MOLECULAR BIOLOGY/GENOMICS Microarray Analysis of Female- and Larval-Specific Gene Expression in the Horn Fly (Diptera: Muscidae) FELIX. D. GUERRERO,1,2 SCOT. E. DOWD,3 YAN SUN,3 LEONEL SALDIVAR,1 GRAHAM B. WILEY,4 SIMONE L. MACMIL,4 FARES NAJAR,4 4 5 BRUCE A. ROE, AND LANE D. FOIL J. Med. Entomol. 46(2): 257Ð270 (2009) ABSTRACT The horn ßy, Haematobia irritans L., is an obligate blood-feeding parasite of cattle, and control of this pest is a continuing problem because the ßy is becoming resistant to pesticides. Dominant conditional lethal gene systems are being studied as population control technologies against agricultural pests. One of the components of these systems is a female-speciÞc gene promoter that drives expression of a lethality-inducing gene. To identify candidate genes to supply this promoter, microarrays were designed from a horn ßy expressed sequence tag (EST) database and probed to identify female-speciÞc and larval-speciÞc gene expression. Analysis of dye swap experiments found 432 and 417 transcripts whose expression levels were higher or lower in adult female ßies, respectively, compared with adult male ßies. Additionally, 419 and 871 transcripts were identiÞed whose expression levels were higher or lower in Þrst-instar larvae compared with adult ßies, respectively. Three transcripts were expressed more highly in adult females ßies compared with adult males and also higher in the Þrst-instar larval lifestage compared with adult ßies. One of these transcripts, a putative nanos ortholog, has a high female-to-male expression ratio, a moderate expression level in Þrst-instar larvae, and has been well characterized in Drosophila. melanogaster (Meigen). In conclusion, we used microarray technology, veriÞed by reverse transcriptase-polymerase chain reaction and massively parallel pyrosequencing, to study life stageÐ and sex-speciÞc gene expression in the horn ßy and identiÞed three gene candidates for detailed evaluation as a gene promoter source for the development of a female-speciÞc conditional lethality system. KEY WORDS Haematobia irritans, microarrays, gene expression analysis, pyrosequencing The horn ßy, Haematobia irritans L., is an obligate and Brazil, respectively (Kunz et al. 1991, Grisi et al. blood-feeding parasite of cattle, and control of this 2002). The primary means of controlling the horn ßy pest is a continuing problem for cattle producers in the is through the application of insecticides, primarily United States and other parts of the world. Feeding from the pyrethroid and organophosphate classes. several times per day, infestations of several thousand However, populations of horn ßies that exhibit sig- ßies per animal have been reported (Bruce 1964). niÞcant resistance to these pesticides are becoming Severe infestations interfere with normal feeding ac- common (Kunz and Schmidt 1985, Kunz et al. 1995, tivity, and economic losses to producers result from Guerrero and Barros 2006), and novel control meth- the reduced weight gain on cattle being prepared for odologies would be an important contribution to the market during the ßy season. Total economic losses cattle industry. attributable to the horn ßy have been estimated at An interesting approach to insect population con- $876 and $150 million annually in the United States trol using a dominant, repressible lethal gene system in Drosophila melanogaster (Meigen)was reported by Thomas et al. (2000). This system requires a sex-spe- Mention of trade names or commercial products in this publication is solely for the purpose of providing speciÞc information and does not ciÞc gene promoter to drive the expression of a re- imply recommendation or endorsement by the U.S. Department of pressible transcription factor. The transcription factor Agriculture. controls the expression of a toxic gene product. This 1 USDAÐARS Knipling-Bushland U.S. Livestock Insects Research control approach and variants on the general theme Laboratory, 2700 Fredericksburg Rd., Kerrville, TX 78028. 2 Corresponding author, e-mail: [email protected]. are also being studied in the agricultural pests Ceratitis 3 USDAÐARS Livestock Issues Research Unit, 1604 E. FM 1294, capitata (Wiedemann) (Fu et al. 2007) and Lucilia Lubbock, TX 79403. cuprina (Wiedemann) (Scott et al. 2004). With the 4 Department of Chemistry and Biochemistry, University of Okla- problems insecticide-resistant horn ßy populations homa, 620 Parrington Oval, Norman, OK 73019. 