Morphological, biometrical and molecular characterization of Archaeopsylla erinacei (Bouché, 1835).

In the present work, we carried out a morphological, biometrical and molecular study of the species Archaeopsylla erinacei (Bouché, 1835) and their subspecies: Archaeopsylla erinacei erinacei (Bouché, 1835) and Archaeopsylla erinacei maura (Jordan & Rothschild, 1912) isolated from hedgehogs (Erinaceus europaeus) from different geographical regions (Seville and Corse). We have found morphological differences in females of A. erinacei from the same geographical origin that did not correspond with molecular differences. We suggest that some morphological characters traditionally used to discriminate females of both subspecies should be revised as well as we set the total length of the spermatheca as a valid criterion in order to discriminate between both subspecies. The Internal Transcribed Spacers 1 and 2 (ITS1, ITS2) and partial 18S rRNA gene, and partial cytochrome c-oxidase 1 (cox1) and cytochrome b (cytb) mtDNA gene sequences were determined to clarify the taxonomic status of these taxa and to assess intra-specific and intra-population similarity. In addition, a phylogenetic analysis with other species of fleas using Bayesian and Maximum Likelihood analysis was performed. All molecular markers used, except 18S, showed molecular differences between populations corresponding with geographical origins. Thus, based on the phylogenetic and molecular study of two nuclear markers (ITS1, ITS2) and two mitochondrial markers (cox1 and cytb), as well as concatenated sequences of both subspecies, we reported the existence of two geographical genetic lineages in A. erinacei corresponding with two different subspecies: A. e. erinacei (Corse, France) and A. e. maura (Seville, Spain), that could be discriminated by polymerase chain reaction-linked random-fragment-length polymorphism.

Acetylcholinesterases of the cat flea Ctenocephalides felis: identification of two distinct genes and biochemical characterization of recombinant and in vivo enzyme activities.

Acetylcholinesterase (AChE, EC3.1.1.7.) is a prime target for insecticides and is the site of action of carbamate and organophosphate drugs used to combat the cat flea Ctenocephalides felis. In this paper we report the identification and cDNA cloning of two AChE-encoding genes from the cat flea, cface1 and cface2. Functional heterologous expression of the catalytic domains in Pichia pastoris shows that both genes encode functional enzymes, CfAChE1 and CfAChE2. Bioinformatical analysis of the predicted translation products and heterologous expression of the full length cDNAs in the human cell line HEK293 demonstrate, that CfAChE1 and CfAChE2 possess glycosylphosphatidylinositol membrane anchors and are transported to the cell surface. Recombinant CfAChE1 and CfAChE2 share high sensitivity towards the anti-flea carbamates propoxur and carbaryl, but can be distinguished by their specificity for different acylthiocholine AChE substrates and, particularly, by their differential sensitivity to the non-covalent inhibitor galanthamine. Comparison of substrate specificities and inhibitor sensitivities of both recombinant enzymes with those of AChE activities extracted from adult fleas suggest that CfAChE1, and not CfAChE2, is the dominant activity in C. felis imagoes. Three-dimensional structure models of CfAChE1 and CfAChE2 reveal similarities, but also differences, and compound docking experiments on these models provide potential rationales for the differential substrate and the inhibitor specificities observed experimentally.

Analysis of Rickettsia typhi-infected and uninfected cat flea (Ctenocephalides felis) midgut cDNA libraries: deciphering molecular pathways involved in host response to R. typhi infection.

