Transcriptome analysis in Ceratitis capitata to unveil genes involved in ageing-maturation process

The sterile insect technique (SIT) is widely used in integrated programmes against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Information on the age distribution of insects, and more particularly, the knowledge of wild female reproductive status (mature or not) at the time of the sterile male release is one of the key factors for the success of the SIT. In recent years, sequencing analysis has become an important tool in molecular biology. In this work we present a genome-wide expression analysis based on SSH (substractive sequence hybridization) and EST (expressed sequence tag) sequencing and macroarray expression analysis to identify signature genes related to the ageing-maturing process in C. capitata, leading to the successful identification of new putative candidate genes of reproductive status in medfly that would serve as molecular markers for ageing. We have sorted out 94 unigenes from 873 single-pass ESTs, of which 57% have homology with known genes. Ageing-maturing process in C. capitata presents a marked expression pattern accompanied by the increase of transcription level of genes involved in reproduction (vitellogenins, chorion proteins and male-specific serum proteins). Other identified cDNAs (43%) with a differential expression pattern would be also candidates but deserve further studies, as they belong to the unknown function class. Additional key words: ageing; expression profile; genome analysis; medfly; macroarray; sexual maturation; SSH. * Corresponding author: bsabater@ivia.es; b.sabater.munyoz@gmail.com Received: 16-01-13. Accepted: 16-07-13. Abbreviations used; EST (expressed sequence tag); GO (gene ontology); RACE RT-PCR (rapid amplification of cDNA ends by reverse transcription-polymerase chain reaction); SCSIE (Servicio Central de Soporte a la Investigación Experimental); SIT (sterile insect technique); SSH (substractive sequence hybridization); Vg (vitelogenin). Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Spanish Journal of Agricultural Research 2013 11(3): 842-854 Available online at www.inia.es/sjar ISSN: 1695-971-X http://dx.doi.org/10.5424/sjar/2013113-3987 eISSN: 2171-9292


Introduction
The Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), is a highly polyphagous insect pest (Liquido et al., 1991;White & Elson-Harris, 1992).Its invasive behaviour has led to its consideration as a major quarantine pest in many countries (Malacrida et al., 2007) and has led to the development of extensive control programmes.In Spain, current medfly control management is mainly based on the use of insecticides; nevertheless the demand for insecticide-free fresh fruit is encouraging the use of environment-friendly methods.The sterile insect technique (SIT) has proven to be an effective and environmentally friendly control method against medfly (which has become a model organism in this technique) and has exponentially grown world-wide in the last years (Hendrichs et al., 2005).As such, an area-wide suppression programme was initiated in 2006 in Spanish Mediterranean citrus areas (San Andrés et al., 2007).The success of a SIT programme depends greatly on the lifespan of mature males released and on the successful mating with wild females.The knowledge of wild female reproductive status (immature, gravid or old) at the time of the sterile male release and the establishment of optimal sexual maturity in sterile released males are key factors for the success of the SIT.The functional maturation of gonads and the onset of mature sexual behaviour are a result of an orderly and timely transition of genome expression through developmental stages.This genome expression orchestration is mainly unknown in the medfly but some biochemical pathways are known in diverse insect species, on which corpus allatum and juvenile hormones play a central role (Moshitzky et al., 2003 and references herein).
Ageing is a process in which the progressive loss of function in multiple tissues (including a reduction in reproduction capacity) results in the increasing probability of death (Kirkwood & Austad, 2000), thus ageing and reproductive status are linked processes.However, ageing is one of the most complex biological processes being determined by both, genetic and environmental factors.In C. capitata sex-specific life span patterns have been described as resulting from an underlying "constitutional" longevity minus the deleterious effects of reproductive biology (i.e.progeny production and effects of reproductive hormones), and sex-related behaviour (i.e.seeking and competing for mates, or territorial defence) (Novoseltsev et al., 2005;Carey et al., 2006Carey et al., , 2008)).Despite the central importance of insect population age structure, relatively few methods have been developed to measure insect age in the field.Both physiological or chronological age could be estimated by the identification of ovarian development in females (Tyndale-Biscoe, 1984), the progressive layering of hydrocarbons in insect cuticles (Desena et al., 1999;Gerade et al., 2004), the use of pteridines (Camin et al., 1991;Mullens & Lehane, 1995;Penilla et al., 2002) or the use of behavioural trait biomarkers (i.e.supine behaviour) (Papadopoulos et al., 2002).More recently, the transcriptional profiling method of ageing, based on genes that display age-dependent expression, have been applied in mosquito (Cook et al., 2006(Cook et al., , 2007)).
Single gene mutations that prolong the longevity are most useful to gain insights into the molecular mechanisms of ageing process and longevity determination with its link with reproductive status.Genetic analyses of yeast, nematode, fly, and mouse have uncovered a number of genes (i.e.superoxide dismutase, methuselah, daf, hsp70) whether mutated or misexpressed, increase the lifespan of these short-lived model organisms and support the general idea of free-radical hypothesis of ageing (Kim, 2008).
Genome-wide expression analysis has emerged as a powerful tool that can be used for the identification of signature genes that behave similarly under particular conditions and time points or under particular conditions, and which can be used in very different ways to analyze large-scale gene expression across species involved in ageing (McCarroll et al., 2004).In a fashion similar to microarrays, the macroarrays have been used to perform this genome-wide expression analysis (Alberola et al., 2004).This analysis requires the knowledge of the target genome or the use of model organisms.If the target organism is not a model one (its genome is probably unknown), or the number of identified genes is low (as in ageing), the expression analysis would become hard to perform.One way to solve this problem is the use of suppressive substractive hybridization technique (SSH) (Diatchenko et al., 1996).The basis of this technique is the comparison of two expression conditions with the objective of identifying differentially expressed genes.Furthermore, SSH can identify rare transcripts (low-abundance differentially expressed cDNAs) and is a highly effective method for generating subtracted cDNA libraries.
We report here on a genome-wide expression analysis -from newly emerged, mature and old non virgin medfly adults-to unveil the medfly genes involved in ageing-maturity by SSH and macroarray hybridization.We used target genes previously described as being related to ageing as well as the new candidate genes obtained by SSH.The final aim is the identification of new putative candidate genes for longevity and/or for determination of reproductive status in medfly.

