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.

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 >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.