RNA-Seq Validation of RNAi Identifies Additional Gene Connectivity in Tribolium castaneum (Coleoptera: Tenebrionidae)

RNA-Seq Validation of RNAi Identifies Additional Gene Connectivity in Tribolium castaneum... RNA interference (RNAi) is a functional genomics tool to correlate genotype and phenotype by delivering tar- geted, gene-specific, and complementary dsRNA into a host via injection, feeding, or other means in order to re- duce gene expression. In the red flour beetle, Tribolium castaneum, RNAi has been successful via injected dsRNA at all life stages. Traditionally, successful transcript knockdown has been quantified by qPCR on a gene- by-gene basis, where only expression of the target gene and normalization genes are evaluated. In this study, RNA-Seq was used to quantify transcript expression in larvae injected with dsRNA for aspartate 1-decarboxyl- ase (ADC), which gives a reliable phenotype of an adult with a black cuticle instead of the wild-type red-brown. ANOVA of control, mock-injected, and ADC-dsRNA injected larvae indicated that target gene expression was significantly (P ¼ 0.002) reduced 4-fold, and the black phenotype was achieved in all adults injected with ADC- dsRNA as larvae. In a pairwise analysis, significant (P< 0.05) differential expression of other genes in ADC-in- jected larvae suggested connections between gene pathways. One gene, dopamine receptor 2, was increased in expression 227-fold (P ¼ 0.025), presumably connected to previous data that showed a reduction in expres- sion of ADC results in increased levels of dopamine. To evaluate the hypothesis that increased dopamine levels can affect mobility, T. castaneum adults injected with ADC-dsRNA as larvae were significantly impaired in movement tests compared to controls, similar to black mutants in Drosophila melanogaster. The data demon- strate that RNA-Seq can reveal gene connectivity and provide more complete data validation and analysis com- pared to qPCR. Key words: Tribolium castaneum, RNAi, ADC, RNA-Seq, gene expression RNA interference (RNAi) is a popular tool in functional genomics dsRNA fragments was sufficient to elicit a response at concentra- and, in pest insects like Tribolium castaneum, a potential strategy tions ranging from 0.0001 to 0.001 lg/ll(Miller et al. 2012). Other for molecular pest control (Baum et al. 2007, Aronstein et al. 2011, studies have demonstrated that injection can be successful in any T. Ulrich et al. 2015). RNAi is an evolutionarily conserved mechanism castaneum life stage (reviewed in Aronstein et al. 2011). that relies on natural cellular pathways to target and degrade double The most common downstream validation of knockdown after stranded RNA (dsRNA) of viruses, as described in nematodes (Fire RNAi is quantitative polymerase chain reaction (qPCR). This type et al. 1998). This natural process can be mimicked by the introduc- of analysis is dependent on gene-specific primers, including onerous tion of dsRNA into an organism (e.g. injection). The endoribonu- optimization that is often circumvented, and generally only used to clease, Dicer, cleaves dsRNA into 21–23 nucleotide short interfering measure the target gene and a normalizer gene as a standard. We RNAs (siRNAs), which are incorporated into an RNA-induced si- suggest this method limits the information gained from RNAi, and lencing complex (RISC) that targets and degrades RNA with com- that RNA transcriptome-sequencing and quantitation (RNA-Seq) is plementary sequence. more informative. RNA-Seq can monitor target gene knockdown RNAi has been used successfully to increase insect mortality or and off-target effects, and provides results similar to qPCR (Morris developmental abnormalities via microinjection or feeding in vitro et al. 2009, Chen et al. 2016). In our hands, RNA-Seq has been ben- or in planta (Baum et al. 2007, Huvenne and Smagghe 2010, eficial in cases where RNAi does not show a visible phenotype, and Aronstein et al. 2011). In T. castaneum, injection of 520 base pair attempts at validation by qPCR are ambiguous. For example, qPCR Published by Oxford University Press on behalf of the Entomological Society of America 2017. This work is written by US Government employees and is in the public domain in the US. 1 Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/57/3573844 by Ed 'DeepDyve' Gillespie user on 17 July 2018 2 Journal of Insect Science, 2017, Vol. 17, No. 2 0 0 validation of knockdown of a major gut cysteine peptidase gene in (2009), forward 5 -AAGGCGAAGGGAACATCAGG-3 and reverse 0 0 T. castaneum was inconclusive by qPCR, and there was no differ- 5 -TCCCCAACCGCTCAATCTC-3 . All secondary PCR primers ence in phenotype (unpublished data). However, using RNA-Seq, had a T7 construct attached to the 5 end (TAATACGACTCAC we demonstrated that knockdown was in fact successful, but closely TATAGGG). Another round of PCR was performed with each sec- related cysteine peptidase as well as serine peptidase genes, were ondary primer set and using the template obtained for each respec- compensating for the loss of target gene expression (Perkin et al. tive gene in the primary reaction as the template, using similar PCR 2017). Thus, sequence similarity and redundancy in gene function, conditions (100 ll reaction; Perkin et al. 2017). Secondary PCR as well as compensation responses embedded in the transcriptome products were evaluated via 1% agarose E-gel for appropriate response, can mask the effects of RNAi, making it difficult to verify length and sufficient amplification. with qPCR alone. The PCR product from the secondary amplification was used to To illustrate that RNA-Seq can be a superior method to validate make dsRNA via a MEGAscript T7 kit (Invitrogen, Life the results of RNAi, we report here the result of knockdown of as- Technologies, Carlsbad, CA), and was purified via a MEGAclear kit partate 1-decarboxylase (ADC)in T. castaneum larvae. ADC is one (ThermoFisher, Waltham, MA; Perkin et al. 2017). Size and quan- of two decarboxylases (the other is DDC, TC013480) needed for tity of products were verified on a TapeStation (Agilent, Santa proper insect cuticle tanning in T. castaneum (sclerotization and pig- Clara, CA) and quantity also was verified by a digital nanophotome- mentation). The process is complex and requires the conjugation ter (Implen, Westlake Village, CA). Negative controls included non- and cross-linking of cuticle proteins, leading to an insoluble, hard, injected larvae (Control) and mock-injected with water and dye and darkened red-brown exoskeleton (Roseland et al. 1987). ADC (Mock). catalyzes the synthesis of b-alanine, which plays a critical role in cu- ticle tanning because of its conjugation with dopamine to produce Micro-injected dsRNA N-b-alanyldopamine (NBAD), a substrate for the phenol oxidase There were three groups in this study: T. castaneum larvae injected laccase that catalyzes the synthesis of the cuticle protein cross- with water/dye (Mock); larvae injected with ADC-dsRNA construct linking agents and pigment precursors (Kramer et al. 1984). (ADC) mixed with dye; and a noninjected (Control) group. Knockdown of ADC leads to a black cuticle phenotype and the ac- Immediately before injection, treatments (water or dsRNA) were cumulation of dopamine (Arakane et al. 2009). mixed with blue dye (FD&C Blue 1, Kroger Food Colors, We demonstrate that RNAi targeting ADC, as verified by RNA- Cincinnati, OH; diluted 1:20) to aid in visualization of the injected Seq and black phenotype, changes the expression of other genes that liquid. Actively feeding, third instar larvae were briefly placed on ice may be interconnected. One affected gene, dopamine receptor 2, led and transferred to double-sided tape on a microscope slide placed us to test whether movement was impaired in T. castaneum adults on a small tissue culture flask ice block (Posnien et al. 2009). injected with ADC-dsRNA as larvae, uncovering a previously un- Briefly, a Drummond Nanoject II (Drummond Scientific Co., known interaction between ADC and genes controlling movement Broomall, PA) with a “bee-stinger” needle was set at 69 nl, and in T. castaneum. dsRNA was diluted to provide 200 ng of dsRNA per larva (Perkin et al. 2017) and loaded into the needle. Needles were made with 3.5 Drummond glass capillary tubes (3-000-203-G) and a micropipette Materials and Methods puller (Sutter Instrument Co. Model P-97, Novato, CA). After injec- tion, each group was allowed to recover for 2 h at room tempera- Insects ture, and then were