Material and methods
Unless otherwise indicated, all molecular techniques and solutions were performed as described by Sambrook et al. (1989).

Medfly time point samples
Adults of C. capitata were collected from a laboratory colony maintained at the IVIA (Valencia, Spain) since 2002.In order to obtain a cohort of adults, approximately 2,000 pupae were separated in a Perspex cage (20 cm × 20 cm × 20 cm) and adults were allowed to emerge.To obtain a cohort of adults (< 24h), newly emerged adults were separated and all unhatched pupae were removed and discarded.After emergence, flies had ad libitum access to a diet consisting of a mixture of sugar and hydrolyzed yeast (Biokar Diagnostics Co., Pantin, France) (4:1; w:w) and water.

Shorting age related genes in medfly
According to the average lifespan of this strain (17 days, unpublished data) and their biological status, the individuals are grouped in three categories: 1) immature adults (< 12 h-old), 2) mature (5 days-old adults), and 3) old (15 days-old adults).Once newly emerged adults were separated, both males and females were kept together in a single ventilated perspex cage (20 cm × 20 cm × 20 cm).For each of the three groups, 20 females and 20 males were fast-frozen and maintained at -80°C until used.Three biological replicates of each biological status course were performed separately in time for the macroarray assay.

Library screening
Each library was plated on selective LB agar plates and white colonies were transferred to v-well plates containing 150 μL of liquid TB-glycerol medium with 50 μg mL -1 ampiciline.Cultures were set at 37°C, overnight without agitation.Plates were subjected to colony PCR (Sabater-Muñoz et al., 2006).PCR products were purified with Sephadex G-50 superfine (GE-Amersham Healthcare, Chalfont St. Giles, UK) and verified by gel electrophoresis on a 2% agarose gel (Pronadisa).Cultured plates were stored at -80°C till used.
Size selected PCR products were directly sequenced at the Servicio Central de Soporte a la Investigación Experimental (SCSIE) from University of Valencia, using Bigdye ® v3.1 chemistry (Applied Biosystems, Foster City, CA, USA) with primer T7 in 1/16 of the recommended reaction volume (single pass sequence reading).