Murine typhus is a flea-borne febrile illness that is caused by the obligate intracellular bacterium, Rickettsia typhi. The cat flea, Ctenocephalides felis, acquires R. typhi by imbibing a bloodmeal from a rickettsemic vertebrate host. To explore which transcripts are expressed in the midgut in response to challenge with R. typhi, cDNA libraries of R. typhi-infected and uninfected midguts of C. felis were constructed. In this study, we examined midgut transcript levels for select C. felis serine proteases, GTPases and defence response genes, all thought to be involved in the fleas response to feeding or infection. An increase in gene expression was observed for the serine protease inhibitors and vesicular trafficking proteins in response to feeding. In addition, R. typhi infection resulted in an increase in gene expression for the chymotrypsin and rab5 that we studied. Interestingly, R. typhi infection had little effect on expression of any of the defence response genes that we studied. We are unsure as to the physiological significance of these gene expression profiles and are currently investigating their potential roles as it pertains to R. typhi infection. To our knowledge, this is the first report of differential expression of flea transcripts in response to infection with R. typhi.

Identification and characterization of two arginine kinases from the parasitic insect Ctenocephalides felis.

Arginine kinase (ATP:l-arginine omega-N-phosphotransferase, EC2.7.3.3.; AK) is an enzyme crucial for the energy metabolism of insects and other invertebrates, that has known allergenic potential in humans and that has been proposed as a pesticidal drug target. Here we report the identification, cDNA cloning, genomic gene structure and functional expression of AK genes from Ctenocephalides (C.) felis (cat flea). In contrast to other insect species investigated so far, C. felis possesses two AK genes, cfak1 and cfak2, encoding the functional enzymes CfAK1 and CfAK2 that can be distinguished by their guanidino substrate specificity and the kinetic parameters for their natural substrates. Molecular modelling on CfAK1 and CfAK2 based on the Limulus polyphemus AK X-ray structure (Zhou et al., 1998) and substrate docking studies provide a potential rational for the observed specificities. Evidence is provided that adult fleas express predominantly CfAK1 as an abundant soluble protein, and that in vivo in C. felis, the AK metabolites are present in concentration ranges relevant for this enzyme.

Molecular characterization of Tunga trimamillata and T. penetrans (Insecta, Siphonaptera, Tungidae): taxonomy and genetic variability.

A new species of the genus Tungo, T. trimamillata has recently been described on the basis of several morphological traits. To explore the taxonomic status of this flea with respect to T. penetrans, we undertook a molecular analysis of cytochrome oxydase II and 16S rDNA mitochondrial genes and of the internal transcribed spacer 2 nuclear marker on samples of both species. Maximum Parsimony evaluations of the three data set indicate a differentiation compatible with a specific rank between the two fleas with very high levels of divergence. Both mitochondrial and nuclear data are in line with a recent bottleneck in the Malagasy population of T. penetrans, possibly due to the recent colonisation of Africa via human transportation. Further, significantly lower mitochondrial variability in the Ecuadorian populations of T. penetrans with respect to the T. trimamillata ones is also evidenced.

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

Allantoinase catalyses the hydrolysis of allantoin to allantoic acid. This reaction is a step in the purine degradation pathway, which produces nitrogenous waste for excretion. A cDNA encoding full-length allantoinase was cloned from a Ctenocephalides felis hindgut and Malpighian tubule (HMT) cDNA library. The cDNA encoded a 483 amino acid protein that had 43% identity with the bullfrog Rana catesbeiana allantoinase and contained the conserved histidine and aspartic acid residues required for zinc-binding and catalytic activity. Unlike the bullfrog allantoinase, the C. felis allantoinase sequence was predicted to contain a 22 amino acid signal sequence, which targets the protein to the secretory pathway. Expression of the mRNA was detected by Northern blot in the first, third, and wandering larval stages as well as in fed and unfed adults, but was not seen in eggs or pupae. In adults, mRNA encoding allantoinase was detected only in the HMT tissues. Immunohistochemistry performed using affinity-purified rabbit immune serum generated against purified recombinant flea allantoinase showed that the native protein localized to the HMT tissues in adult fleas. The anti-allantoinase serum recognized two proteins in an adult flea soluble protein extract, one migrating at 56 kDa and the other at 53 kDa. The two proteins were separated by gel filtration chromatography and were both associated with allantoinase activity. The difference in size appeared to be due to a difference in glycosylation of the proteins. The 53 kDa protein was further purified to near homogeneity by affinity chromatography and retained allantoinase activity. A comparison of the sizes of the native and recombinant C. felis proteins indicated that the 53 kDa native protein may be the product of a post-translational cleavage event, possibly at the putative 22 amino acid signal sequence at the N-terminus of the protein.