covered with diet (95% wheat flour, 5% The T. castaneum lab strain was originally collected from a grain Brewer’s yeast) and kept at 28 C, 75% relative humidity, 0:24 bin in Kansas and has been reared at the Center for Grain and (L:D) h. All injections in a single replicate were done on the same Animal Health Research (CGAHR, Agricultural Research Service, day, with a total of three independent replicates per treatment. United States Department of Agriculture, Manhattan, KS) for over 20 yr. Insects are maintained on a diet of 95% wheat flour and 5% Brewer’s yeast at 28 C, 75% relative humidity, 0:24 (L:D) h. mRNA Extraction From each treatment group, eight T. castaneum larvae were ran- domly selected at 7-d postinjection, ground in liquid nitrogen with a Primers and dsRNA disposable pellet pestle, and total RNA was obtained using a Qiagen Primers were designed via Primer-BLAST (http://www.ncbi.nlm.nih. RNeasy Plus kit (Qiagen, Hilden, Germany). The Plus version of gov/tools/primer-blast/) to the ADC gene in T. castaneum using this kit utilizes a “gDNA Eliminator” spin column as a pretreatment default parameters. All primers were specific, and selected primer to further diminish any contamination of the RNA with DNA, and sets were unique to the ADC gene. The first round of PCR amplified 0 an optional step of on-column DNase digestion also was used. the entire gene using the primary primer sets: ADC forward 5 - 0 0 Quantity and quality at each step of mRNA collection were eval- AAGGCGAAGGGAACATCAGG-3 and reverse 5 -CTCCCC uated by TapeStation, and quantity was verified by a nanophotome- AACCGCTCAATCTC-3 . PCR reactions were 25 ll total volume ter (Implen, Westlake Village, CA). Aliquots were stored at 80 C. using genomic DNA template from the lab strain and standard ther- mal cycle conditions (Perkin et al. 2017). The product was assessed on a 1% agarose E-gel (ThermoFisher, Waltham, MA) to ensure the RNAseq amplified region was the correct size. A stranded mRNA-Seq Kit (KAPA Biosystems, Wilmington, MA) A second set of primers was designed to amplify within the first with appropriate Ion Torrent primers, adapters, and bar codes amplification region, and the products were used for dsRNA ampli- (Integrated DNA Technologies, Coralville, IA) was used for library fication. Primers were designed to target each sequence towards the preparation of 4 mg of total RNA from each biological replicate from 3 end, according to previous data that suggested this region was each treatment. A final concentration of adapter and barcodes of best for specific and maximal knockdown (Whyard et al. 2009, 100 nM was used without optimization of the concentration. Eight Perkin et al. 2017). ADC-dsRNA primers were as in Arakane et al. equimolar barcoded libraries from all treatment groups of a Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/57/3573844 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 3 biological replicate were pooled based on concentration estimates from a KAPA Library Quantification Kit for Ion Torrent platform (KK4827), and were placed in an Ion Chef (ThermoFisher, Grand Island, NY) for template preparation and loading onto an Ion PI Chip v2 for sequencing on the Ion Proton sequencer (Perkin et al. 2017). Data Analysis Transcripts were mapped to the Tcas5.2 genome build (NCBI) in SeqManNGen (DNAStar, Madison, WI), and differential gene expression was analyzed in ArrayStar (DNAStar version 14) using default parameters. Genes were normalized using Reads Per Kilobase per Million (RPKM; Mortazavi et al. 2008) and filtered to transcripts>8-fold change between groups in Student’s t-test com- parisons, and later were filtered to a 90% confidence interval after correction for multiple comparisons with False Discovery Rate (FDR) analysis (Benjamini and Hochberg 1995), with exceptions as noted. We also used gene expression data from different develop- mental stages (egg, larvae, pupae, and adult) of T. castaneum,as ADC Control Mock detailed in Perkin et al. (2016). Nonannotated differentially expressed genes were submitted to Treatments BLAST2GO PRO (Valencia, Spain) to elucidate possible function. From this analysis, additional functions were identified in Rfam Fig. 1. ADC expression (average total RPKM) differences between larvae injected with dsRNA targeting ADC (ADC), the noninjected control (Control), (Nawrocki et al. 2015). and the mock-injected (Mock) group. Error bars denote SE. Each replicate had>7 million reads with a total of>31 million reads per sample (Supp Table 1 [online only]). Sequences from this in larvae, and we used RNA-Seq to validate knockdown. ADC is a study were deposited into NCBI Sequence Read Archive gene commonly used as a positive RNAi phenotypic control. Most PRJNA302304. previous validation experiments for RNAi in T. castaneum have been by qPCR, but we chose RNA-Seq because we wanted to exam- Behavior Assays and Analysis ine other potential effects on reduced transcript levels of ADC. To assess movement and behavioral patterns in ADC-injected lar- vae, 25 late instar larvae were collected from ADC, Mock, and ANOVA of All Treatment Groups Control groups and placed into 1 oz solo cups with holes in the lid. All T. castaneum larvae injected with ADC-dsRNA in all replicates Media composed of 90% stabilized wheat germ and 10% flour was developed into adults with a black cuticle compared to the wild-type added. Each group was monitored daily, and pupae were collected red/brown cuticle, presumably due to manipulation of the pigmenta- and placed in individual wells of a 24-well plate and allowed to tion and sclerotization pathways (Arakane et al. 2009). In T. casta- emerge as adults. neum larvae injected with ADC-dsRNA, ADC expression was At 3- to 12-d posteclosion, individual beetles were placed in the reduced 4.25-fold compared to a water-injected control (Mock; center of a 90-mm diameter plastic petri dish using vacuum suction, Fig. 1). An ANOVA comparing Control, Mock and ADC treatments facing the same direction at the start of each assay. After the lid was indicated that ADC expression was significantly different between placed on the dish, individuals were allowed to acclimate for 10 s treatments (P ¼ 0.002; Table 1). Other genes were also differentially before a video recording (Sony Handycam HDR-XR520V) was expressed (>2-fold) in ADC-dsRNA injected larvae, many related to taken of each beetle. After 5 min, the recording was terminated and gene regulation. The most highly up-regulated gene (7.39-fold) enc- beetles were removed from the petri dish and discarded. There were odes a leucine-rich repeat-containing protein (LOC103314786), 18 individuals from each treatment group (ADC-injected and which is annotated in insects, mollusks, and chordates (Supp Fig. 2 Mock) and control that were recorded for movement behavior. [online only]). In UniProt, the gene is annotated in Caenorhabditis Videos were analyzed using Ethovision XT (Version 8.0, Noldus elegans and Trichinella spp. (parasitic roundworms), but most func- Information Technology, Wageningen, The Netherlands). Video tional data is from C. elegans where it was expressed in multiple tis- tracks were analyzed for Distance Moved (cm); Mobile, Immobile, sues in embryo through larval development, and was important in or Highly Mobile states (defined as the changes in body position maintaining apical extracellular matrix integrity (Mancuso et al. between frames at 10 frames per second, where 0%, 20%, and 60% 2012, Finn et al. 2016). However, the relationship of this gene to of the body changes position) in sec; and velocity (distance traveled ADC is not apparent. Additionally, a gene encoding a hypothetical per time between frames, measured as the mean in cm/s). Distance, noncoding RNA (LOC107397781) was dramatically down- Velocity, and the three mobility states were analyzed using individ- regulated (974-fold). ual PROC GLM statements in SAS (SAS Institute, Cary NC, version 9.4), where strain was the main effect in the model and pairwise dif- ferences were Tukey HSD adjusted. Pairwise Analysis of ADC-injected Versus Mock-injected We compared gene expression of ADC-injected T. castaneum larvae to the mock-injected control (Table 2 and Supp Fig. 1 [online only]). Results The expression of dopamine receptor 2 (LOC661535) was increased In this study, we reduced the expression of the T. castaneum aspar- 227-fold in ADC compared to Mock (P ¼ 0.025), but the expression tate 1-decarboxylase (ADC, LOC100124592, TC034596) via RNAi of dopamine receptor 1 was unchanged (data not shown). Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/57/3573844 by Ed 'DeepDyve' Gillespie user on 17 July 2018 ADC larval expression (RPKM) 4 Journal of Insect Science, 2017, Vol. 17, No. 2 Table 1. ANOVA of control, mock-injected, and ADC-dsRNA injected T. castaneum larvae, filtered to linear RPKM> 1 in at least one of the groups (values to right for each group),>2-fold change in Mock versus ADC, and P< 0.05 (FDR Benjamini and Hochberg ) Name Description Fold change P value ADC RPKM Control RPKM Mock RPKM LOC103314786 Leucine-rich repeat-containing protein egg-6-like 7.385 0.044600 7.067 0.802 0.957 LOC107397729 Pfam : Retrotransposon gag protein, Reverse transcriptase 6.253 0.014700 2.695 0.559 0.431 LOC103315154 Leucine-rich repeat-containing protein let-4-like 3.267 0.035500 4.513 1.958 1.381 LOC658512 Carboxylesterase 1E 2.383 0.001280 52.77 22.45 22.15 LOC656320 Trypsin I-P1 isoform X1 2.180 0.000184 47.91 25.74 21.97 LOC103314471 Ribulose-phosphate 3-epimerase 2.424 0.012800 33.22 66.39 80.52 LOC664050 Serine/threonine-protein kinase RIO3 2.771 0.001390 9.336 25.40 25.88 LOC107398585 CPDF : protein kinase 3.815 0.031300 0.380 0.696 1.449 LOC100124592 Aspartate 1-decarboxylase 4.245 0.001800 0.563 2.123 2.390 LOC107397781 pfam: ncRNA 974.2 0.000001 0.001 0.001 1.106 Target gene (ADC) is shaded. Benjamini and Hochberg, 1995. Fold change in ADC-injected larvae. pfam: protein families database (Finn et al. 2016). CPDF: Conserved Protein Domain Family, NCBI. Table 2. Significant differentially expressed genes in a pairwise comparison of ADC-dsRNA injected T. castaneum larvae to mock-injected larvae (> 8-fold change,>90% confidence interval, Student’s t-test P values with Benjamini and Hochberg FDR correction, with linear total RPKM values on the right) b b Name Description Fold change P value Mock RPKM ADC RPKM LOC107398333 Uncharacterized 245.2 0.0290 0.001 0.278 LOC661535 Dopamine receptor 2 227.0 0.0250 0.001 0.258 LOC659605 Pfam : Protein of unknown function (DUF229), alkaline phosphatase 141.4 0.0044 0.001 0.161 LOC103313283 Pfam: Domain of unknown function (DUF4550) 66.93 0.0338 0.076 0.001 LOC107398197 Uncharacterized 81.96 0.0301 0.093 0.001 LOC657226 Glutamate dehydrogenase, mitochondrial-like 90.06 0.0290 0.112 0.001 LOC107398565 Odorant receptor 85a-like 136.7 0.0250 0.155 0.001 LOC107398092 CPDF : 7tm chemosensory receptor 146.3 0.0250 0.166 0.001 LOC107398783 Uncharacterized 176.0 0.0250 0.200 0.001 LOC663271 Pfam: haemolymph juvenile hormone binding protein (JHBP) 257.7 0.0003 0.293 0.001 LOC100142608 Pfam: 7tm odorant receptor (OR139) 240.6 0.0145 0.273 0.001 LOC107398253 Pfam: (ncRNA/snoRNA U3) 593.3 0.0081 0.674 0.001 LOC657178 odorant binding protein 18 (OBP18/OBP4A) 789.3 0.0250 0.896 0.001 LOC107397781 Pfam: ncRNA 974.2 0.0244 1.106 0.001 LOC100216358 Allatotropin I preprohormone isoform X1 1,411 0.0011 1.603 0.001 Benjamini and Hochberg, 1995. Fold change in ADC-injected larvae. Pfam: protein families database (Finn et al. 2016). CPDF: Conserved Protein Domain Family, (Marchler-Bauer et al. 2015). The expression of two other genes also increased, an uncharacter- Movement Assays ized gene (245-fold; P ¼ 0.029) and a protein with unknown func- The black cuticle phenotype of beetles injected with ADC-dsRNA tion with an alkaline phosphatase motif (141-fold; P ¼ 0.004). has been demonstrated to associated with an accumulation of dopa- Injection of ADC-dsRNA also significantly decreased the expres- mine (Arakane et al. 2009). However, this is the first demonstration sion of 12 additional genes compared to the mock-injected control that a reduction in ADC leads to an increase in expression of a dop- (P< 0.03; Table 2). The most severely repressed gene was amine receptor gene. Previous work in D. melanogaster indicated LOC100216358, a gene encoding allatotropin I preprohormone, that body color mutants often have neurological phenotypes that was decreased 1,411-fold (P ¼ 0.001). Several chemosensory (Wittkopp et al. 2003), including black mutants that show reduced genes (LOC107398565, LOC107398092, LOC100142608, activity at the larval/pupal boundary or “wandering stage” (Phillips LOC657178), three of which have a conserved odorant receptor et al. 2005). Therefore, we conducted a movement behavior assay domain, were significantly decreased 137- to 240-fold (P< 0.03). on T. castaneum adults injected with ADC-dsRNA as larvae to eval- There were two other uncharacterized genes (LOC107397781 and uate if increased expression of dopamine receptor 2 results in a simi- LOC107398253) that decreased 974- and 593-fold, respectively, lar neurological phenotype. and both had motifs similar to ncRNAs. Further analysis with Rfam Tribolium castaneum larvae injected with dsRNA targeting found the latter is part of the small nucleolar RNA (snoRNA) U3 ADC were significantly slower and moved less often as adults than family. This family is predicted to guide site-specific cleavage of both Mock beetles and Control beetles (Table 3 and Supp Video 1 ribosomal RNA (rRNA) during pre-rRNA processing (Clery et al. [online only]). Slowed movement was demonstrated by a reduction 2007). in mean velocity (cm/s) and total distance traveled in the Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/57/3573844 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 5 Table 3. Statistical data of movement patterns of T. castaneum adults injected as larvae with ADC-dsRNA compared to Control and Mock Trait Type III SS Mean square F value Pr> F Tukey’s HSD P value Distance moved 8,042 4,020 6.68 0.003 0.018 Velocity 0.089 0.045 6.68 0.003 0.018 High mobility 2.580 1.290 1.74 0.190 0.210 Immobile 609.4 304.7 5.44 0.007 0.011 Mobile 532.7 266.3 5.47 0.007 0.011 Values are ANOVA for each movement metric; source of ANOVA variation is from the main effect of strain (DF ¼ 2 for all comparisons). Distance moved is the total distance moved in cm for a 5-min assay; Velocity is the mean velocity in cm/s as measured by distance moved between frames; High Mobility refersto when 60% or more of the body changes location between frames, at 10 frames per second; Immobile means 0% of the body has moved between frames, whereas Mobile means 20% of the body is changing per frame. The far right column includes the Tukey HSD adjusted P values for the ADC/Mock comparison. Fig. 2. Box plots showing the distance moved (A) and velocity (B) of ADC-, Control-, and Mock-injected beetles. Solid black lines indicate the median distance and velocity. The vertical edges of the boxes represent 25% and 75% of the data distribution (Interquartile Range or IQR) with whiskers representing 1.5  (IQR), a measure of the maximum and minimum values in the dataset. ADC-injected beetles compared to Mock and Control (Fig. 2). Over us to evaluate possible behavioral differences. Dopamine is a neuro- the 5-min period of uninhibited movement, ADC-injected beetles transmitter important for a variety of life functions in vertebrates traveled on an average 23.11 cm less than Mock beetles (P ¼ 0.018) and invertebrates, and many human disorders have been attributed and had an average velocity 0.076 cm/s slower than Mock beetles to disruptions in dopamine pathways (reviewed in Beaulieu and (P ¼ 0.018). Overall, neither ADC-injected nor mock beetles had Gainetdinov 2011). Dopamine receptors are G protein-coupled long bouts of highly mobile activity (where 60% of body position receptors separated into D1 and D2 classes. In vertebrates, D1-class changes per frame) and the durations were not significantly different dopamine receptors include D1 and D5, and they stimulate cyclic- (ADC-injected ¼ 0.32 s; Mock ¼ 0.82 s; P ¼ 0.21). However, ADC- AMP production by adenylate cyclase, whereas the D2-class recep- injected beetles spent significantly more time in an immobile state tors include D2, D3, and D4, and they inhibit adenylate cyclase and (where 0% of body changes position per frame at 10 frames per sec- thus lead to a decrease in cyclic AMP (reviewed in Civelli et al. ond). ADC-injected beetles spent 297.4 s in an immobile state, while 1993). In humans, the D2 receptor is associated with different struc- Mock spent 289.9 s immobile (P ¼ 0.011; Table 3). tures in the brain, including olfaction. Insects have orthologs of dop- amine receptors 1 and 2, and there are genes encoding dopamine receptors 1 and 2 in T. castaneum (Hauser et al. 2008, Tribolium Genome Sequencing Consortium 2008). Our data suggests that Discussion expression of