Unigene set establishment
Electropherograms were checked and assembled into contigs by using Staden package software 1.6.0version (Staden et al., 2000) for each subtracted library.Each consensus sequence was compared to GenBank by blast using tblastx as implemented in NCBI web page to assign gene function (Altschul et al., 1997).Sequences were also compared to those EST (expressed sequence tags) available of C. capitata in dbEST (GenBank: FG068301−FG089553; Gomulski et al., 2012).Gene ontology (GO) was established by assignment of each identified gene function into GO browser.The sequences reported in this work have been deposited into GenBank (dbEST) at NCBI under accession number TSA117765.
All identified gene, gene product or putative ORF (open reading frame) were compared to all other by aligning sequences two by two with bl2seq algorithm at NCBI web page.Consensus sequences from same or different substracted libraries that coded for the same gene were aligned to the D. melanogaster ortholog or to the corresponding gene from medfly.
After these comparisons, a medfly unigene set was established and cultured again in v-well plates as des-  cribed.This new gene collection was used as source for probes for the expression assay.

Macroarray printing, hybridization and data analysis
Additional probes were generated by RT-PCR with specific primers from some of the characterized genes from medfly which are involved in ageing process in model organisms.These genes are: SOD1, SOD2, hsp70 and catalase.Two medfly genes, RpO and RpS21, were used as references − see Table 1 for specific primers sequence and amplicon sizes.Catalase probe was obtained from a cDNA clone generated during this work by using RACE RT-PCR kit (Invitrogen) following manufacturer instructions with a degenerated primer (catDIR-d1: 5' ATT CCR GAR CGW GTN GTR CAY GC-3') designed from an alignment of catalase genes from Drosophila melanogaster (X52286), Apis melifera (AF436842), Bombix mori (AB164195) and Anopheles gambiae (AY505416).The 1.2 kb PCR fragment was cloned into pGEM-T Easy (Promega) following manufacturer instructions.After clone sequencing, primers (Table 1) were designed to amplify the medfly catalase gene from the pGEM-T cloned copy to be used as probe in the macroarray.SSH probes were generated by PCR using the unigene collection created which contained 94 clones (see Table 2 and 3).
BioGrid (BioRobotics Li., Cambridge, UK) was used as the spotting robot.Macroarrays were made by printing the PCR products (without purification) onto a positively charged nylon membrane (Amersham Hybond N+).Printing was done with a 384-pinhead printer, consisting of regular 4 × 4 spots per pin, yielding a total of 360 available positions distributed in 12 a GO: gene ontology.b GenBank accesion number of the most similar sequence to the described EST.c NSC: number of sequences per contig.
gene probes, 57 for control probes and 9 remained empty.Membranes were kept humid by setting them onto three Hybond blotting-paper sheets soaked in denaturing solution (1.5 M NaCl, 0.5 M NaOH).After printing, the membrane was neutralized with 1.5 M NaCl, 0.5 M Tris/HCl (pH 7.2), 1 mM EDTA (pH 8.0) for 1 min.Membranes were kept on filter paper until complete dryness.Total RNA extraction from each time point for each biological replicate of medfly females and males were performed as described previously.Three medflies were used for each time point.Radioactive sample labelling including spikes, macroarray printing and hybridization, scanning and analysis were performed at SCSIE (Alberola et al., 2004).A total of 6 membranes were printed and 18 hybridizations performed.Membranes were sweep rotated for each time point, and all samples from the same time point were hybridized at the same time.
Reproducibility of the replicates was tested using ARRAYSTAT software (Imaging Research Inc.) considering the data as independent and allowing the program to take a minimum number of valid replicates of two in order to calculate mean values for each cDNA.Application of a Z-test for independent data provided the differences in individual cDNA expression between two time points.A p-value of 0.05 and the false discovery rate method were used to monitor the overall false-positive error rate.Only those signif icantly expressed cDNAs were considered to perform a cluster analysis in order to arrange cDNAs according to similarity in pattern of gene expression.Cluster analysis was performed with the Self Organising Tree Algorithm SOTArray tool included in the Gene Expression Pattern Analysis Suite v 3.1 (GEPAS) (Herrero et al., 2003) available at the CIPF Bioinformatics Unit (http://gepas3.bioinfo.cipf.es)using the linear correlation coefficient among the six-variables vectors as distance between genes.

