Trypanosomatid protozoa: survey of acetylornithinase and ornithine acetyltransferase

Trypanosomatid protozoa (Crithidia deanei, C. deanei aposymbiotic, C. oncopelti, C. fasciculata, C. acanthocephali, Leptomonas seymouri, L. collosoma, L. samueli, Herpetomonas samuelpessoai, H. sp., H. megaseliae, H. muscarum muscarum, Leishmania donovani, L. braziliensis, Trypanosoma cruzi, T. conorhini and T. mega) were examined for the presence of acetylornithinase (EC 3.5.1.16) and ornithine acetyltransferase (EC 2.3.1.35). As a rule, species of the genus Crithidia presented one of the two enzymes for the conversion of acetylornithine into ornithine. Crithidia fasciculata and C. acanthocephali presented acetylornithinase, while C. deanei and C. oncopelti, species harboring symbionts, presented ornithine acetyltransferase. The enzyme was absent in the aposymbiotic strain of C. deanei, which suggests that the enzyme belongs to the symbiont. Among the other trypanosomatids examined only Herpetomonas samuelpessoai presented acetylomithinase. The participation of acetylornithinase and ornithine acetyltransferase in the metabolism of trypanosomatids is discussed in the light of their nutritional requirements and possession of enzymes of the arginineornithine metabolism.     

First Defined Media for Leptomonas spp. from Insects*

SYNOPSIS. Defined media for Leptomonas collosoma, L. mirabilis, and L. sp. from Dysdercus are described. In addition to factors required by Crithidia spp., including a source of Crithidia factor, all 3 organisms require glycine. Leptomonas collosoma and L. sp. from Dysdercus require choline. All trypanosomatids thus far studied require calcium pantothenate but L. collosoma has an especially large requirement. Leptomonas mirabilis but not L. sp. from Dysdercus can synthesize methionine from homocysteine thiolactone during growth. Homocysteine thiolactone is toxic to L. sp. from Dysdercus. Methionine plus vitamin B₁₂ partly annul homocysteine thiolactone toxicity but only if glycine is also present.     

Cytochemical Analysis at the Fine-Structural Level of Trypanosomatids Stained with Phosphotungstic Acid*

SYNOPSIS The ethanolic phosphotungstic acid (PTA) technic was used to detect, at the fine-structural level, basic proteins in various developmental stages of pathogenic Trypanosoma cruzi, and nonpathogenic Herpetomonas samuelpessoai, Leptomonas samueli, and Crithidia deanei, trypanosomatids. Reactions were observed in the nucleus of all stages. In the kinetoplast of epimastigote and promastigote forms reactions were noted mainly at the periphery. In trypomastigotes and choanomastigotes forms, however, an intense reaction was observed throughout the kinetoplast. Reactions were present in cytoplasmic vesicles related to protein storage in T. cruzi and in membrane-bounded peroxisome-like organelles of H. samuelpessoai, L. samueli and C. deanei. The network of filaments which forms the paraxial rod did not react. In the flagellum, reaction was noted only at the peripheral doublet microtubules. PTA reacts also with structures related to the junction between the flagellar and cell body membranes.     

Leptomonas seymouri sp. n. from the cotton stainer Dysdercus suturellus*

SYNOPSIS. Leptomonas seymouri sp. n., isolated from Dysdercus suturellus (Hemiptera: Pyrrhocoridae) from Florida, is described and distinguished from other species of Leptomonas from closely related hosts.     