ADC and dopamine receptor 2 are diametrically Our approach demonstrates that RNA-Seq documents target knock- opposed in T. castaneum. down by RNAi of ADC in T. castaneum through normalized gene To understand how dopamine and cuticle pigmentation path- expression data, even in small differences of expression. All ADC- ways may interact, we turned to the abundant literature on D. mela- injected larvae resulted in the expected phenotype, adults with a nogaster pigmentation. D. melanogaster pigmentation is more black cuticle and a knockdown of 4.25-fold decrease. The black complicated than T. castaneum due to multiple color patterns and cuticle phenotype was the result of decreased ADC leading to an sexual dimorphism. However, most D. melanogaster pigment genes accumulation of dopamine upstream in the cuticle-tanning pathway have orthologs in T. castaneum. For example, the D. melanogaster in epidermal cells, which may be the predominant site of expression black gene is orthologous to the T. castaneum ADC gene, both genes of this gene (Arakane et al. 2009). encoding aspartate 1-decarboxylase, and contributing to the black RNA-Seq as an RNAi validation method also revealed differen- phenotype in mutants for this gene. Many studies have shown that pig- ces in expression of genes not previously associated with the target mentation gene mutants in D. melanogaster,suchas black, ebony,and gene, ADC. Most significant was the increased expression of dopa- tan not only have body color phenotypes, but also neurological pheno- mine receptor 2 (LOC661535) in the ADC RNAi treatment that led types (Wittkopp et al. 2003). For example, D. melanogaster black Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/57/3573844 by Ed 'DeepDyve' Gillespie user on 17 July 2018 6 Journal of Insect Science, 2017, Vol. 17, No. 2 mutants have reduced activity at the larval/pupal boundary or We propose that, based on our data, a decrease in ADC results in “wandering stage”. In the adult stage, black null mutants had sig- the accumulation of dopamine and an increased expression of dopa- nificantly reduced number of initiated walks compared to wild- mine receptor 2, and consequently decreased adenylate cyclase and type (Phillips et al. 2005). In T. castaneum black mutants, the loss production of cAMP. As both dopamine receptor 2 and allatotropin I of ADC was attributed to lower transcript levels (Arakane et al. are expressed in the T. castaneum brain, we speculate that decreased 2009), similar to what we achieved through RNAi of wild-type expression of allatotropin I was due to decreased cAMP. While it is insects. We examined the movement patterns of ADC-dsRNA- known that allatotropin I expression results in increased cAMP in T. injected T. castaneum and T. castaneum mutants with black phe- castaneum (Vuerinckx et al. 2011), our data suggests that expression notype (data not shown) and observed reduction in distance of allatotropin I may also be sensitive to decreased cAMP levels in a moved, overall velocity, and mobility compared to Mock and feedback loop mechanism; this hypothesis remains to be tested. Control. Thus, T. castaneum adults with a disrupted ADC gene Finally, we note that in the examination of RNA-Seq data, we function similarly to D. melanogaster black mutants, suggesting began our analyses with all differentially expressed genes, to exam- that T. castaneum mutants with black phenotype may also have a ine those that may be functionally related even if genes do not meet mutation in the ADC gene and/or reduction in ADC expression. the criteria for inclusion (i.e., statistical or minimal expression val- Expression of two predicted ncRNAs was significantly altered in ues). In the pairwise analysis, the RPKM values of both ADC- the ADC RNAi treatment. These RNAs are transcribed from DNA injected or Mock-injected were mostly below the threshold of 1 but are not translated to proteins, hence noncoding. They are pro- (Table 1), but the data was still useful in making relevant biological posed to serve as epigenetic regulators and include miRNA, siRNA, predictions, as was the case for increased dopamine receptor 2 and piRNA, and lncRNA. ADC-dsRNA induced a 974-fold decrease of decreased mobility. Our data analysis and statistical significance ncRNA LOC107397781; this sequence is upstream of the gene may be improved with additional coverage. However, while we white (TC007047), an eye pigment gene. However, the expression understand the need to carefully examine RNA-Seq data, significant of white was not significantly changed (data not shown). The other information may be missed in situations where the data is less than differentially expressed ncRNA was LOC107398253, which is part optimal (i.e., low RPKM values) but with statistically significant, of the snoRNA U3 family. These ncRNAs are found in the nucleo- large changes in expression with adequate biological replicates. lus, and instead of functioning as an epigenetic regulator, are In summary, we propose that RNA-Seq is superior to qPCR to proposed to guide site-specific cleavage of rRNA during the pre- quantify RNAi knockdown, because differential expression of other processing of rRNA (Clery et al. 2007). The data suggest that ADC genes may provide new information on gene interconnectivity. Our RNAi not only reduced targeted transcript expression, but also may results demonstrated a connection between ADC and dopamine have affected the expression of other genes controlling the process- receptor 2 genes in T. castaneum with additional evidence via func- ing of RNA through changes in expression of ncRNA. tional tests, as well as other possible gene connections that remain Other genes identified in the study include four chemosensory- to be tested. related genes that were significantly decreased in the ADC-dsRNA treatment group, which also may indicate that expression of these Supplementary Data genes is affected by increased dopamine. Expression of Supplementary data are available at Journal of Insect Science online. LOC657178, odorant binding protein 18 (OBP18), was the most significantly reduced, 789-fold in ADC-injected larvae. OBP18 (also known as OBP4A) was highly expressed in the T. castaneum larval Acknowledgments head and mouthparts compared to the body (Dippel et al. 2014). Three chemosensory receptor genes belong to the 7tm receptor gene We would like to thank technicians Ken Friesen and Tom Morgan for their contribution, injecting larvae and RNA extraction and library preparation, class, LOC100142608, LOC107398092, and LOC107398565. respectively, Sierra Upton for video editing, and Dr Subbaratnam Engsontia et al. (2008) found that LOC100142608, also referred to Muthukrishnan for his comments on an earlier version of the manuscript. as OR139, is part of a T. castaneum-specific clade in an odorant Mention of trade names or commercial products in this publication is solely receptor gene tree consisting of sequences from Tribolium, for the purpose of providing specific information and does not imply recom- Drosophila, Apis, and Heliothis, and expression was isolated to the mendation or endorsement by the U.S. Department of Agriculture. USDA is adult and larval head. While less is known about the latter two 7tm an equal opportunity provider and employer. receptor genes, the data suggest that these chemoreceptors may have functional similarity, and that they may be responsive to dopamine levels in the head. References Cited ADC-dsRNA injection also decreased the expression of a gene Abdel-Latief, M., and H. K. Hoffmann. 2014. 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D., Singh, S., Wong 2009. Ingested double-stranded RNAs can and M. V. Sundaram. 2012. Extracellular leucine-rich repeat proteins are re- act as species-specific insecticides. Insect Biochem. and Mol. Biol. 39: quired to organize the apical extracellular matrix and maintain epithelial 824–832. junction integrity in C. elegans. Development 139: 979–990. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/57/3573844 by Ed 'DeepDyve' Gillespie user on 17 July 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Insect Science Oxford University Press

RNA-Seq Validation of RNAi Identifies Additional Gene Connectivity in Tribolium castaneum (Coleoptera: Tenebrionidae)

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Published by Oxford University Press on behalf of the Entomological Society of America 2017. This work is written by US Government employees and is in the public domain in the US.
