Environmental Entomology Advance Access published April 10, 2015 INSECT-SYMBIONT INTERACTIONS
Do Bacterial Symbionts Govern Aphid’s Dropping Behavior? OMER LAVY,1,2 NOA SHER,3 ASSAF MALIK,3 AND ELAD CHIEL1
Environ. Entomol. 1–5 (2015); DOI: 10.1093/ee/nvv044
ABSTRACT Defensive symbiosis is amongst nature’s most important interactions shaping the ecology and evolution of all partners involved. The pea aphid, Acyrthosiphon pisum Harris (Hemiptera: Aphididae), harbors one obligatory bacterial symbiont and up to seven different facultative symbionts, some of which are known to protect the aphid from pathogens, natural enemies, and other mortality factors. Pea aphids typically drop off the plant when a mammalian herbivore approaches it to avoid incidental predation. Here, we examined whether bacterial symbionts govern the pea aphid dropping behavior by comparing the bacterial fauna in dropping and nondropping aphids of two A. pisum populations, using two molecular techniques: high-throughput profiling of community structure using 16 S reads sequenced on the Illumina platform, and diagnostic polymerase chain reaction (PCR). We found that in addition to the obligatory symbiont, Buchnera aphidicola, the tested colonies of A. pisum harbored the facultative symbionts Serratia symbiotica, Regiella insecticola and Rickettsia, with no significant differences in infection proportions between dropping and nondropping aphids. While S. symbiotica was detected by both techniques, R. insecticola and Rickettsia could be detected only by diagnostic PCR. We therefore conclude that A. pisum’s dropping behavior is not affected by its bacterial symbionts and is possibly affected by other factors. KEY WORDS symbiotica
Acyrthosiphon pisum, defensive mutualism, Regiella insecticola, Rickettsia, Serattia
Defensive symbiosis, a mutualistic relationship in which one partner benefits from its symbiont protection in exchange for hosting services or other goods (Offenberg 2001), is a well-described phenomenon that stretches across all major taxa and habitats. Fundamental examples include endophytic fungi–grasses (Waller et al. 2005), ants–acacia trees (Janzen 1966), and sea anemone–clown fishes (Mariscal 1970). Defensive mutualism is considered a key player in the evolution of symbiotic partners, altering their behavior and traits to maximize potential benefits of both sides (Offenberg, 2001, Oliver et al. 2014). Symbiotic interactions between insects and vertically transmitted microorganisms are widespread. Some of these interactions are obligatory as the symbiont(s) produces nutrients that are absent in the host’s diet. Other interactions are facultative as hosts do not depend on the symbionts for their normal development, yet facultative symbionts may still have crucial roles in host fitness under certain ecological circumstances, as will be detailed hereinafter (Moran et al. 2008). Both obligatory and facultative symbionts are intracellular and are vertically transmitted with high fidelity. Because the fitness of symbiont and host are linked in vertically transmitted associations, symbionts which provide net benefits can invade insect populations and spread beneficial traits (Russell et al. 2013b). 1 Department of Biology and Environment, University of Haifa, Oranim, Tivon 36006, Israel. 2 Corresponding author, e-mail:
[email protected]. 3 Bioinformatics Service Unit, University of Haifa, Haifa, Israel.
One of the classic model organisms in the field of microbial–insect symbiosis is the pea aphid, Acyrthosiphon pisum Harris (Hemiptera: Aphididae). All aphid species, including the pea aphid, carry an obligate bacterial symbiont, Buchnera aphidicola, that completes the aphid’s dietary needs by producing essential amino acids lacking in the phloem sap (Russell et al., 2013a). Additionally, A. pisum populations may harbor one or more facultative bacterial symbionts – Hamiltonella defensa, X-type symbiont, Regiella insecticola, Rickettsia sp., Rickettsiella sp., Spiroplasma sp., and Serratia symbiotica (Russell et al. 2013b), some of which contribute to the aphids’ fitness in various ways, including pathogen, parasitoid, and predator tolerance (Costopoulos et al. 2014, Łukasik et al. 2013, Oliver et al. 2014, Parker et al. 2013). Facultative symbionts may also alter the behavior of their host. For instance, H. defensa increases surviving chances of pea aphids attacked by parasitic wasps (Oliver et al. 2003); consequently, H. defensa-infected aphids behave less aggressively toward parasitic wasps and show less escaping attempts than noninfected aphids (Dion et al. 2011). However, this behavior may also be costly, as complacent H. defensa-infected aphids suffer higher predation rates from ladybird beetles (Polin et al. 2014). In another study, aphids that carry the symbiont Spiroplasma and are infected with an entomopathogenic fungus drop off the host plant in higher numbers than noninfected aphids before the fungus sporulates, thereby decreasing the risk of infecting their sister aphids in the vicinity (Łukasik et al. 2013). R. insecticola is associated with increased fecundity of aphids