Substractive EST collection
The SSH performed resulted in a total of 3,456 independent cDNA clones isolated from the six cDNA libraries from which 873 clones were single-pass sequenced.Table 2 shows the characteristics of each library.
The known or putative functional assignments were based on the similarity of the deduced protein sequences to previously-described molecules deposited in the nr or EST database at NCBI.
The 518 ESTs obtained from all libraries were assembled into 94 unigenes after eliminating sequences corresponding to D. melanogaster C virus or to ribosomal RNAs.The number of ESTs per contig ranged from 1 to 87.The majority of the contigs were formed by less than 5 ESTs, being the singleton the most abundant class.
Globally, the largest set of genes was assigned to the unknown function or low homology class.Forty EST unigenes (43%) did not show similarity with known proteins (named as low homology class or unknown function); the 57% of the unigenes exhibited significant protein homology to previously identif ied or putative proteins in the database.The ESTs with significant protein homology were sorted by their biological process into 11 groups as the result of GO annotation: cell development (19%), response to chemical stimulus (17%), cellular metabolic process (7%), unknown function (5%), cell motion (4%), regulation of biological characteristic (2%), cellular adhesion (2%), protein transport (2%), cell communication (1%), biosynthetic process (1%), and localization (1%).These ESTs were classified differently among libraries according to their GO.

Transcriptional profiling
The new consensus sequences obtained by the comparison of the entire consensus ESTs by aligning sequences two by two with bl2seq algorithm at NCBI web page resulted in a new medfly unigen set collection which was used as probes in the macroarray (Table 3).All ESTs were submitted to the EST division of GenBank with accession number TSA grp117765 and can be accessed at the NCBI EST database.
The Z-score normalization (which preserves the original relative dispersion) allowed the comparison of all these genes for common profiles.Age-regulated ESTs were def ined as those showing signif icant differences with the Z-score test using the age point zero (< 12 hr, newly emerged) as reference for the comparisons between the age points.This test indicated that 87.6 and 78.4% were significantly differential expressed in females and males respectively.The rela- tive expression level rates of each gene/EST are referenced in grey scale in Fig. 2. Within these differential expressed genes, one of the internal controls, the RpS21 gene, presented a differential expression pattern in females whereas the RpO showed a constant pattern.Hierarchical clustering analysis of the expression pattern profiles showed differences between the males and females (Fig. 2a and 2b).With the SOTA of z-score vectors 5 main clusters with different expression patterns were constructed.Males and females showed different expression pattern for the same gene (EST).In males, for the three first clusters the transcripts were down-regulated.In the cluster 1 (7 transcripts), the lowest expression peak was reached by old flies.In this cluster, a vitellogenin-2 precursor (CC3P1C10) and a myosin heavy chain 3 (CC9P29D11) were included.In cluster 2 (13 cDNAs), which included a hexamerin (CC1P5G3) and a chorion protein s38 (CC3P9D4), the Shorting age related genes in medfly 849  lowest expression was obtained for mature flies and then increased but slightly.The most abundant cluster (cluster 3), composed with 31 transcripts, included a cuticle protein (CC1P4A10), a hexamerin (CC1P5A2), a vitellogenin 1γ (CC3P10B12), a trypsin-like serine protease precursor (CC3P11E1), two chorion proteins (CC3P11H2 and CC3P14H5), the actin A3 (CC7P21E3) and the male specif ic serum protein (mssp) β1 (CC9P30D3) genes.The genes RpS21 and the SOD2 were part of this cluster too.The expression of these transcripts remained constant between the immature and old flies decreasing from the interval between 0 and 5 days and then increasing.Cluster 4 (14 cDNAs) was composed by a transferrin precursor (CC11P33D7), a MSP (CC11P35F10), a cytochrome b (CC11P37A3), the CG5284 gene product Cl channel (CC5P19D5), and a ATP synthase F0 s8 (CC7P23C9).The expression of these transcripts decreased sharply at 5 day point but then increased reaching at 15 days old time point the same level expression than at 0 days.In this cluster, SOD1 was also found.The last cluster (11 transcripts) was the only up-regulated.For the two first time point, the expression was almost the same, but peaked considerably at 15 days old time point.Three mssp (β3, β2, α1) (CC11P3C8, CC11P35B12, CC11P35F8), the ATP synthase F0 s6 and the hsp70 were part of this group.
In females, 10 ESTs (cluster 1) including 2 vitellogenins (CC3P1C10-vitellogenin precursor and CC3P10B12vitellogenin 1 γ), 2 chorion proteins (CC3P11H2chorion protein s19 and CC3P3H5-chorion protein s36), and the gene SOD2 peaked at the 5 day time point followed by a slight decrease.The expression level of 7 other transcripts (cluster 2), including a transferring precursor (CC11P33D7) and the genes SOD1 and hsp70, had a similar expression pattern but less marked between time points.In the cluster 3 (the biggest one), transcripts presented a constant pattern expression for the two f irst time point, and then decreasing.This group, formed by 45 transcripts, included the 5 mssp gene products (CC11P3C8-mssp β3, CC11P35B12mssp β2, CC11P35F8-mssp α1, CC11P35F10-mssp, CC9P30D3-mssp β1) and the elongation factor EF2 (CC3P1C12).In the cluster 4 (18 transcripts), the transcripts had their lowest peak at 15 days old time point.Two hexamerins (CC1P5A2 and CC1P5G3) and a flotillin were included.Finally, in the last cluster, the 5 ESTs displayed a similar decreasing expression pattern.An elongation factor product (CC3P12H1) belonged to this cluster.