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Abstract

RNA interference (RNAi) is a functional genomics tool to correlate genotype and phenotype by delivering tar- geted, gene-specific, and complementary dsRNA into a host via injection, feeding, or other means in order to re- duce gene expression. In the red flour beetle, Tribolium castaneum, RNAi has been successful via injected dsRNA at all life stages. Traditionally, successful transcript knockdown has been quantified by qPCR on a gene- by-gene basis, where only expression of the target gene and normalization genes are evaluated. In this study, RNA-Seq was used to quantify transcript expression in larvae injected with dsRNA for aspartate 1-decarboxyl- ase (ADC), which gives a reliable phenotype of an adult with a black cuticle instead of the wild-type red-brown. ANOVA of control, mock-injected, and ADC-dsRNA injected larvae indicated that target gene expression was significantly (P ¼ 0.002) reduced 4-fold, and the black phenotype was achieved in all adults injected with ADC- dsRNA as larvae. In a pairwise analysis, significant (P< 0.05) differential expression of other genes in ADC-in- jected larvae suggested connections between gene pathways. One gene, dopamine receptor 2, was increased in expression 227-fold (P ¼ 0.025), presumably connected to previous data that showed a reduction in expres- sion of ADC results in increased levels of dopamine. To evaluate the hypothesis that increased dopamine levels can affect mobility, T. castaneum adults injected with ADC-dsRNA as larvae were significantly impaired in movement tests compared to controls, similar to black mutants in Drosophila melanogaster. The data demon- strate that RNA-Seq can reveal gene connectivity and provide more complete data validation and analysis com- pared to qPCR. Key words: Tribolium castaneum, RNAi, ADC, RNA-Seq, gene expression RNA interference (RNAi) is a popular tool in functional genomics dsRNA fragments was sufficient to elicit a response at concentra- and, in pest insects like Tribolium castaneum, a potential strategy tions ranging from 0.0001 to 0.001 lg/ll(Miller et al. 2012). Other for molecular pest control (Baum et al. 2007, Aronstein et al. 2011, studies have demonstrated that injection can be successful in any T. Ulrich et al. 2015). RNAi is an evolutionarily conserved mechanism castaneum life stage (reviewed in Aronstein et al. 2011). that relies on natural cellular pathways to target and degrade double The most common downstream validation of knockdown after stranded RNA (dsRNA) of viruses, as described in nematodes (Fire RNAi is quantitative polymerase chain reaction (qPCR). This type et al. 1998). This natural process can be mimicked by the introduc- of analysis is dependent on gene-specific primers, including onerous tion of dsRNA into an organism (e.g. injection). The endoribonu- optimization that is often circumvented, and generally only used to clease, Dicer, cleaves dsRNA into 21–23 nucleotide short interfering measure the target gene and a normalizer gene as a standard. We RNAs (siRNAs), which are incorporated into an RNA-induced si- suggest this method limits the information gained from RNAi, and lencing complex (RISC) that targets and degrades RNA with com- that RNA transcriptome-sequencing and quantitation (RNA-Seq) is plementary sequence. more informative. RNA-Seq can monitor target gene knockdown RNAi has been used successfully to increase insect mortality or and off-target effects, and provides results similar to qPCR (Morris developmental abnormalities via microinjection or feeding in vitro et al. 2009, Chen et al. 2016). In our hands, RNA-Seq has been ben- or in planta (Baum et al. 2007, Huvenne and Smagghe 2010, eficial in cases where RNAi does not show a visible phenotype, and Aronstein et al. 2011). In T. castaneum, injection of 520 base pair attempts at validation by qPCR are ambiguous. For example, qPCR Published by Oxford University Press on behalf of the Entomological Society of America 2017. This work is written by US Government employees and is in the public domain in the US. 1 Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/57/3573844 by Ed 'DeepDyve' Gillespie user on 17 July 2018 2 Journal of Insect Science, 2017, Vol. 17, No. 2 0 0 validation of knockdown of a major gut cysteine peptidase gene in (2009), forward 5 -AAGGCGAAGGGAACATCAGG-3 and reverse 0 0 T. castaneum was inconclusive by qPCR, and there was no differ- 5 -TCCCCAACCGCTCAATCTC-3 . All secondary PCR primers ence in phenotype (unpublished data). However, using RNA-Seq, had a T7 construct attached to the 5 end (TAATACGACTCAC we demonstrated that knockdown was in fact successful, but closely TATAGGG). Another round of PCR was performed with each sec- related cysteine peptidase as well as serine peptidase genes, were ondary primer set and using the template obtained for each respec- compensating for the loss of target gene expression (Perkin et al. tive gene in the primary reaction as the template, using similar PCR 2017). Thus, sequence similarity and redundancy in gene function, conditions (100 ll reaction; Perkin et al. 2017). Secondary PCR as well as compensation responses embedded in the transcriptome products were evaluated via 1% agarose E-gel for appropriate response, can mask the effects of RNAi, making it difficult to verify length and sufficient amplification. with qPCR alone. The PCR product from the secondary amplification was used to To illustrate that RNA-Seq can be a superior method to validate make dsRNA via a MEGAscript T7 kit (Invitrogen, Life the results of RNAi, we report here the result of knockdown of as- Technologies, Carlsbad, CA), and was purified via a MEGAclear kit partate 1-decarboxylase (ADC)in T. castaneum larvae. ADC is one (ThermoFisher, Waltham, MA; Perkin et al. 2017). Size and quan- of two decarboxylases (the other is DDC, TC013480) needed for tity of products were verified on a TapeStation (Agilent, Santa proper insect cuticle tanning in T. castaneum (sclerotization and pig- Clara, CA) and quantity also was verified by a digital nanophotome- mentation). The process is complex and requires the conjugation ter (Implen, Westlake Village, CA). Negative controls included non- and cross-linking of cuticle proteins, leading to an insoluble, hard, injected larvae (Control) and mock-injected with water and dye and darkened red-brown exoskeleton (Roseland et al. 1987). ADC (Mock). catalyzes the synthesis of b-alanine, which plays a critical role in cu- ticle tanning because of its conjugation with dopamine to produce Micro-injected dsRNA N-b-alanyldopamine (NBAD), a substrate for the phenol oxidase There were three groups in this study: T. castaneum larvae injected laccase that catalyzes the synthesis of the cuticle protein cross- with water/dye (Mock); larvae injected with ADC-dsRNA construct linking agents and pigment precursors (Kramer et al. 1984). (ADC) mixed with dye; and a noninjected (Control) group. Knockdown of ADC leads to a black cuticle phenotype and the ac- Immediately before injection, treatments (water or dsRNA) were cumulation of dopamine (Arakane et al. 2009). mixed with blue dye (FD&C Blue 1, Kroger Food Colors, We demonstrate that RNAi targeting ADC, as verified by RNA- Cincinnati, OH; diluted 1:20) to aid in visualization of the injected Seq and black phenotype, changes the expression of other genes that liquid. Actively feeding, third instar larvae were briefly placed on ice may be interconnected. One affected gene, dopamine receptor 2, led and transferred to double-sided tape on a microscope slide placed us to test whether movement was impaired in T. castaneum adults on a small tissue culture flask ice block (Posnien et al. 2009). injected with ADC-dsRNA as larvae, uncovering a previously un- Briefly, a Drummond Nanoject II (Drummond Scientific Co., known interaction between ADC and genes controlling movement Broomall, PA) with a “bee-stinger” needle was set at 69 nl, and in T. castaneum. dsRNA was diluted to provide 200 ng of dsRNA per larva (Perkin et al. 2017) and loaded into the needle. Needles were made with 3.5 Drummond glass capillary tubes (3-000-203-G) and a micropipette Materials and Methods puller (Sutter Instrument Co. Model