Analysis of expressed sequence tags from subtracted and unsubtracted Ctenocephalides felis hindgut and Malpighian tubule cDNA libraries

Abstract

Insect hindgut and Malpighian tubule (HMT) tissues regulate the contents of the haemolymph through the excretion of waste products and the specific reabsorption of nutrients. As such, they perform a role that is essential for survival and may contain molecular targets for insect control strategies. In order to discover genes expressed in the HMT tissues of the cat flea, Ctenocephalides felis, expressed sequence tags (ESTs) were generated from an unsubtracted HMT cDNA library and from a subtracted HMT cDNA library that had been enriched for HMT-specific cDNAs. A total of 4844 ESTs were analysed from both libraries: 3657 from the subtracted library and 1187 from the unsubtracted library. Of the 1418 distinct ESTs identified from both libraries, 953 had significant similarity to other sequences reported in the GenBank database. A comparison of the results from the two libraries confirmed that the percentages of genes likely to be involved with metabolism, cell structure, and digestion were reduced by the subtraction procedure, whereas genes likely to be involved with ion transport were enriched. Analysis of the prevalence of three individual cDNAs in each library revealed that the actin cDNA was reduced in the subtracted library whereas the cDNAs encoding allantoinase and a peritrophin-like protein were greatly enriched in the subtracted library. Northern blot analysis demonstrated that the actin cDNA was expressed in both the HMT and carcass tissues, whereas the allantoinase and peritrophin-like cDNAs were detected exclusively in the HMT tissues. In total, 97 distinct ESTs that appear to encode proteins involved with ion transport were analysed. Some of these proteins may be directly involved with diuresis or the specific reabsorption of salts and nutrients, and thus may be potential molecular targets for flea control strategies.

Gene expression in the gut of keratin-feeding clothes moths (Tineola) and keratin beetles (Trox) revealed by subtracted cDNA libraries

“…These TUGs lacking annotations were either EST clusters, some containing as many as 78 ESTs, or singletons (23 singletons out of 60 TUGs in Tineloa and 32 singletons out of 61 TUGs in Trox). This high proportion of TUGs without apparent database match is similar to previous studies on insect guts (Gaines et al, 2002;Eigenheer et al, 2003;Pedra et al, 2003;Siegfried et al, 2005) suggesting that numerous genes expressed in the guts of Tineola and Trox do not have close equivalents in the comparatively distant species with substantial available sequence data in GenBank or, alternatively, they were too short or covering regions too poorly conserved to detect matches. The remaining TUGs in Tineola mainly had best matches to insect sequences (87%), specifically 45% to Lepidoptera (Fig.…”

“…For example, in the D. melanogaster genome, serine proteinases constitute a large number of genes coding for 199 trypsin-like and 178 chymotrypsin-like proteins (Rubin et al, 2000). Large multigene serine protease families have also been identified in the sheep blowfly, Lucilia cuprina (Casu et al, 1994), the old-world screwworm, Chrysomya bezziana (Muharsini et al, 2001), as well as A. grandis (Oliveira-Neto et al, 2004), Mamestra configurata (Hegedus et al, 2003), H. armigera (Gatehouse et al, 1997) and the flea, Ctenophalides felis (Gaines et al, 2002), to name a few.…”

“…We therefore used ESTs to catalogue the battery of digestive enzymes expressed in the gut of Tineola and Trox. To avoid the sequencing of genes that are not specifically expressed in the gut, we used a subtracted cDNA library approach (Gaines et al, 2002). This procedure greatly enriches those mRNAs that are specifically expressed in a target tissue but not elsewhere using a PCR-based hybridization technique, by removing the non-target genes commonly expressed in a 'tester' (i.e.…”

Insect allantoinase: cDNA cloning, purification, and characterization of the native protein from the cat flea, Ctenocephalides felis

“…A cDNA (BF731795) encoding a partial-length allantoinase protein was labeled with [a-32 P]ATP using the Megaprime 2 DNA Labeling Kit (Amersham Pharmacia Biotech) following the manufacturer's protocol, and was used for nucleic acid hybridization screening of the HMT lambda phage cDNA library using standard methods (Gaines et al, 2002). A 2056 base pair cDNA (AY151136) was isolated that encoded a fulllength allantoinase protein 483 amino acids in length.…”

Gene expression profile of human chondrocyte HCS-2/8 cell line by EST sequencing analysis

“…Each cDNA library is constructed from total RNA or poly (A) RNA derived from specific tissues or cells, and represents genes expressed in the original cellular population. The sequencing of large numbers of randomly selected cDNA provides a profile of gene expression in a specific condition of an organism (Verdun et al, 1998) which can lead to a better understanding of its physiology (Gaines et al, 2002).…”