DOI: 10.5061/dryad.fxpnvx0v9
title: No evidence of bacterial symbionts influencing host specificity in Aphis gossypii Glover (Hemiptera: Aphididae)

description: The cotton-melon aphid, Aphis gossypii Glover, is a polyphagous insect
 pest with many host-specialized biotypes, such as Cucurbitaceae- and
 Malvaceae-specialized (CU and MA) biotype. Bacterial symbionts were
 reported to determine host range in some aphids. Whether this is the case
 in A. gossypii remains unknown. Here, we tested host specificity of CU and
 MA biotype and compared host specificity between wingless and winged morph
 within the same biotype, and analyzed the composition of bacterial
 symbionts. The reproduction of CU and MA biotype reduced by 66.67% and
 82.79% respectively on non-native hosts, compared with that on native
 hosts. The composition of bacterial symbionts was not significantly
 different between CU and MA biotype, with Buchnera abundance &gt;95%
 in both biotypes. While, winged morph produced significantly more nymphs
 than wingless morph on non-native hosts, and Buchnera abundance in winged
 morph was only about 10% of that in wingless morph. There seemed to be a
 relationship between Buchnera abundance and host specificity. We regulated
 Buchnera abundance by temperature and antibiotics, but did not find that
 low Buchnera abundance resulted in high reproduction on non-native hosts.
 We conclude that host specificity of A. gossypii is not controlled by
 specific bacterial symbionts or by Buchnera abundance.
description: Bacterial symbionts
 of Cucurbitaceae- and Malvaceae-specialized biotype (CU and
 MA biotype) were analyzed by 16S rRNA gene sequencing. To obtain aphids of
 the same age, aphids of CU and MA biotype were cultured on detached
 cucumber and cotton leaves in Petri dishes, respectively. Ten wingless
 adults were introduced into each Petri dish to give birth to nymphs and
 removed 24 h late. The nymphs were cultured in an artificial climate
 chamber at 20 ± 2 °C with a 16L:8D photoperiod, and developed into new
 wingless adults in 6 d. The wingless adults were extracted for DNA using a
 universal DNA extraction kit (Takara, Japan) according to the
 manufacturer’s protocol after surface sterilization in 75% ethanol for 30
 s. The final DNA concentration and purification were determined using a
 NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, USA), and
 DNA quality was checked by running a 1% agarose gel electrophoresis. The
 V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified
 with primers 341F (5′- CCTAYGGGRBGCASCAG -3′) and 806R (5′-
 GGACTACNNGGGTATCTAAT -3′) using the GeneAmp 9700 thermal cycler PCR system
 (Applied Biosystems, Foster City, USA). The resulting PCR products were
 extracted from a 2% agarose gel and further purified using the AxyPrep™
 DNA Gel Extraction Kit (Axygen Biosciences, Union City, USA) and
 quantified using QuantiFluor™-ST Fluorometer (Promega, Madison, USA).
 Purified amplicons were pooled in equimolar quantities and paired-end
 sequenced (2×250) using the Illumina HiSeq 2500 System (Illumina, San
 Diego, USA). Raw FASTQ files were demultiplexed, quality-filtered using
 Trimmomatic, and merged using FLASH software
 (http://cbcb.umd.edu/software/flash). Operational taxonomic units (OTUs)
 were constructed to estimate the diversity of bacterial
 symbionts.