P-97, Novato, CA). After injec- tion, each group was allowed to recover for 2 h at room tempera- Insects ture, and then were covered with diet (95% wheat flour, 5% The T. castaneum lab strain was originally collected from a grain Brewer’s yeast) and kept at 28 C, 75% relative humidity, 0:24 bin in Kansas and has been reared at the Center for Grain and (L:D) h. All injections in a single replicate were done on the same Animal Health Research (CGAHR, Agricultural Research Service, day, with a total of three independent replicates per treatment. United States Department of Agriculture, Manhattan, KS) for over 20 yr. Insects are maintained on a diet of 95% wheat flour and 5% Brewer’s yeast at 28 C, 75% relative humidity, 0:24 (L:D) h. mRNA Extraction From each treatment group, eight T. castaneum larvae were ran- domly selected at 7-d postinjection, ground in liquid nitrogen with a Primers and dsRNA disposable pellet pestle, and total RNA was obtained using a Qiagen Primers were designed via Primer-BLAST (http://www.ncbi.nlm.nih. RNeasy Plus kit (Qiagen, Hilden, Germany). The Plus version of gov/tools/primer-blast/) to the ADC gene in T. castaneum using this kit utilizes a “gDNA Eliminator” spin column as a pretreatment default parameters. All primers were specific, and selected primer to further diminish any contamination of the RNA with DNA, and sets were unique to the ADC gene. The first round of PCR amplified 0 an optional step of on-column DNase digestion also was used. the entire gene using the primary primer sets: ADC forward 5 - 0 0 Quantity and quality at each step of mRNA collection were eval- AAGGCGAAGGGAACATCAGG-3 and reverse 5 -CTCCCC uated by TapeStation, and quantity was verified by a nanophotome- AACCGCTCAATCTC-3 . PCR reactions were 25 ll total volume ter (Implen, Westlake Village, CA). Aliquots were stored at 80 C. using genomic DNA template from the lab strain and standard ther- mal cycle conditions (Perkin et al. 2017). The product was assessed on a 1% agarose E-gel (ThermoFisher, Waltham, MA) to ensure the RNAseq amplified region was the correct size. A stranded mRNA-Seq Kit (KAPA Biosystems, Wilmington, MA) A second set of primers was designed to amplify within the first with appropriate Ion Torrent primers, adapters, and bar codes amplification region, and the products were used for dsRNA ampli- (Integrated DNA Technologies, Coralville, IA) was used for library fication. Primers were designed to target each sequence towards the preparation of 4 mg of total RNA from each biological replicate from 3 end, according to previous data that suggested this region was each treatment. A final concentration of adapter and barcodes of best for specific and maximal knockdown (Whyard et al. 2009, 100 nM was used without optimization of the concentration. Eight Perkin et al. 2017). ADC-dsRNA primers were as in Arakane et al. equimolar barcoded libraries from all treatment groups of a Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/57/3573844 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 3 biological replicate were pooled based on concentration estimates from a KAPA Library Quantification Kit for Ion Torrent platform (KK4827), and were placed in an Ion Chef (ThermoFisher, Grand Island, NY) for template preparation and loading onto an Ion PI Chip v2 for sequencing on the Ion Proton sequencer (Perkin et al. 2017). Data Analysis Transcripts were mapped to the Tcas5.2 genome build (NCBI) in SeqManNGen (DNAStar, Madison, WI), and differential gene expression was analyzed in ArrayStar (DNAStar version 14) using default parameters. Genes were normalized using Reads Per Kilobase per Million (RPKM; Mortazavi et al. 2008) and filtered to transcripts>8-fold change between groups in Student’s t-test com- parisons, and later were filtered to a 90% confidence interval after correction for multiple comparisons with False Discovery Rate (FDR) analysis (Benjamini and Hochberg 1995), with exceptions as noted. We also used gene expression data from different develop- mental stages (egg, larvae, pupae, and adult) of T. castaneum,as ADC Control Mock detailed in Perkin et al. (2016). Nonannotated differentially expressed genes were submitted to Treatments BLAST2GO PRO (Valencia, Spain) to elucidate possible function. From this analysis, additional functions were identified in Rfam Fig. 1. ADC expression (average total RPKM) differences between larvae injected with dsRNA targeting ADC (ADC), the noninjected control (Control), (Nawrocki et al. 2015). and the mock-injected (Mock) group. Error bars denote SE. Each replicate had>7 million reads with a total of>31 million reads per sample (Supp Table 1 [online only]). Sequences from this in larvae, and we used RNA-Seq to validate knockdown. ADC is a study were deposited into NCBI Sequence Read Archive gene commonly used as a positive RNAi phenotypic control. Most PRJNA302304. previous validation experiments for RNAi in T. castaneum have been by qPCR, but we chose RNA-Seq because we wanted to exam- Behavior Assays and Analysis ine other potential effects on reduced transcript levels of ADC. To assess movement and behavioral patterns in ADC-injected lar- vae, 25 late instar larvae were collected from ADC, Mock, and ANOVA of All Treatment Groups Control groups and placed into 1 oz solo cups with holes in the lid. All T. castaneum larvae injected with ADC-dsRNA in all replicates Media composed of 90% stabilized wheat germ and 10% flour was developed into adults with a black cuticle compared to the wild-type added. Each group was monitored daily, and pupae were collected red/brown cuticle, presumably due to manipulation of the pigmenta- and placed in individual wells of a 24-well plate and allowed to tion and sclerotization pathways (Arakane et al. 2009). In T. casta- emerge as adults. neum larvae injected with ADC-dsRNA, ADC expression was At 3- to 12-d posteclosion, individual beetles were placed in the reduced 4.25-fold compared to a water-injected control (Mock; center of a 90-mm diameter plastic petri dish using vacuum suction, Fig. 1). An ANOVA comparing Control, Mock and ADC treatments facing the same direction at the start of each assay. After the lid was indicated that ADC expression was significantly different between placed on the dish, individuals were allowed to acclimate for 10 s treatments (P ¼ 0.002; Table 1). Other genes were also differentially before a video recording (Sony Handycam HDR-XR520V) was expressed (>2-fold) in ADC-dsRNA injected larvae, many related to taken of each beetle. After 5 min, the recording was terminated and gene regulation. The most highly up-regulated gene (7.39-fold) enc- beetles were removed from the petri dish and discarded. There were odes a leucine-rich repeat-containing protein (LOC103314786), 18 individuals from each treatment group (ADC-injected and which is annotated in insects, mollusks, and chordates (Supp Fig. 2 Mock) and control that were recorded for movement behavior. [online only]). In UniProt, the gene is annotated in Caenorhabditis Videos were analyzed using Ethovision XT (Version 8.0, Noldus elegans and Trichinella spp. (parasitic roundworms), but most func- Information Technology, Wageningen, The Netherlands). Video tional data is from C. elegans where it was expressed in multiple tis- tracks were analyzed for Distance Moved (cm); Mobile, Immobile, sues in embryo through larval development, and was important in or Highly Mobile states (defined as the changes in body position maintaining apical extracellular matrix integrity (Mancuso et al. between frames at 10 frames per second, where 0%, 20%, and 60% 2012, Finn et al. 2016). However, the relationship of this gene to of the body changes position) in sec; and velocity (distance traveled ADC is not apparent. Additionally, a gene encoding a hypothetical per time between frames, measured as the mean in cm/s). Distance, noncoding RNA (LOC107397781) was dramatically down- Velocity, and the three mobility states were analyzed using individ- regulated (974-fold). ual PROC GLM statements in SAS (SAS Institute, Cary NC, version 9.4), where strain was the main effect in the model and pairwise dif- ferences were Tukey HSD adjusted. Pairwise Analysis of ADC-injected Versus Mock-injected We compared gene expression of ADC-injected T. castaneum larvae to the mock-injected control (Table 2 and Supp Fig. 1 [online only]). Results The expression of dopamine receptor 2 (LOC661535) was increased In this study, we reduced the expression of the T. castaneum aspar- 227-fold in ADC compared to Mock (P ¼ 0.025), but the expression tate 1-decarboxylase (ADC, LOC100124592, TC034596) via RNAi of dopamine receptor 1 was unchanged (data not shown). Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/57/3573844 by Ed 'DeepDyve' Gillespie user on 17 July 2018 ADC larval expression (RPKM) 4 Journal of Insect Science, 2017, Vol. 17, No. 2 Table 1. ANOVA of control, mock-injected, and ADC-dsRNA injected T. castaneum larvae, filtered to linear RPKM> 1 in at least one of the groups (values to right for each group),>2-fold change in Mock versus ADC, and P< 0.05 (FDR Benjamini and Hochberg ) Name Description Fold change P value ADC RPKM Control RPKM Mock RPKM LOC103314786 Leucine-rich repeat-containing protein egg-6-like 7.385 0.044600 7.067 0.802 0.957 LOC107397729 Pfam : Retrotransposon gag protein, Reverse transcriptase 6.253 0.014700 2.695 0.559 0.431 LOC103315154 Leucine-rich repeat-containing protein let-4-like 3.267 0.035500 4.513 1.958 1.381 LOC658512 Carboxylesterase 1E 2.383 0.001280 52.77 22.45 22.15 LOC656320 Trypsin I-P1 isoform X1 2.180 0.000184 47.91 25.74 21.97 LOC103314471 Ribulose-phosphate 3-epimerase 2.424 0.012800 33.22 66.39 80.52 LOC664050 Serine/threonine-protein kinase RIO3 2.771 0.001390 9.336 25.40 25.88 LOC107398585 CPDF : protein kinase 3.815 0.031300 0.380 0.696 1.449 LOC100124592 Aspartate 1-decarboxylase 4.245 0.001800 0.563 2.123 2.390 LOC107397781 pfam: ncRNA 974.2 0.000001 0.001 0.001 1.106 Target gene (ADC) is shaded. Benjamini and Hochberg, 1995. Fold change in ADC-injected larvae. pfam: protein families database (Finn et al. 2016). CPDF: Conserved Protein Domain Family, NCBI. Table 2. Significant differentially expressed genes in a pairwise comparison of ADC-dsRNA injected T. castaneum larvae to mock-injected larvae (> 8-fold change,>90% confidence interval, Student’s t-test P values with Benjamini and Hochberg FDR correction, with linear total RPKM values on the right) b b Name Description Fold change P value Mock RPKM ADC RPKM LOC107398333 Uncharacterized 245.2 0.0290 0.001 0.278 LOC661535 Dopamine receptor 2 227.0 0.0250 0.001 0.258 LOC659605 Pfam : Protein of unknown function (DUF229), alkaline phosphatase 141.4 0.0044 0.001 0.161 LOC103313283 Pfam: Domain of unknown function (DUF4550) 66.93 0.0338 0.076 0.001 LOC107398197 Uncharacterized 81.96 0.0301 0.093 0.001 LOC657226 Glutamate dehydrogenase, mitochondrial-like 90.06 0.0290 0.112 0.001 LOC107398565 Odorant receptor 85a-like 136.7 0.0250 0.155 0.001 LOC107398092 CPDF : 7tm chemosensory receptor 146.3 0.0250 0.166 0.001 LOC107398783 Uncharacterized 176.0 0.0250 0.200 0.001 LOC663271 Pfam: haemolymph juvenile hormone binding protein (JHBP) 257.7 0.0003 0.293 0.001 LOC100142608 Pfam: 7tm odorant receptor (OR139) 240.6 0.0145 0.273 0.001 LOC107398253 Pfam: (ncRNA/snoRNA U3) 593.3 0.0081 0.674 0.001 LOC657178 odorant binding protein 18 (OBP18/OBP4A) 789.3 0.0250 0.896 0.001 LOC107397781 Pfam: ncRNA 974.2 0.0244 1.106 0.001 LOC100216358 Allatotropin I preprohormone isoform X1 1,411 0.0011 1.603 0.001 Benjamini and Hochberg, 1995. Fold change in ADC-injected larvae. Pfam: protein families database (Finn et al. 2016). CPDF: Conserved Protein Domain Family, (Marchler-Bauer et al. 2015). The expression of two other genes also increased, an uncharacter- Movement Assays ized gene (245-fold; P ¼ 0.029) and a protein with unknown func- The black cuticle phenotype of beetles injected with ADC-dsRNA tion with an alkaline phosphatase motif (141-fold; P ¼ 0.004). has been demonstrated to associated with an accumulation of dopa- Injection of ADC-dsRNA also significantly decreased the expres- mine (Arakane et al. 2009). However, this is the first demonstration sion of 12 additional genes compared to the mock-injected control that a reduction in ADC leads to an increase in expression of a dop- (P< 0.03; Table 2). The most severely repressed gene was amine receptor gene. Previous work in D. melanogaster indicated LOC100216358, a gene encoding allatotropin I preprohormone, that body color mutants often have neurological phenotypes that was decreased 1,411-fold (P ¼ 0.001). Several chemosensory (Wittkopp et al. 2003), including black mutants that show reduced genes (LOC107398565, LOC107398092, LOC100142608, activity at the larval/pupal boundary or “wandering stage” (Phillips LOC657178), three of which have a conserved odorant receptor et al. 2005). Therefore, we conducted a movement behavior assay domain, were significantly decreased 137- to 240-fold (P< 0.03). on T. castaneum adults injected with ADC-dsRNA as larvae to eval- There were two other uncharacterized genes (LOC107397781 and uate if increased expression of dopamine receptor 2 results in a simi- LOC107398253) that decreased 974- and 593-fold, respectively, lar neurological phenotype. and both had motifs similar to ncRNAs. Further analysis with Rfam Tribolium castaneum larvae injected with dsRNA targeting found the latter is part of the small nucleolar RNA (snoRNA) U3 ADC were significantly slower and moved less often as adults than family. This family is predicted to guide site-specific cleavage of both Mock beetles and Control beetles (Table 3 and Supp Video 1 ribosomal RNA (rRNA) during pre-rRNA processing (Clery et al. [online only]). Slowed movement was demonstrated by a reduction 2007). in mean velocity (cm/s) and total distance traveled in the Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/57/3573844 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 5 Table 3. Statistical data of movement patterns of T. castaneum adults injected as larvae with ADC-dsRNA compared to Control and Mock Trait Type III SS Mean square F value Pr> F Tukey’s HSD P value Distance moved 8,042 4,020 6.68 0.003 0.018 Velocity 0.089 0.045 6.68 0.003 0.018 High mobility 2.580 1.290 1.74 0.190 0.210 Immobile 609.4 304.7 5.44 0.007 0.011 Mobile 532.7 266.3 5.47 0.007 0.011 Values are ANOVA for each movement metric; source of ANOVA variation is from the main effect of strain (DF ¼ 2 for all comparisons). Distance moved is the total distance moved in cm for a 5-min assay; Velocity is the mean velocity in cm/s as measured by distance moved between frames; High Mobility refersto when 60% or more of the body changes location between frames, at 10 frames per second; Immobile means 0% of the body has moved between frames, whereas Mobile means 20% of the body is changing per frame. The far right column includes the Tukey HSD adjusted P values for the ADC/Mock comparison. Fig. 2. Box plots showing the distance moved (A) and velocity (B) of ADC-, Control-, and Mock-injected beetles. Solid black lines indicate the median distance and velocity. The vertical edges of the boxes represent 25% and 75% of the data distribution (Interquartile Range or IQR) with whiskers representing 1.5  (IQR), a measure of the maximum and minimum values in the dataset. ADC-injected beetles compared to Mock and Control (Fig. 2). Over us to evaluate possible behavioral differences. Dopamine is a neuro- the 5-min period of uninhibited movement, ADC-injected beetles transmitter important for a variety of life functions in vertebrates traveled on an average 23.11 cm less than Mock beetles (P ¼ 0.018) and invertebrates, and many human disorders have been attributed and had an average velocity 0.076 cm/s slower than Mock beetles to disruptions in dopamine pathways (reviewed in Beaulieu and (P ¼ 0.018). Overall, neither ADC-injected nor mock beetles had Gainetdinov 2011). Dopamine receptors are G protein-coupled long bouts of highly mobile activity (where 60% of body position receptors separated into D1 and D2 classes. In vertebrates, D1-class changes per frame) and the durations were not significantly different dopamine receptors include D1 and D5, and they stimulate cyclic- (ADC-injected ¼ 0.32 s; Mock ¼ 0.82 s; P ¼ 0.21). However, ADC- AMP production by adenylate cyclase, whereas the D2-class recep- injected beetles spent significantly more time in an immobile state tors include D2, D3, and D4, and