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feeding on clover (Tsuchida et al. 2004) and the symbiont S. symbiotica increases the aphid’s probability of surviving heat shock stress (Montllor et al. 2002). Facultative symbionts, then, are a key component in the ecology and evolution of A. pisum. Nonetheless, because carrying facultative symbionts has metabolic costs, symbiont-carrying hosts will be gradually replaced by symbiont-free hosts in the absence of a selection pressure (i.e., parasitic wasps, pathogens, etc.; Oliver et al. 2008). Being a tiny plant-dwelling insect, the pea aphid is at constant risk of being ingested by a mammalian herbivore feeding on the plant. To avoid such incidental predation, aphids tend to drop off the host plant when a mammalian herbivore approaches it. Aphids drop when they sense reliable cues of mammalian breath, a combination of warm and humid air stream. However, not all aphids drop off the plant in response to the same breath signals, and dropping rates are lower as ambient temperature rises (Gish et al. 2012, 2011). Given the variability of secondary symbiont assemblages between and within pea aphid populations and the assorted defensive effects of symbionts on pea aphids, we hypothesized that symbionts may also be involved in the aphids’ dropping behavior, i.e., dropping and nondropping pea aphids may differ in their symbiotic assemblage. Accordingly, the goals of the current study were to characterize the fauna of facultative bacterial symbionts in local populations of A. pisum and to determine whether there is a difference in secondary symbiont assemblage between dropping and nondropping individuals. Materials and Methods Aphids. Two A. pisum colonies were tested: A laboratory-reared colony grown on broad bean seedlings (Vicia faba L.) under conditions of 22 6 1 C, 36 6 2% relative humidity, and a photoperiod of 18:6 (L:D) h, as described in Gish et al. (2012), and a second population that was grown on Medicgo sp. seedlings in an open greenhouse under changing ambient conditions, correlated to the humidity, temperature, and photoperiod of Mt. Carmel area, Israel, (Mediterranean climate) during September 2013. Dropping Behavior Assay. The dropping behavior assay was performed according to Gish et al. (2011). Ten adult wingless A. pisum were transferred to each of the six clean broad bean seedlings and were given 2 h to adjust and settle. After the 2-h adjustment, we shook each plant gently for 2 s and then blew on it for 4 s from a distance of 10 cm to induce dropping (Gish et al. 2012). We then collected the droppers and the nondroppers into two separate 95% ethanol-filled test tubes to be kept until DNA extraction. Because pea aphids are viviparous and reproduce rapidly, by the time the dropping assay was initiated (the 2-h adjustment time), there were >10 aphids on each plant (see Table 1). The proportions of dropping and nondropping aphids were analyzed by t-tests after arcsine transformations to normalize data, Leven’s test to verify equality of variances and Shapiro–Wilk test to verify
normality of distributions (IBM SPSS statistics 20, Chicago, IL). DNA Extraction. DNA was extracted by squashing the aphids individually in 100 ml of proteinase K buffer solution, incubating the sample for 45 min in 35 C and then for 10 min in 95 C. The extractions were diluted 4 in polymerase chain reaction (PCR)-grade water and kept at 20 C until further use. DNA was extracted from a total of 60 laboratory-reared A. pisum (30 droppers and 30 nondroppers) and 40 greenhousereared A. pisum (20 droppers and 20 nondroppers). In addition, the DNA of six more greenhouse-reared aphids (three droppers and three nondroppers) was extracted using the DNeasy Blood & Tissue Kit (Qiagen) to assess the aphid microbial assemblage on the Illumina platform, as described in the following section. Analyzing Symbiont Assemblage. Symbiotic communities were analyzed by two complementary methods: high-throughput sequencing and diagnostic PCR. High-Throughput Sequencing. DNA was extracted separately from three dropping and three nondropping greenhouse-reared aphids, using the Qiagen DNeasy Blood & Tissue Kit, Germantown, Maryland; USA. 16 S library construction and sequencing was performed by the Michigan State Genomics Core Facility using a modified version of the protocol presented by Caporaso et al. (2011) adapted for the Illumina MiSeq platform. The V4 region of the 16 S rRNA gene was amplified in a one-step PCR at an annealing temperature of 50 C with region-specific primers (515F/806R) that included the Illumina flowcell adapter sequences. A negative control was used during the PCR and verified blank by agarose gel electrophoresis. The final libraries were normalized and pooled using a PicoGreen assay and subsequently quantified using the Kappa qPCR kit. After cluster formation on the MiSeq instrument, the amplicons were sequenced with custom primers designed to be complimentary to the V4 amplification primers to avoid sequencing of the primers, and the barcode was read using a third sequencing primer in an additional cycle. The amplification primers were adapted from Caporaso et al.’s (2012) protocol to include nine extra bases in the adapter region of the forward amplification primer to support multiplexed paired-end sequencing on the MiSeq. Sequencing was performed using v2 chemistry and sequenced using a 2 250 bp run with 5% phiX spike-in. Paired-end (PE) reads were quality filtered using trimmomatic (Bolger et al. 2014; filtering parameters: LEADING:30 TRAILING:30 SLIDINGWINDOW: 10:25 CROP:200 MINLEN:200), and filtered read quality was inspected using Fastqc (http://www.bioinfor matics.babraham.ac.uk/projects/fastqc/, accessed 23 February 2015). PE reads were truncated to include only the first 200 bp (as the last 50-bp region usually contains more sequencing error), and then assembled into 250-bp amplicons using PANDASeq (Masella et al. 2012). PANDAseq sequences were mapped to the phiX genome (NC_001422) using bowtie2 (Langmead and Salzberg, 2012) to remove reads coming from the
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LAVY ET AL.: BACTERIAL SYMBIONTS GOVERN APHID’S DROPPING BEHAVIOR?