Anisakis,Phocanema, Contracaecum, and Sulcascaris Spp.: Electrophoresis and thermostability of alcohol and malate dehydrogenases from larvae

Electrophoretic surveys were conducted on individual larvae of four anisakine nematode genera: Anisakis, Phocanema, Contracaecum, and Sulcascaris. The larval worms were obtained from a variety of fish and molluscan hosts from widely dispersed geographic regions. Of several enzymes detected, constant and apparently species-specific electrophoretic patterns were obtained for alcohol dehydrogenase (ADH, alcohol:NAD oxidoreductase, EC 1.1.1.1) and malate dehydrogenase (MDH, l-malate: NAD oxidoreductase, EC 1.1.1.37). ADH, in all but Sulcascaris sp., possessed two isozymes, the slower of which was sensitive to temperature and inhibitors. Failure of preelectrophoretic treatment with NAD to cause interconversion of these isozymes suggests that they are products of separate genetic loci. Both isozymes were maximally active with isopropanol, sec-butanol, and amyl alcohol. Within a given species, ADH showed negligible variation (i.e., apparent genetic polymorphism) with respect to individual larvae, site of larvae in the host, or geographical origin of the host. MDH from Anisakis, Sulcascaris, and Phocanema spp. possessed one, two, and three bands of activity, respectively; MDH is highly thermostable in Anisakis sp. but not in the other species.     

Comparative Morphology of Atalodera spp. and Thecavermiculatus spp. (Heteroderidae) with Scanning Electron Microscopy

SEM examination of second-stage juveniles (J2) and adults of Atalodera ucri, A. lonicerae (syn. Sherodera lonicerae), Thecavermiculatus sp. (undescribed new species), T. andinus, and T. crassicrustatus revealed new characters. A primitive en face pattern with six separate lips occurs in J2 of Thecavermiculatus spp. examined and in about half the polymorphic A. lonicerae. A derived en face pattern with fused adjacent submedial lips occurs in the other half of A. lonicerae and all A. ucri. Posteriorly, the J2 head of all species is annulated. The primitive en face pattern also occurs in males of A. lonicerae and Thecavermiculatus spp., and posteriorly the head of these species consists of plates. Fewer plates occur rarely in males of A. ucri. Males of A. ucri have a derived en face pattern where lips are fused and the head is annulated. Fusion of lips occurs rarely in males of A. lonicerae. Females of all species have similar derived en face patterns. En face patterns of J2 and males o f Atalodera and Thecavermiculatus may aid in species identification and to elucidate intergeneric relationships, but en face characters shared by the two genera are primitive and are not useful for demonstrating monophyly. Perineal region of females indicates the closeness of the vulval-anal distance, as a derived character, which is shared by Atalodera and most Thecavermiculatus spp. suggesting possible monophyly. T. andinus, while having a similar en face pattern to J2 of other Thecavermiculatus species, lacks the derived character of the perineal region. Phasmid openings were not observed in adults of any of the species examined.     

Intranuclear Parasitism of the Ciliate Euplotes by a Trypanosomatid Flagellate

A trypanosomatid flagellate, Leptomonas sp., develops and multiplies in the macronucleus (only) of natural and laboratory-reared populations of the ciliate Euplotes. Up to 90% of the natural populations of Euplotes in our test pond had such nuclear infections. Laboratory infections were transmitted to this ciliate by feeding it liberated parasites. Paramecium resisted infections. All laboratory-induced infections were lethal to Euplotes, while control clones of the uninfected ciliates remained viable. This leptomonad, unlike Leptomonas karyophilus (found in Paramecium), shows no leishmanial forms in its several ciliate hosts and shows a varied pattern of locomotion.     

Trypanosoma brucei: activities and subcellular distribution of glycolytic enzymes from differently disrupted cells

The effect of disruption procedure on the subcellular distribution and the activities of 11 enzymes catalyzing the glycolytic pathway in Trypanosoma brucei has been studied. The activities of the enzymes varied with the lytic procedure used. Maximum specific enzyme activity values were obtained after treatment with saponin whereas digitonin treatment gave the lowest results. The intracellular location of the enzymes was examined by means of differential centrifugation following cell lysis with saponin, Triton X-100, digitonin, or by freezing and thawing. Irrespective of the method of cell lysis employed, the six enzymes, hexokinase, phosphofructokinase, aldolase, phosphoglycerate kinase, glycerol phosphate dehydrogenase, and glycerokinase, were particulate. Of the remaining 5 enzymes, digitonin liberates only phosphoglycerate mutase (partially); saponin or Triton X-100 liberates phosphoglucose isomerase, phosphoglycerate mutase, enolase, and pyruvate kinase but not glyceraldehyde 3-phosphate dehydrogenase; freezing and thawing acts like saponin or Triton X-100 except that it fails to liberate phosphoglucose isomerase, while cell grinding with silicon carbide liberates only glyceraldehyde phosphate dehydrogenase (partially), phosphoglycerate mutase, enolase, and pyruvate kinase. The relative maximal activities of the enzymes suggest that the rate-limiting steps in glycolysis in T. brucei are the reactions catalyzed by aldolase and phosphoglycerate mutase.     