Discussion
The set of genes obtained from the libraries suggests that a large number of genes are involved in ageing and/or maturity response pathways, although only a few of them have a known function.As result of libraries construction, the 57% of the unigenes obtained had protein homology to genes from the database; some of the identified ESTs are related to reproduction in females (i.e.vitellogenin, chorion protein), or in males (i.e.male specific serum protein) and their corresponding observed expression pattern are related to fertility status.Thus, the results obtained support the validity of the substracted libraries used, and these results were conf irmed by macroarray expression analysis.Some of the genes that have been reported as ageing-related genes, such as SOD1, SOD2, hsp70 or catalase (Aigaki et al., 2002) were not found in our libraries, but were sorted out by a differential expression pattern in the macroarray experiments.This could be due to the experimental design and medfly strain used, or due to the number of EST clones tested.In our experimental design for the EST libraries construction, the maturation process is more prone to be unveiled, as mating was allowed.Despite this limitation our results could be more realistic as some biological and cellular processes are also modulated by mating status, as demonstrated by Moshitzky et al. (2003) or by Gomulski et al. (2012).These authors have demonstrated that mating can modulate the biosynthesis of juvenile hormones in medfly females (the male specific serum proteins and peptides are the responsible of down-regulation of the juvenile hormones in the mated females when compared to virgin females of the same age).Regardless of common characteristics emerged between males and females libraries, some differences were also observed between them, suggesting the existence of sex-related expressed genes.
By using a combination of SSH and cDNA macroarray approach, we have successfully identified a set of genes with a modified expression pattern through ageing in term of reproduction in the medfly.The majority of the identified genes with known function in the databases were included in the cell development and response to chemical stimulus categories.Cell development class was mainly formed by vitellogenins and chorion proteins and response to chemical stimulus class, by male specific serum proteins.
In most animals, longevity is achieved at the expense of fertility (Carey, 2003).Vitellogenins (Vgs) are syn-thesized by insects in the fat body and transported to the developing oocytes via the hemolymph (Wyatt & Davey, 1996).The primary function of this protein is to provide amino acids, carbohydrates, lipids for the developing embryo, but it has been also described as oxidative stress reducer, by scavenging free radicals and thereby prolonging life span (Seehuus et al., 2006).Moreover, in some species Vgs are also produced by female immature stages, or even by males (Satyanarayana et al., 1994;Yano et al., 1994;Bellés, 2004;Raikhel et al., 2004).The pattern of transcription obtained for the males and the females was the expected.In females, the production of Vgs increases during sexual maturation.Nevertheless, at 15 days-old a decrease was observed.In males, these genes are down-regulated, and the increased expression detected in 5 day-old adults could be due to their implication on other biological process (Bellés, 2004).Other group of transcripts included in the functional category cell development, present in female libraries, was those encoding for chorion genes.Changes in the expression of these genes in females are a general response to sexual maturity in insects.The egg represents a crucial stage of any insect life cycle and is linked to successful reproductive strategies.The organization, developmental regulation and evolution of clustered chorion gene family encoding chorion proteins have been extensively studied in Diptera.Six major chorion genes have been isolated from medfly located in two separate clusters (s36 and s38, s16, s19, s15 and s18 genes) (Konsolaki et al., 1990;Tolias et al., 1990;Vlachou et al., 1997).These genes are under strict tissue regulation; both in space and time (see Vlachou et al., 1997 and the references cited therein).The response to chemical stimulus class was only formed by male specific serum proteins (mssp).The mssp gene family in medfly consists of seven members classified in three subgroups according to the degree of the deduced polypeptide similarity: two mssp α, three mssp β, and two mssp γ.Five mssps have been characterized in the medfly (Katsoris et al., 1990;Thymianou et al., 1995).The synthesis of the mssps has been described to take place mainly during the first 5 days after adults' emergence (Thymianou et al., 1998;Scolari et al., 2012).Our data reveal that the main expression of these genes took place between the 5 and the 15 day after emergence, suggesting that male maturity in this medfly strain is reached later.We have also found that the ageing-maturing process in C. capitata females presents a marked expression pattern accompanied by the increase of transcription level of genes involved in reproduction followed by a reduction (vitellogenins and chorion genes), reaching the maximum peak expression at 5 days-old.In males, an increase in the expression level of some genes involved in reproduction (male specific serum proteins) is also observed, but it stars later in time (5 days-old adults) and the maximum has not been determined, because the maximum expression is obtained with 15 day old adults.The study of the expression pattern of the novel expressed genes obtained from the libraries as well as the expression pattern of the ageing-related genes included in this work, different from those described in other organisms and/or experimental conditions, may offer important clues in understanding the complexity of this process.Moreover, although we have been unable to assign a specific function to about 40 genes reported here, they would be of great interest when the medfly genome would be released by assigning a potential function to some of them.
Altogether, the use of the known genes, vitellogenins, chorion and male specific serum proteins, and these new candidates, would allow us to select in a future a transcriptional profiling protocol to determine the reproductive status and putative age of medflies early in spring (to get the first medfly generation of the year), as advisable for the SIT programmes.In this context, the data obtained could help us also in determination of optimal release age for sterile medfly males, but further research is needed.
The experimental design of this work, a medfly cohort in free-mating conditions to simulate the first medfly generation in wild population, has allowed us to sort out some age-maturation related genes as described, and these results have been confirmed by expression analysis by using macroarrays.Ageing-maturation is characterized by highly dynamic changes in the expression of many genes, which provides a powerful molecular description of the normal sources of variation in expression from experimental ageing process.The data reported in this study offer novel insights into the genes implied in maturity in medfly, some of them sex specific.Nevertheless, further studies on the biology and genes implicated in these processes are needed to designate candidate genes for reproduction-age biomarker test by a transcriptional profiling protocol.This work can be considered as the threshold for a more accurate differential expression pattern study, when the data of the ongoing C. capitata whole-genome sequencing will be available.The data provided will pave the way for new researches by given more information of the "low homology" genes.All this information will allow developing a deeper expression evaluation by qRT-PCR at different ages and with wild flies.

