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Background:   Bemisia tabaci  species ( B. tabaci ), or whiteflies, are the world’s most devastating insect pests. They cause billions of dollars (US) of damage each year, and are leaving farmers in the developing world food insecure. Currently, all publically available transcriptome data for  B. tabaci  are generated from pooled samples, which can lead to high heterozygosity and skewed representation of the genetic diversity. The ability to extract enough RNA from a single whitefly has remained elusive due to their small size and technological limitations.    Methods:  In this study, we optimised the single whitefly RNA extraction procedure, and sequenced the transcriptome of four individual adult Sub-Saharan Africa (SSA1)  B. tabaci.  Transcriptome sequencing resulted in 39-42 million raw reads.  De novo  assembly of trimmed reads yielded between 65,000-162,000 transcripts across  B. tabaci  transcriptomes.    Results:  Bayesian phylogenetic analysis of mitochondrion cytochrome I oxidase (mtCOI) grouped the four whiteflies within the SSA1 clade. BLASTn searches on the four transcriptomes identified five endosymbionts; the primary endosymbiont  Portiera   aleyrodidarum  and four secondary endosymbionts:  Arsenophonus, Wolbachia, Rickettsia,  and  Cardinium spp.  that were predominant across all four SSA1 B.  tabaci  samples with prevalence levels between 54.1-75%. Amino acid alignments of the  Nus G gene of  P. aleyrodidarum  for the SSA1  B. tabaci  transcriptomes of samples WF2 and WF2b revealed an eleven amino acid residue deletion that was absent in samples WF1 and WF2a. Comparison of the protein structure of the  Nus G protein from  P. aleyrodidarum  in SSA1 with known  Nus G structures showed the deletion resulted in a shorter D loop.    Conclusions:  The use of field-collected specimens means time and money will be saved in future studies using single whitefly transcriptomes in monitoring vector and viral interactions. Our method is applicable to any small organism where RNA quantity has limited transcriptome studies.

The first transcriptomes from field-collected individual whiteflies (Bemisia tabaci, Hemiptera: Aleyrodidae)