Post-translational processing, metabolic stability and catalytic efficiency of oat arginine decarboxylase expressed in Trypanosoma cruzi epimastigotes

Trypanosoma cruzi epimastigotes are auxotrophic for polyamines because they are unable to synthesize putrescine de novo. This deficiency is due to the absence of ornithine and arginine decarboxylase genes in the parasite genome. We have been able to obtain transgenic T. cruzi expressing heterologous genes coding for these enzymes. Since arginine decarboxylase normal expression in oat requires a post-translational proteolytic cleavage of an enzyme precursor, we have investigated whether a similar processing occurs inside the transformed protozoa expressing oat arginine decarboxylase or the same enzyme attached to a C-terminal (his)₆-tag. We were able to demonstrate that the post-translational processing also takes place inside the transgenic parasites. This cleavage is probably the result of a general proteolytic activity of T. cruzi acting on a protease-sensitive region of the protein. Interestingly, the (his)₆-tagged enzyme expressed in the transformed parasites showed considerably increased metabolic stability and catalytic efficiency.     

Comparative Disc-Electrophoretic Protein Analyses of Selected Meloidogyne,Ditylenchus, Heterodera and Aphelenchus spp.

Disc-electrophoretic separation of soluble proteins from whole nematode homogenates yielded band profiles useful for distinguishing selected species of Meloidogyne and Ditylenchus, and the genera Heterodera, and Aphelenchus. Certain protein bands were common to all the species of Meloidogyne, whereas other bands were specific. Meloidogyne spp. and Heterodera glycines shared some protein similarities, but other genera differed distinctly. Protein profiles of Meloidogyne spp. were not significantly altered by the host on which the nematode was cultured.     

Immunological Cross-Reactivities of Adenosine-5′-Phosphosulfate Reductases from Sulfate-Reducing and Sulfide-Oxidizing Bacteria

Crude extracts from 14 species of sulfate-reducing bacteria comprising the genera Desulfovibrio, Desulfotomaculum, Desulfobulbus, and Desulfosarcina and from three species of sulfide-oxidizing bacteria were tested in an enzyme-linked immunosorbent assay with polyclonal antisera to adenosine 5′-phosphosulfate reductase from Desulfovibrio desulfuricans G100A. The results showed that extracts from Desulfovibrio species were all highly cross-reactive, whereas extracts from the other sulfate-reducing genera showed significantly less cross-reaction. An exception was Desulfotomaculum orientis, which responded more like Desulfovibrio species than the other Desulfotomaculum strains tested. Extracts from colorless or photosynthetic sulfur bacteria were either unreactive or exhibited very low levels of reactivity with the antibodies to the enzyme from sulfate reducers. These results were confirmed by using partially purified enzymes from sulfate reducers and the most cross-reactive sulfide oxidizer, Thiobacillus denitrificans. Two types of monoclonal antibodies to adenosine 5′-phosphosulfate reductase were also isolated. One type reacted more variably with the enzymes of the sulfate reducers and poorly with the Thiobacillus enzyme, whereas the second reacted strongly with Desulfovibrio, Desulfotomaculum orientis, and Thiobacillus enzymes.     

