

DNA extraction and PCR

DNA was extracted using the Chelex® method: A 5% Chelex® 100-resin (Bio-Rad, Germany) solution was made using UltraPure water (Invitrogen, Germany) and heated to c. 60 °C. 10 μl Chelex® solution and 1.5 μl Proteinase K (20 mg L⁻¹, Macherey-Nagel GmbH & Co. KG, Germany) were added to each sample. Aphids were homogenised using sterile pipette tips and mummies with a sterile micropestle. After homogenisation, 80 μl Chelex® solution was added, the sample was vortexed and incubated for 1.5 h at 56 °C. After incubation, samples were centrifuged at max speed for 5 minutes and the solution was transferred to a clean Eppendorf tube, diluted 1:2 with TE buffer (10 mM Tris-HCl pH 7.5; 1 mM EDTA pH 8.0), and stored at −20 °C. An extraction blank was included with each batch of DNA extractions.

Successful DNA extraction was confirmed using a PCR marker for the obligate endosymbiont B. aphidicola. The presence of facultative endosymbionts was determined using multiplex assays (Beekman et al., 2022). Assays were conducted in a Biometra TRIO 48 Thermocycler (Analytik Jena, Germany). Primer and thermocycling details are described in Table S1. Successful amplification was detected by product separation on a 1% agarose gel stained with GelRed® (Biotium, Germany).

Results and discussion

From the 140 sampling regions (14 fields × 10 quadrats) we sampled 114 aphid populations and 20 mummies, with DNA successfully extracted from 119 of these samples (indicated by detection of B. aphidicola; Table 1). Aphid samples primarily comprised M. persicae (Table 1). Our results indicate that M. persicae and B. brassicae rarely harboured facultative endosymbionts. From the 116 M. persicae populations (109 successful aphid extractions + seven successful mummy extractions; Table 1) we detected facultative endosymbionts in four populations – a 3.6% infection frequency. Endosymbionts included one single Ricketsiella spp. infection, two single H. defensa infections, and one co-infection (S. symbiotica and Rickettsia spp.). Previous studies characterising endosymbionts in M. persicae populations found similarly low levels of endosymbiont prevalence. In the Netherlands no facultative endosymbionts were detected in 780 M. persicae samples collected from peppers (Beekman et al., 2022), a global study found <2% facultative endosymbiont infection frequencies (screening for H. defensa, R. insecticola, and S. symbiotica) in 50 M. persicae populations sampled from a range of crop plants (Henry et al., 2015), a survey of M. persicae from pepper crops in Colombia found no facultative endosymbiont associations (Gallo-Franco et al., 2019), and a recent global survey found no endosymbiont associations in 52 M. persicae populations sampled across Australia, Europe, USA, and Chile (Yang et al., 2023b). Alongside our current observations, these findings indicate that M. persicae rarely form facultative endosymbiotic associations (Henry et al., 2015; Gallo-Franco et al., 2019; Beekman et al., 2022).

Table 1. Details on the total number of samples (aphid or mummy) collected for each field alongside endosymbiont prevalence

Symbiont abbreviations: B.a (B. aphidicola; essential primary endosymbiont – used to confirm DNA extraction), Spi (Spiroplasma spp.), R.i. (Regiella insecticola), H.d. (Hamiltonella defensa), R-siella (Rickettsiella sp.), F.s. (Fukatsuia symbiotica), S.s. (Serratia symbiotica), R-tsia (Rickettsia spp.), and Ars. (Arsenophonus spp).

Conversely, facultative endosymbionts have been detected at much higher frequencies in M. persicae sampled from China (Xu et al., 2021; Yang et al., 2023b). A greater frequency and diversity of endosymbionts were reported by Xu et al. (2021) where the main endosymbionts present in aphid populations were Wolbachia spp. (present in 57% of samples), Rickettsia spp., Arsenophonus spp., and S. symbiotica (16% of samples), R. insecticola (13% of samples), H. defensa (4% of samples), and Spiroplasma spp. (3% of samples). In Yang et al. (2023b) only two endosymbionts were detected, namely Rickettsia spp (present in 19% of samples). and Spiroplasma spp. (4% of samples), but these were at a higher prevalence than observed in aphids surveyed from other regions. The endosymbiont frequencies described in Xu et al. (2021), Yang et al. (2023b) are greater than those detected in M. persicae sampled from other geographical areas, including Europe, Australia, USA, and Chile (Henry et al., 2015; Gallo-Franco et al., 2019; Beekman et al., 2022). This indicates that the endosymbiont communities in M. persicae might differ depending on bio-geographic location, as previously suggested for cereal aphids (Guo et al., 2019).

There are several potential explanations for this consistent observation of greater diversity and prevalence of endosymbionts in M. persicae sampled from China when compared with aphid populations from other regions (this study; Henry et al., 2015; Xu et al., 2021; Beekman et al., 2022; Yang et al., 2023b). Variation in endosymbiont communities between native vs invasive populations is one explanation that has recently been suggested (Yang et al., 2023b). Yang et al., 2023b observed that M. persicae sampled in their putative native habitat (China) had a greater diversity, with regards to both genetic and microbial diversity, than invasive M. persicae populations sampled from Europe, Australia, or America. This is in-line with observations in similar aphid species where native populations can have a greater microbial diversity when compared with invasive (exotic) populations (Desneux et al., 2018). Although this phenomena might be species-specific as little difference was detected between natural and invasive soybean aphid, Aphis glycines Matsumura, populations (Bansal et al., 2014).