5 Louisiana State University, Department of Entomology, 404 Life pose to current control programs, we initiated a Sciences Building, Baton Rouge, LA 70803. project aimed at designing a dominant, repressible 258 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 46, no. 2 female-speciÞc lethal gene system tailored for the (NanoDrop Technologies, Wilmington, DE), and horn ßy. Rather than use genetic components from the quality was conÞrmed by electrophoresis. D. melanogaster laboratory strainÐderived system Microarray Design. Using an expressed sequence (Thomas et al. 2000), we sought to identify speciÞc tag (EST) collection described previously (Guerrero orthologues and gene promoters from a natural pop- et al. 2008), a unigene set for horn ßy was generated ulation of the horn ßy. Initially, it was necessary to by assembly of the ESTs at 96% similarity. This unigene acquire an adequate database of expressed gene se- set was used for BlastX search against SwissProt/ quences, because there were very few GenBank en- Tremble to identify those genes that were in forward tries for the horn ßy (Guerrero et al. 2008). A critical or reverse mRNA orientation based on the largest component of this system is the sex-speciÞc gene pro- ORF with e-value top hits Ͻ1 ϫ 10Ϫ112. Those unigene moter that must drive the expression of the lethality- set members with adequate information to determine inducing gene product. An obvious choice for a pro- the mRNA orientation were included into the array moter source would be the yolk protein genes, of design after putting the sequencing into mRNA sense which there are at least three in Musca domestica L. orientation. Unigene sequences without adequate in- (White and Bownes 1997). Sequences with signiÞcant formation were included in the array design in both BlastX similarity to yolk proteins 2 and 3 have been orientations. A total of 6,505 horn ßy tentative con- reported from the horn ßy (Guerrero et al. 2004). sensus (hfTC) sequences were entered into a custom However, at least one of the M. domestica yolk protein script and used to design 60-mer oligonucleotide genes, yp3, has detectable expression in male ßies probes with two probes sought for each hfTC. A total (White and Bownes 1997). Thus, microarrays were of 12,806 probes were identiÞed. These probes were designed from the recently reported horn ßy se- uploaded to E-array 4.5 in an 8 by 15K high-density quence database (Guerrero et al. 2008) and probed to format (Agilent Technologies, Santa Clara, CA). A characterize female-speciÞc gene expression and total of 536 control genes (including positive and neg- identify several candidate genes that could supply ative control probes) were also included in the design promoters with tight female-speciÞc expression. An of the array, and the Agilent two color spike-in kit was additional desirable trait we sought in the gene pro- used for technical normalizations. moter activity proÞle for the repressible female-spe- Microarray Protocol. For each sample, 10 ␮g of total ciÞc lethal gene system was expression at an early RNA and a 50-fold dilution of the Agilent two color stage in the life cycle. Ideally, lethality would be in- spike-in control RNA kit (Agilent Technologies) were duced in the embryonic or early larval stage to prevent labeled with either CyDye3-dCTP or CyDye5-dCTP devoting resources to mass rear individuals destined to (Amersham Biosciences, Piscataway, NJ) using the become females only to be killed later in the process. LabelStar kit (Qiagen, Valencia, CA) and oligo-dT and To identify early gene expression, the microarrays random nonamers (Sigma-Aldrich, St. Louis, MO). In were probed with labeled cDNA from Þrst-instar horn short, RNA from female horn ßies was labeled with ßy larvae and larval expression data compared with CyDye3-dCTP and hybridized against CyDye5- that from labeled cDNA from adult ßies. dCTPÐlabeled RNA from male horn ßies. As a second replicate to control for gene-speciÞc dye bias, the female RNA was labeled with CyDye5-dCTP and hy- bridized against CyDye3-dCTPÐlabeled RNA from Materials and Methods male ßies. Similarly, RNA from Þrst-instar larvae was Insect Material. Adult ßies were collected on a sin- labeled and hybridized against a labeled 50:50 mix of gle collection date from pastured cattle at the Loui- RNA from adult male and female ßies using the dye siana State University Agricultural Center, St. Gabriel swap design. All labeling, hybridization, and washing Research Station (St. Gabriel, LA) by aerial nets and procedures were performed according to the respec- transferred to Erlenmeyer ßasks at 30ЊC and total tive manufacturersÕ protocols. Labeled cDNA was hy- darkness for 1.5 h to facilitate egg collection (Lysyk bridized to the Agilent microarrays using the Gene 1991). Collected eggs were divided into two samples, Expression Hybridization kit (Agilent Technologies) and adult ßies were frozen at Ϫ80ЊC and sexed while following the manufacturerÕs protocols. Arrays were on dry ice. One egg sample was frozen at Ϫ80ЊC, and washed with Gene Expression Wash Buffer kit (Agi- the second egg sample was transferred to moist Þlter lent Technologies). A total of four arrays (two inde- paper in petri plates and maintained at room temper- pendent RNA extractions per ßy tissue type and one ature to induce egg hatch. The following day, the dye swap per extraction) were used in the male to wandering Þrst-instar larvae were collected and fro- female comparison, and four arrays in the larval to zen at Ϫ80ЊC. adult comparison were used to obtain genes that were RNA Extraction.