In silico annotation of unreviewed acetylcholinesterase (AChE) in some lepidopteran insect pest species reveals the causes of insecticide resistance

Lepidoptera is the second most diverse insect order outnumbered only by the Coeleptera. Acetylcholinesterase (AChE) is the major target site for insecticides. Extensive use of insecticides, to inhibit the function of this enzyme, have resulted in the development of insecticide resistance. Complete knowledge of the target proteins is very important to know the cause of resistance. Computational annotation of insect acetylcholinesterase can be helpful for the characterization of this important protein. Acetylcholinesterase of fourteen lepidopteran insect pest species was annotated by using different bioinformatics tools. AChE in all the species was hydrophilic and thermostable. All the species showed lower values for instability index except L. orbonalis, S. exigua and T. absoluta. Highest percentage of Arg, Asp, Asn, Gln and Cys were recorded in P. rapae. High percentage of Cys and Gln might be reason for insecticide resistance development in P. rapae. Phylogenetic analysis revealed the AChE in T. absoluta, L. orbonalis and S. exigua are closely related and emerged from same primary branch. Three functional motifs were predicted in eleven species while only two were found in L. orbonalis, S. exigua and T. absoluta. AChE in eleven species followed secretory pathway and have signal peptides. No signal peptides were predicted for S. exigua, L. orbonalis and T. absoluta and follow non secretory pathway. Arginine methylation and cysteine palmotylation was found in all species except S. exigua, L. orbonalis and T. absoluta. Glycosylphosphatidylinositol (GPI) anchor was predicted in only nine species.     

Cytochemical and biochemical observations on the digestive tracts of digenetic trematodes. 8. Acid phosphatase activity in Schistosoma mansoni and Schistosomatium douthitti

At the light microscope level, nonspecific acid phosphatase (AcPase) (EC 3.1.3.2) and N-acetyl glucosaminidase (NAGase) (EC 3.2.1.29) activities are in the esophageal gland cells of Schistosoma mansoni and Schistosomatium douthitti and in the gastrodermis of S. mansoni. The gastrodermis of S. douthitti is negative for these two enzymes. At the electron microscope level, AcPase activity in the esophageal gland cells of both species is observed in cytoplasmic vesicles. In S. mansoni, AcPase activity is also observed associated with the infoldings of the basal plasma membranes of the esophagus and the gastrodermis. It is hypothesized that this enzyme(s) is involved with membrane transport. AcPase activity is also associated with “droplets” and vesicles in the gastrodermis of S. mansoni. It is believed that the digestion of foodstuffs in both species occurs extracellularly.     

A New Oligonucleotide Microarray for Detection of Pathogenic and Non-Pathogenic Legionella spp.

Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp.     

Acetylcholinesterase secretion by parasitic nematodes. II. Trichostrongylus spp

Unusually high levels of acetylcholinesterase (AChE) were found in the nematode parasites Trichostrongylus axei, T. colubriformis and T, retortaeformis. In T. colubriformis the enzyme was located in the oesophageal and excretory glands of the parasitic stages. The highest level/unit wt was found in the fourth-stage larvae, which per worm had a comparable level to that in adult worms because the excretory gland was fully developed in the fourth-stage larvae. In acrylamide gel electrophoresis, T. axei and T. colubriformis AChE and esterases were similar but differed from those present in T. retortaeformis. Globulins prepared from the sera of sheep and guinea-pigs infected with T. colubriformis complexed with T. colubriformis and T. axei AChE, but not with esterases nor with AChE from T. retortaeformis, Nippostrongylus brasiliensis, Oesophagostomum radiatum or O. venulosum. Complexing of AChE to globulins did not inhibit the enzymic function of this enzyme.     

Morphology,toxicity and molecular characterization of Gambierdiscus spp. towards risk assessment of ciguatera in south central Cuba

