Development and immunity-related microRNAs of the lepidopteran model host Galleria mellonella

Abstract

Background: MicroRNAs (miRNAs) are small non-coding RNAs that act as key players in the post-transcriptional regulation of protein synthesis. Although little is known about their role in complex physiological processes such as development and immunity, our knowledge is expanding rapidly, thanks to the use of model systems. The larvae of the greater wax moth Galleria mellonella are now established as model hosts for pathogens that infect insects or humans. To build on our previously-reported comprehensive G. mellonella transcriptome, here we describe the identification and analysis of development and immunity-related miRNAs, thus providing valuable additional data to promote the use of this model host for the analysis of complex processes.

Results: To screen for miRNAs that are differentially expressed in G. mellonella (1) during metamorphosis or (2) following infection with the entomopathogenic bacterium Serratia entomophila or (3) with the parasitic fungus Metarhizium anisopliae, we designed a microarray containing more than 2000 insect miRNA probe sequences. We identified miRNAs that were significantly expressed in pre-pupae (16), pupae (22) and last-instar larvae infected with M. anisopliae (1) in comparison with untreated last-instar larvae which were used as a reference. We then used our transcriptomic database to identify potential 3' untranslated regions that form miRNA-mRNA duplexes by considering both base pair complementarity and minimum free energy hybridization. We confirmed the co-expression of selected miRNAs (such as miR-71, miR-263a and miR-263b) with their predicted target mRNAs in last-instar larvae, pre-pupae and pupae by RT-PCR. We also identified miRNAs that were expressed in response to infection with bacterial or fungal pathogens, and one miRNA that may act as a candidate mediator of trans-generational immune priming.

Conclusions: This is the first study to identify miRNAs that are predicted to regulate genes expressed during metamorphosis or in response to infection in the lepidopteran model host G. mellonella.

Figure 1

Expression profiling of G. mellonella miRNAs. The microarray heat map was generated following microarray hybridization, statistical analysis and hierarchical clustering. The heat map highlights a set of differentially-expressed miRNAs (infected vs non-infected, pre-pupae vs larvae, pupae vs larvae, and pupae vs pre-pupae. Key: red = upregulated; green = downregulated. The log score of each fold change is indicated.

Figure 2

Distribution of expressed miRNAs in pupae, pre-pupae and parasitized G. mellonella larvae. The miRNAs were selected from miRBase v18 for arthropods and their expression levels were determined by microarray analysis. For the individual miRNAs presented here, the fold difference in expression was significant (p < 0.05) compared to the expression levels in untreated last-instar G. mellonella larvae.

Figure 3

Venn diagram showing the differential expression of miRNAs in G. mellonella pupae, pre-pupae and larvae infected with M. anisopliae , including the miRNAs that are unique to individual to or shared among particular sample types. The miRNA sequences were sourced from miRBase v18 for arthropods and differential expression was confirmed by microarray analysis. The fold-difference in expression level for all miRNAs presented here (compared to naïve last-instar G. mellonella larvae) was statistically significant (p < 0.05).

Figure 4

Schematic illustration of the strategy used to predict miRNA targets.

Figure 5

The three best minimum free energy (MFE) duplexes formed between ame-miR-71 and the 3′-UTRs of G. mellonella mRNAs (the 5′ ends are marked) are shown. The targets are (A) contig 15133_1.exp, (B) GME-string_contig_292.0 and (C) contig 16779_1.exp. The alignment shows the complete seed region of the miRNA hybridized to the target UTRs. Each UTR was only searched for one optimal hit.

Figure 6

The three best minimum free energy (MFE) duplexes formed between api-miR-263a and the 3′-UTRs of G. mellonella mRNAs (the 5′ ends are marked) are shown. The targets are (A) contig 00981_1.f1.exp, (B) contig 16425_1.f1.exp and (C) contig 21732_1.exp. The alignment shows the complete seed region of the miRNA hybridized to the target UTRs. Each UTR was only searched for one optimal hit.

Figure 7

The two best minimum free energy (MFE) duplexes formed between ame-miR-263b and the 3′-UTRs of G. mellonella mRNAs (the 5′ ends are marked) are shown. The targets are (A) contig 05432_1.f1.exp and (B) contig 20004_1.f1.exp. The alignment shows the complete seed region of the miRNA hybridized to the target UTRs. Each UTR was only searched for one optimal hit.

Figure 8

The three best minimum free energy (MFE) duplexes formed between dps-miR-210b and the 3′-UTRs of G. mellonella mRNAs (the 5′ ends are marked) are shown. The targets are (A) contig 15648_1.f1.exp, (B) contig 19765_1.exp and (C) contig 15841_1.exp. The alignment shows the complete seed region of the miRNA hybridized to the target UTRs. Each UTR was only searched for one optimal hit.

Figure 9

Differential expression of miRNAs and predicted target mRNAs in G. mellonella pupae and pre-pupae. The miRNAs identified by microarray analysis and their predicted mRNA targets were validated by RT-PCR in order to confirm differential expression in pupae and pre-pupae. They include (A) ame-miR-71 and (B) contig 15133; (C) api-miR-263a and (D) contig 21732; (E) ame-miR-263b and (F) contig 20004. The relative fold changes indicated for the miRNAs and mRNAs have been normalized to aae-miR-252 and 18S rRNA as the internal reference control. (*p < 0.05, ***p < 0.0005, ns = not significant).

Figure 10

Differential expression of miRNA and predicted target mRNAs following infection with M. anisopliae . The miRNAs identified by microarray analysis and their predicted mRNA targets were validated by RT-PCR in order to confirm differential expression in infected insects. They include (A) dps-miR-210b and (B) contigs 19765 and 15841. The relative fold changes indicated for the miRNAs and mRNAs have been normalized to aae-miR-252 and 18S rRNA as the internal reference control. (*p < 0.05, ***p < 0.0005.

Figure 11

Transgenerational expression analysis of miRNA in G. mellonella following exposure to bacteria contaminated diet. Expression of api-miR-263a in midgut tissue (A) and rest of the body (B) of larvae fed either with E. coli or S. entomophila. Expression of api-miR-263a in eggs laid by female imagoes that were fed as larvae with E. coli or S. entomophila (C). The transcription of this miRNA gene is given relative to that observed in larvae fed with an uncontaminated diet. Values were normalized against aae-miR-252.