Identification of genes for engineering the male germline of Aedes aegypti and Ceratitis capitata

Abstract

Background: Synthetic biology approaches are promising new strategies for control of pest insects that transmit disease and cause agricultural damage. These strategies require characterised modular components that can direct appropriate expression of effector sequences, with components conserved across species being particularly useful. The goal of this study was to identify genes from which new potential components could be derived for manipulation of the male germline in two major pest species, the mosquito Aedes aegypti and the tephritid fruit fly Ceratitis capitata.

Results: Using RNA-seq data from staged testis samples, we identified several candidate genes with testis-specific expression and suitable expression timing for use of their regulatory regions in synthetic control constructs. We also developed a novel computational pipeline to identify candidate genes with testis-specific splicing from this data; use of alternative splicing is another method for restricting expression in synthetic systems. Some of the genes identified display testis-specific expression or splicing that is conserved across species; these are particularly promising candidates for construct development.

Conclusions: In this study we have identified a set of genes with testis-specific expression or splicing. In addition to their interest from a basic biology perspective, these findings provide a basis from which to develop synthetic systems to control important pest insects via manipulation of the male germline.

Keywords: Aedes aegypti; Ceratitis capitata; Male germline; Pest insect; RNA-seq; Synthetic biology.

Fig. 1

Importance of pre-meiotic protein expression in bipartite synthetic genetic systems. If transcription is repressed from meiosis onwards, post-meiotic translation of the transcription factor in a bipartite expression system is not adequate for expression of the target transgene (a). Expression of the transgene requires translation of the transcription factor before meiosis such that the target transgene is transcribed before transcriptional repression at meiosis (b)

Fig. 2

Gels showing PCR results for Ae. aegypti expression candidates. a Candidates for which no band of the expected size for the testis sample could be seen in non-testis samples. b Candidates for which a band of the expected size for the testis sample could be seen in a non-testis sample, but it was faint and in the cases indicated by asterisks, could have resulted from contaminating gDNA. Expected PCR product sizes are indicated with arrows. In some cases bands of other sizes are of the expected size for products amplified from contaminating gDNA. Other bands of unexpected sizes may represent isoforms that were not predicted, or non-specific amplification

Fig. 3

Gels showing PCR results for C. capitata expression candidates. Presented as for Fig. 2

Fig. 4

Relative expression levels in different tissues for Ae. aegypti expression candidates, determined using qRT-PCR. Results for AAEL012239 are shown inset, as the expression level for this gene was too low to view at the same scale as for the other genes. * Primers could also have amplified from gDNA, so apparent low expression in non-testis tissues could be a result of gDNA contamination

Fig. 5

Relative expression levels in different tissues for C. capitata expression candidates, determined using qRT-PCR

Fig. 6

RT-PCR testing of candidate testis-specifically spliced genes. Expression of the predicted testis-specific splice form was assessed using primers designed to span the predicted testis-specific exon-exon junction. Expression of other splice forms was assessed using additional primers targeting either multiple splice forms – both the predicted testis-specific splice form and other splice forms – but yielding products of different sizes (a), or other splice forms only (b). Note that primers amplifying splice forms other than the predicted testis-specific splice form may still yield a product in testis samples, as these splice forms may be expressed in the testis in addition to the testis-specific splice form. The splice forms illustrated here are simplified examples

Fig. 7

Gels showing PCR results for Ae. aegypti splicing candidates. a Candidates for which no band of the expected size for the predicted testis-specific splice form could be seen in non-testis samples. b Candidates for which a band of the expected size for the predicted testis-specific splice form could be seen in a non-testis sample, but it was only faint. Expected PCR product sizes are indicated with arrows. Bands of unexpected sizes may represent other splice forms that were not predicted, or non-specific amplification

Fig. 8

Gels showing PCR results for C. capitata splicing candidates. Presented as for Fig. 7

Fig. 9

Relative expression levels in different tissues for predicted testis-specific and other splice forms of Ae. aegypti splicing candidates, determined using qRT-PCR. Where expression levels in the testis are too low to view at the same scale as for the other splice forms, results for testis are shown inset. The relative expression value of the testis-specific splice form is set at 1 in all cases. Error bars show +/− standard error of the mean for two technical replicates

Fig. 10

Relative expression levels in different tissues for predicted testis-specific and other splice forms of C. capitata splicing candidates, determined using qRT-PCR. Presented as for Fig. 9

Fig. 11

Microscope images illustrating preparation of staged testis samples. a Whole testis from A. aegypti pupa. b Apical region of A. aegypti testis after bisection; used to generate “early” sample. c Basal region of A. aegypti testis after bisection; used to generate “late” sample. d Whole testis from C. capitata pupa. e Isolated C. capitata early spermatocytes. f Isolated C. capitata late spermatocytes. g Isolated C. capitata early spermatids. h Isolated C. capitata late spermatids. Scale bar is 100 μm in all panels

Fig. 12

Computational pipeline for identification of candidate testis-specifically spliced genes. RNA-seq reads were mapped to the relevant reference genome using TopHat. Transcript assemblies were generated using Cufflinks. Transcript expression was quantified using Cuffdiff. The output of these steps, along with user-defined threshold FPKM values, was used as input for a custom Python program. Custom Python scripts in combination with bedtools were used to output a list of candidates with associated information used for further filtering, such as exon-exon junction coverage and expression values, as well as sequences in a convenient format for primer design – intron flanking sequences, and alignments of all splice forms for each gene