Host plant diversity is another potential explanation. Recent controlled environment studies have shown that host plant diversity can influence and modulate the microbiome of M. persicae (He et al., 2021); host plant diversity could also influence the microbiome of M. persicae in natural aphid populations. Most surveys have focussed on sampling M. persicae from agricultural systems, where host plant diversity is relatively low (this study; Henry et al., 2015; Beekman et al., 2022; Yang et al., 2023b). However, Xu et al., 2021 collected M. persicae from a diverse range of host plants, not solely from agricultural fields. This could potentially explain the high diversity reported for M. persicae in Xu et al., 2021, particularly when compared with a slightly lower endosymbiont prevalence in M. persicae sampled from agricultural fields in China (Yang et al., 2023b). However, this requires further testing.

We detected no endosymbionts in the three B. brassicae populations, and a similar survey of aphid populations from Northern Britain also found no facultative endosymbiont occurrence in B. brassicae (Clark et al., 2012). Several studies have employed 16S rDNA sequencing methods to identify uncharacterised, or cryptic, species beyond the main facultative endosymbionts. Where additional bacteria have been detected, these were primarily environmental microbes, e.g., Klebsiella spp., Escherichia spp., Pseudomonas spp. (Clark et al., 2012; Beekman et al., 2022), indicating that endosymbiotic prevalence in European M. persicae and B. brassicae populations is low.

Endosymbionts can provide strong protective traits, including resistance against parasitoid wasps (von Burg et al., 2008; Vorburger et al., 2009). It has been assumed that the presence of these endosymbionts will have detrimental effects on sustainable pest management practices, with the presence of endosymbiont-infected aphids disrupting biocontrol success. Field-based research using H. defensa-infected and uninfected A. fabae in bean fields has provided experimental evidence showing that endosymbiont infection reduces parasitism rate under field conditions (Rothacher et al., 2016). However, as indicated by our study and others (Henry et al., 2015; Xu et al., 2021; Beekman et al., 2022), the prevalence of these potentially protective endosymbionts in natural aphid populations is low, indicating that the impact of endosymbionts on biocontrol success under natural conditions could be minimal (Beekman et al., 2022). The role of other traits that might influence biocontrol success, such as aphid genotype, also needs to be examined in more detail.

Conversely, recent research has indicated that endosymbionts could also be used as a novel method of aphid biocontrol (Gu et al., 2023; Soleimannejad et al., 2023). Artificial transfection of aphids with a Ricketsiella symbiont (R. viridis) was shown to contribute towards the suppression of M. persicae populations (Gu et al., 2023), with R. viridis infection reducing aphid fecundity. R. viridis is thought to also be able to follow a horizontal transfer mechanism via plant-mediated transfer and can be spread throughout an aphid colony (Gu et al., 2023). The interaction between this aphid-suppressing endosymbiont and the parasitoid D. rapae was recently assessed, with results suggesting that D. rapae preferred to probe aphids infected with R. viridis (Soleimannejad et al., 2023). The authors proposed that this could potentially contribute an additional horizontal transfer mechanism for R. viridis (Soleimannejad et al., 2023); although the successful rate of horizontal transfer of facultative endosymbionts by parasitoids is minimal (Gehrer and Vorburger, 2012). These mechanisms of R. viridis-mediated aphid suppression and D. rapae-mediated transfer of R. viridis could be combined into a novel biocontrol method (Soleimannejad et al., 2023). However, several points need to considered: (1) Currently these observations are made for M. persicae that have been artificially transfected with R. viridis derived from the pea aphid, Acyrthosiphon pisum Harris (Hemiptera:Aphididae), and the potential role of M. persicae-derived Rickettsiella spp. needs to be considered; (2) When transferred horizontally R. viridis is only stable for c. two generations (Soleimannejad et al., 2023), therefore the potential impact this short retention might have on aphid suppression under natural conditions needs to be considered; (3) The interactions between R. viridis and naturally occurring endosymbionts also needs to be examined in more detail, particularly the interaction with endosymbionts that confer resistance against parasitism and the potential impact of this on D. rapae-mediated horizontal transfer.

Conclusion

Our results add further evidence that facultative endosymbiont associations are rarely formed in M. persicae and B. brassicae aphids across Europe (Clark et al., 2012; Henry et al., 2015; Beekman et al., 2022). Contrasting variation between endosymbiont frequencies in M. persicae from Europe (this study; Henry et al., 2015; Beekman et al., 2022; Yang et al., 2023b) and China (Xu et al., 2021; Yang et al., 2023b) suggest that endosymbiont frequencies could be influenced by other environmental or bio-geographical factors; geographical variation has been detected in the endosymbionts of cereal aphids (Guo et al., 2019). Recent research has suggested that native aphid populations are potentially more diverse than invasive populations (Yang et al., 2023b) and highlighted the role the host plant plays in influencing endosymbiont composition (He et al., 2021). Exploring these in greater detail would be interesting avenues for future research.