Identification, characterization, and cloning of an immunoglobulin degrading enzyme in the cat flea, Ctenocephalides felis.

The degradation of cat immunoglobulin G (IgG) in blood-fed adult C. felis midguts was examined. SDS-PAGE analysis of dissected midgut extracts obtained from C. felis that had been blood fed for various times between 0 to 44 h revealed that by 24 h most of the high molecular weight proteins, including the heavy chain of IgG, were digested. A 31-kDa serine protease with IgG degrading activity was purified from fed C. felis midguts by benzamidine affinity chromatography, hydrophobic interaction chromatography, and cation exchange chromatography. Three primary cleavage products between 30- and 40-kDa were observed when the purified protease was incubated with protein A purified cat IgG. N-terminal amino acid sequence analysis of the products revealed that the IgG degrading protease cleaves after specific cysteine and lysine residues within the hinge region of IgG. The enzyme is also capable of degrading other immunoglobulins, serum albumin, and hemoglobin, suggesting that it may have roles in both combating the host's immune system and providing nutrients for the flea. A cDNA clone encoding the 265 amino acid IgG degrading protease proenzyme was isolated. When expressed in a baculovirus/insect cell expression system, the recombinant protein had the same N-terminus as the processed 237 amino acid mature native protein and possessed IgG degrading activity indistinguishable from the native protein. Arch. Insect Biochem.

Preliminary characterisation of esterase and platelet-activating factor (PAF)-acetylhydrolase activities from cat flea (Ctenocephalides felis) salivary glands.

Naphthyl esterase and platelet-activating factor (PAF)-acetylhydrolase activities were detected in the salivary glands of the cat flea, Ctenocephalides felis. Salivary naphthyl esterase activity is disgorged during exploratory probing. Whole extracts of salivary glands contain esterase activity against the short-chain naphthyl esters alpha-naphthyl acetate (approximately 210pmol/min/gland pair; 10.0micromol/min/mg specific activity; K(m) approximately 59microM) and beta-naphthyl acetate (approximately 110pmol/min/gland pair; 5.2micromol/min/mg specific activity; K(m) approximately 132microM). Salivary gland extracts have PAF-acetylhydrolase activity (approximately 5pmol/min/gland pair; 0.24micromol/min/mg specific activity) but do not have detectable acetylcholinesterase activity. Native-PAGE and IEF resolve three and six salivary gland naphthyl esterase bands, respectively, and both patterns are different from carcass esterases. Salivary gland naphthyl esterase activity binds reversibly to Concanavalin A, and enzymatic deglycosylation with glycopeptidase F produced a new, fast-migrating salivary gland naphthyl esterase band on Native-PAGE. Renaturation of esterase activity after SDS-PAGE gave approximately 56kDa, approximately 57kDa and approximately 58kDa naphthyl-esterase-positive bands. On gel filtration naphthyl esterase and PAF-acetylhydrolase activities co-elute as a single peak with an apparent molecular weight of approximately 59kDa. This partially purified pool of enzyme had esterase activity against a series of short-chain alpha- and beta-naphthyl esters. The heterogeneity of salivary gland esterases, their relationship to PAF-acetylhydrolase, and the possible physiological functions of salivary gland PAF-acetylhydrolase activity are discussed.

Cloning of a family of serine protease genes from the cat flea Ctenocephalides felis.

Serine protease gene fragments approximately 480 nucleotides in length were amplified from Ctenocephalides felis larval and adult cDNA libraries using degenerate oligonucleotide PCR primers. Partial clones of thirty-eight distinct serine protease encoding sequences were isolated, and nineteen different full-length cDNAs encoding mature serine proteases were subsequently cloned and sequenced. All of the mature proteases contained the histidine, aspartic acid and serine amino acids of the catalytic triad characteristic of serine proteases. The mature C. felis serine proteases had amino acid sequences that were at most 29-53% identical to those known insect and arachnid serine proteases. Two of the C. felis gene sequences had similarity with the Drosophila melanogaster developmental genes snake and stubble. mRNA expression of selected serine protease genes was examined in different life stages, tissues, genders, and in response to bloodfeeding.