854 Figure 1 .
Figure 1.Medfly SSH libraries construction.Each library is constructed using one age condition (indicated in hours or days for each gender) as tester and another age point as driver.The driver source is indicated by the arrow head.Names of libraries are close to their corresponding arrow.

Figure 2 .Figure 2
Figure 2. Hierarchical clustering of differentially expressed C. capitata ESTs in males (A) and females (B) at 3 different ages (0, 5 and 15 days) obtained by the SOTArray method is represented by these binary trees, in which the most similar patterns are clustered in a hierarchy of nested subsets.Grey scale designates relative transcript abundance in a given chronological age, with grey and white being the up and down-regulated, respectively.

Table 1 .
Shorting age related genes in medfly845 Primers and probe size for ageing-related genes and controls used, specific for Ceratitis capitata Primers and amplification conditions as inMagaña et al. (2008).b Obtained by RACE RT-PCR with degenerate primers, cDNA cloned into pGEM-T Easy and sequenced.Primers were designed on the obtained sequence. a

Table 2 .
Summary of the C. capitata EST libraries.The total number of high quality sequences after trimming of vector and short or poor quality sequences, ribosomal or not genomic medfly origin, the sequence length average, the number of singletons ("unique" clones), contigs (more than one clone with contiguous nucleotide sequences), and unigenes (the number of singletons and contigs) per library, as well as the number of ESTs that had BLASTX matches to the NCBI non-redundant database a > 500 bp, b after trimming.c unigenes represent putative unique sequences.

Table 3 .
Unigen set printed in macroarray membranes blocks of 3 columns and 10 rows.Solid pins of 0.4mm diameter were used.These pins spot 20 nL each time.PCR products were spotted five times so that each spot contained 20-30 ng DNA in a diameter of approximately 0.6 mm.Gene probes were printed in triplicate.The chips contained as positive control spots and spikes 5 triplicates of pGIBS-THR, pGIBS-PHE and pGIBS-LYS (cloned genes from Bacillus subtilis, from American type culture collection ATCC 87484, 87483 and 87482 respectively).Negative control spots included four replicates of 0.2 ng Escherichia coli genomic DNA.Therefore, 294 positions were used for