they inhibit adenylate cyclase and (where 0% of body changes position per frame at 10 frames per sec- thus lead to a decrease in cyclic AMP (reviewed in Civelli et al. ond). ADC-injected beetles spent 297.4 s in an immobile state, while 1993). In humans, the D2 receptor is associated with different struc- Mock spent 289.9 s immobile (P ¼ 0.011; Table 3). tures in the brain, including olfaction. Insects have orthologs of dop- amine receptors 1 and 2, and there are genes encoding dopamine receptors 1 and 2 in T. castaneum (Hauser et al. 2008, Tribolium Genome Sequencing Consortium 2008). Our data suggests that Discussion expression of ADC and dopamine receptor 2 are diametrically Our approach demonstrates that RNA-Seq documents target knock- opposed in T. castaneum. down by RNAi of ADC in T. castaneum through normalized gene To understand how dopamine and cuticle pigmentation path- expression data, even in small differences of expression. All ADC- ways may interact, we turned to the abundant literature on D. mela- injected larvae resulted in the expected phenotype, adults with a nogaster pigmentation. D. melanogaster pigmentation is more black cuticle and a knockdown of 4.25-fold decrease. The black complicated than T. castaneum due to multiple color patterns and cuticle phenotype was the result of decreased ADC leading to an sexual dimorphism. However, most D. melanogaster pigment genes accumulation of dopamine upstream in the cuticle-tanning pathway have orthologs in T. castaneum. For example, the D. melanogaster in epidermal cells, which may be the predominant site of expression black gene is orthologous to the T. castaneum ADC gene, both genes of this gene (Arakane et al. 2009). encoding aspartate 1-decarboxylase, and contributing to the black RNA-Seq as an RNAi validation method also revealed differen- phenotype in mutants for this gene. Many studies have shown that pig- ces in expression of genes not previously associated with the target mentation gene mutants in D. melanogaster,suchas black, ebony,and gene, ADC. Most significant was the increased expression of dopa- tan not only have body color phenotypes, but also neurological pheno- mine receptor 2 (LOC661535) in the ADC RNAi treatment that led types (Wittkopp et al. 2003). For example, D. melanogaster black Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/57/3573844 by Ed 'DeepDyve' Gillespie user on 17 July 2018 6 Journal of Insect Science, 2017, Vol. 17, No. 2 mutants have reduced activity at the larval/pupal boundary or We propose that, based on our data, a decrease in ADC results in “wandering stage”. In the adult stage, black null mutants had sig- the accumulation of dopamine and an increased expression of dopa- nificantly reduced number of initiated walks compared to wild- mine receptor 2, and consequently decreased adenylate cyclase and type (Phillips et al. 2005). In T. castaneum black mutants, the loss production of cAMP. As both dopamine receptor 2 and allatotropin I of ADC was attributed to lower transcript levels (Arakane et al. are expressed in the T. castaneum brain, we speculate that decreased 2009), similar to what we achieved through RNAi of wild-type expression of allatotropin I was due to decreased cAMP. While it is insects. We examined the movement patterns of ADC-dsRNA- known that allatotropin I expression results in increased cAMP in T. injected T. castaneum and T. castaneum mutants with black phe- castaneum (Vuerinckx et al. 2011), our data suggests that expression notype (data not shown) and observed reduction in distance of allatotropin I may also be sensitive to decreased cAMP levels in a moved, overall velocity, and mobility compared to Mock and feedback loop mechanism; this hypothesis remains to be tested. Control. Thus, T. castaneum adults with a disrupted ADC gene Finally, we note that in the examination of RNA-Seq data, we function similarly to D. melanogaster black mutants, suggesting began our analyses with all differentially expressed genes, to exam- that T. castaneum mutants with black phenotype may also have a ine those that may be functionally related even if genes do not meet mutation in the ADC gene and/or reduction in ADC expression. the criteria for inclusion (i.e., statistical or minimal expression val- Expression of two predicted ncRNAs was significantly altered in ues). In the pairwise analysis, the RPKM values of both ADC- the ADC RNAi treatment. These RNAs are transcribed from DNA injected or Mock-injected were mostly below the threshold of 1 but are not translated to proteins, hence noncoding. They are pro- (Table 1), but the data was still useful in making relevant biological posed to serve as epigenetic regulators and include miRNA, siRNA, predictions, as was the case for increased dopamine receptor 2 and piRNA, and lncRNA. ADC-dsRNA induced a 974-fold decrease of decreased mobility. Our data analysis and statistical significance ncRNA LOC107397781; this sequence is upstream of the gene may be improved with additional coverage. However, while we white (TC007047), an eye pigment gene. However, the expression understand the need to carefully examine RNA-Seq data, significant of white was not significantly changed (data not shown). The other information may be missed in situations where the data is less than differentially expressed ncRNA was LOC107398253, which is part optimal (i.e., low RPKM values) but with statistically significant, of the snoRNA U3 family. These ncRNAs are found in the nucleo- large changes in expression with adequate biological replicates. lus, and instead of functioning as an epigenetic regulator, are In summary, we propose that RNA-Seq is superior to qPCR to proposed to guide site-specific cleavage of rRNA during the pre- quantify RNAi knockdown, because differential expression of other processing of rRNA (Clery et al. 2007). The data suggest that ADC genes may provide new information on gene interconnectivity. Our RNAi not only reduced targeted transcript expression, but also may results demonstrated a connection between ADC and dopamine have affected the expression of other genes controlling the process- receptor 2 genes in T. castaneum with additional evidence via func- ing of RNA through changes in expression of ncRNA. tional tests, as well as other possible gene connections that remain Other genes identified in the study include four chemosensory- to be tested. related genes that were significantly decreased in the ADC-dsRNA treatment group, which also may indicate that expression of these Supplementary Data genes is affected by increased dopamine. Expression of Supplementary data are available at Journal of Insect Science online. LOC657178, odorant binding protein 18 (OBP18), was the most significantly reduced, 789-fold in ADC-injected larvae. OBP18 (also known as OBP4A) was highly expressed in the T. castaneum larval Acknowledgments head and mouthparts compared to the body (Dippel et al. 2014). Three chemosensory receptor genes belong to the 7tm receptor gene We would like to thank technicians Ken Friesen and Tom Morgan for their contribution, injecting larvae and RNA extraction and library preparation, class, LOC100142608, LOC107398092, and LOC107398565. respectively, Sierra Upton for video editing, and Dr Subbaratnam Engsontia et al. (2008) found that LOC100142608, also referred to Muthukrishnan for his comments on an earlier version of the manuscript. as OR139, is part of a T. castaneum-specific clade in an odorant Mention of trade names or commercial products in this publication is solely receptor gene tree consisting of sequences from Tribolium, for the purpose of providing specific information and does not imply recom- Drosophila, Apis, and Heliothis, and expression was isolated to the mendation or endorsement by the U.S. Department of Agriculture. USDA is adult and larval head. While less is known about the latter two 7tm an equal opportunity provider and employer. receptor genes, the data suggest that these chemoreceptors may have functional similarity, and that they may be responsive to dopamine levels in the head. References Cited ADC-dsRNA injection also decreased the expression of a gene Abdel-Latief, M., and H. K. Hoffmann. 2014. 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