Characterization of apyrase activity from the salivary glands of the cat flea Ctenocephalides felis.

Apyrase activity (ATP diphosphohydrolase, EC 3.6.1.5) was detected in salivary glands of the cat flea Ctenocephalides felis. Whole extracts of salivary glands contain approximately 21 ng of protein, 145 U/mg ADP'ase and 158 U/mg ATP'ase activity; AMP is not hydrolysed by salivary gland extracts. DEAE-Sepharose CL-6B anion exchange chromatography, and Cibacron Blue affinity chromatography each give a single coincident peak of ADP/ATP'ase activity. Biogel P-100 gel filtration of salivary gland homogenates made in buffer containing Triton and protease inhibitors, separated enzymatic activity into 57 kD and 44 kD peaks of ADP/ATP'ase activity. Partially purified ADP/ATP'ases are dependent on divalent cations and activation increases between 0.125 mM and 5.0 mM calcium. At 5 mM, magnesium is almost equally effective as calcium in activating ADP/ATP'ase but manganese and zinc are less so, and EDTA abolishes activity. ADP/ATP'ases have a pH optima of 7-9. The Km for ADP hydrolysis by whole extracts and partially purified enzyme is approximately 66 microM ADP. The co-purification of ADP'ase and ATP'ase activity by three physiochemical techniques and parallelism between ADP and ATP hydrolysis under varying conditions of pH and activating cation indicates enzymatic activity is attributable to true apyrase(s).

[Studies on esterase isoenzyme of three species of flea].

The patterns of esterase (Est) isoenzyme of 3 flea species were studied by vertical slab polyacrylamide gel electrophoresis (PAGE). The samples prepared from the third larval instar, female and male (the unsucking and sucked one) were run in the same gel slab. The result was as follows: Xenopsylla cheopis showed 14 bands, with 5-6 major bands, Ctenocephalides felis felis showed 9 bands with 4-5 major bands and Nosopsyllus laeviceps kuzenkovi showed 9 bands with 4-5 major bands. These three species have their common bands as well as respective specific bands. The difference in Est isoenzymogram among families was greater than among genera, displaying a species-specific feature.

Characterization of the salivary apyrase activity of three rodent flea species.

1. Salivary gland lysates of the adult female fleas Oropsylla bacchi, Orchopea howardi and Xenopsylla cheopis hydrolyse ATP and ADP, but not AMP, thus characterizing the existence of a salivary apyrase activity. 2. In all species Mg++ or Ca++ function as activators, and a pH optimum between 7 and 8 is observed. 3. Salivary gland lysates of male fleas contain significantly smaller amounts of the enzyme activity than do those of female fleas. 4. Immediately following a blood meal, apyrase activity and protein content of female X. cheopis salivary glands are 2-3-fold less than that of unfed fleas, indicating that salivary apyrase activity is secreted during feeding. 5. It is suggested that, as in other arthropods, salivary apyrase may facilitate blood location and blood feeding by preventing ADP-induced platelet aggregation at the site of the bite.

[Comparative characteristics of the digestive enzymes of blood-sucking insects. II. Invertase and lipase]. / K sravnitel'noi kharakteristike pishchevaritel'nykh fermentov krovososushchikh nasekomykh. II. Invertaza i lipaza.

The activity of invertase and lipase in the midguts of 11 hematophagous insect species have been determined. The highest invertase activity at pH 7-10. Insects feeding on carbohydrate meals as well as with blood have the greatest invertase activity, the obligatory blood feeders have the least one. The lipase activity does not depend on trophic peculiarities of the blood-suckling insects.