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Table 1. Mean percentages ( 6 S.E) of dropping and nondropping aphids in the bioassay Population
n
Droppers
Nondroppers
t-test values
Laboratory Greenhouse
6; 13 6 0.7 10; 14 6 2.4
43.6 6 9.5% 54.2 6 7.4% t1 ¼ 0.78, P ¼ 0.44
56.4 6 9.5% 45.8 6 7.4% t1 ¼ 0.86, P ¼ 0.4
t1 ¼ 0.93; P ¼ 0.375 t1 ¼ 0.73; P ¼ 0.47
N ¼ number of plants used; average number of aphids 6 S.E on each plant (see text for details).
Table 2. Symbiont-specific 16 S rRNA primers that were used in this study Symbiont H. defensa R. insecticola S. symbiotica Rickettsia Spiroplasma Rickettsiella X-type
Primers 10F/419R 1279F/35R 16SA1/PASScmp 16A1/Rick 16SR 16SA1/TKSSsp 211F/470R 10F/420R
Product size 409 1,244 480 200 510 259 410
phiX spike-in to the MiSeq run. The remaining PANDAseq sequences were further truncated to a constant length of 250 bp, and then were subsampled (rarefied) to perform analysis with an identical number of reads on all samples. The UPARSE pipeline, part of the USEARCH program, was used for operational taxonomic unit (OTU) clustering (Edgar, 2013), following the recommended pipeline (http://www.drive5.com/ usearch/manual/uparse_pipeline.html, accessed 23 February 2015). Specifically, this pipeline includes: read de-replication, abundance sorting and singleton discarding (a minimum of five sequences was required per OTU), OTU clustering and chimera filtering. The resultant representative sequences of each OTU were compared with the Silva database to identify the representative genus of each OTU. The relative abundance of B. aphidicola and S. symbiotica were compared and analyzed by Mann–Whitney U-test (IBM SPSS statistics 20). Diagnostic PCR. To verify the high-throughput sequencing results, we also performed diagnostic PCR using known aphid symbiont-specific 16 S rRNA primers at the specified PCR conditions (Table 2). Each reaction contained 0.5 ml of each primer, 1 ml of an aphid lysate, 1 ml of MgCl2, 5 ml of master mix (Thermo Scientific 2 ReddyMix), and 2.5 ml of PCR-grade water for a total volume of 10 ml. The PCR products were visualized on ethidium bromide-containing agarose gel (1.5%). The proportions of aphids infected with R. insecticola and Rickettsia in dropping versus nondropping aphids were analyzed by Fisher’s exact test based on a chi-square distribution (IBM SPSS statistics 20). Results Dropping Assay. In each of the tested populations, approximately half of the aphids dropped off the plant (Table 1). The proportions of dropping and nondropping did not differ significantly between the laboratory population and the greenhouse population (statistical analyses detailed in Table 1).
Annealing temp; time
57 C; 1 min 57 C; 1 min 55 C; 30 s 55 C; 30 s 55 C; 30 s 64 C; 30 s 64 C; 30 s
Reference (Russell et al., 2013b) (Russell et al. 2013b) (Moran et al. 2005) (Tsuchida et al. 2002) (Tsuchida et al. 2002) (Russell et al., 2013b) (Ferrari et al. 2012)
Table 3. Composition of symbiont fauna in dropping and nondropping pea aphids, as sequenced by Illumina platform Symbiont
Dropping (n ¼ 3)
Nondropping (n ¼ 3)
B. aphidicola S. symbiotica Pseudomonas Staphylococcus Methylophaga
0.854 6 0.032 0.146 6 0.032