Ciguatera poisoning is caused by the consumption of reef fish or shellfish that have accumulated ciguatoxins, neurotoxins produced by benthic dinoflagellates of the genera Gambierdiscus or Fukuyoa. Although ciguatera constitutes the primary cause of seafood intoxication in Cuba, very little information is available on the occurrence of ciguatoxins in the marine food web and the causative benthic dinoflagellate species. This study conducted on the south-central coast of Cuba reports the occurrence of Gambierdiscus and Fukuyoa genera and the associated benthic genera Ostreopsis and Prorocentrum. Gambierdiscus/Fukuyoa cells were present at low to moderate abundances depending on the site and month of sampling. This genus was notably higher on Dictyotaceae than on other macrophytes. PCR analysis of field-collected samples revealed the presence of six different Gambierdiscus and one Fukuyoa species, including G. caribaeus, G. carolinianus, G. carpenteri, G. belizeanus, F. ruetzleri, G. silvae, and Gambierdiscus sp. ribotype 2. Only Gambierdiscus excentricus was absent from the eight Gambierdiscus/Fukuyoa species known in the wider Caribbean region. Eleven clonal cultures were established and confirmed by PCR and SEM as being either G. carolinianus or G. caribaeus. Toxin production in each isolate was assessed by a radioligand receptor binding assay and found to be below the assay quantification limit. These novel findings augment the knowledge of the ciguatoxin-source dinoflagellates that are present in Cuba, however further studies are needed to better understand the correlation between their abundance, species-specific toxin production in the environment, and the risk for fish contamination, in order to develop better informed ciguatera risk management strategies.     

Almeidea A. St.-Hil. Belongs to Conchocarpus J.C. Mikan (Galipeinae,Rutaceae): Evidence from Morphological and Molecular Data,with a First Analysis of Subtribe Galipeinae

Subtribe Galipeinae (tribe Galipeeae, subfamily Rutoideae) is the most diverse group of Neotropical Rutaceae, with 28 genera and approximately 130 species. One of its genera is Almeidea, whose species are morphologically similar to those of the genus Conchocarpus. Species of Almeidea occur in the Atlantic Rain Forest of Eastern Brazil, with one species (Almeidea rubra) also present in Bolivia. The objective of this study was to perform a phylogenetic analysis of Almeidea, using a broader sampling of Galipeinae and other Neotropical Rutaceae, the first such study focused on this subtribe. To achieve this objective, morphological data and molecular data from the nuclear markers ITS-1 and ITS-2 and the plastid markers trnL-trnF and rps16 were obtained. Representatives of eight genera of Galipeinae and three genera of Pilocarpinae (included also in Galipeeae) and Hortia (closely related to Galipeeae) were used. Five species of Almeidea and seven of Conchocarpus were included, given the morphological proximity between these two genera. Individual (for each molecular marker) and combined phylogenetic analyses were made, using parsimony and Bayesian inference as optimization criteria. Results showed Galipeinae as monophyletic, with the species of Almeidea also monophyletic (supported by the presence of pantocolporate pollen) and nested in a clade with a group of species of Conchocarpus, a non-monophyletic group. Additionally, C. concinnus appeared in a group with Andreadoxa, Erythrochiton, and Neoraputia, other members of Galipeinae. As a result, Conchocarpus would be monophyletic only with the exclusion of a group of species related to C. concinnus and with the inclusion of all species of Almeidea with the group of species of Conchocarpus that includes its type species, C. macrophyllus. Thus, species of Almeidea are transferred to Conchocarpus, and the new combinations are made here.     

Temperature and metabolism in Leishmania. 3. Some dehydrogenases of L. donovani, L. mexicana, and L. tarentolae

The effects of temperature on four dehydrogenases in homogenates of promastigotes of Leishmania donovani (several strains), L. mexicana, and L. tarentolae were studied.     

Exeristes,Itoplectis, Aphaereta,Brachymeria, and Hyposoter species: In vitro glyceride synthesis and regulation of fatty acid composition

Microsomes from two species of parasitic Hymenoptera, Exeristes roborator and Itoplectis conquisitor, exhibited little or no de novo glyceride synthesis but actively acylated endogenous mono- and diacylglycerides. It is suggested that this lack of de novo synthesis is related to the fact that the fatty acid composition of these parasitoid species closely resembles that of the hosts on which they are reared. Microsomes from three other species of parasitic Hymenoptera, Aphaereta pallipes, Brachymeria lasus, and Hyposoter exigua, whose fatty acid compositions are little influenced by the host species, exhibited active de novo glyceride synthesis as well as acylation of endogenous mono- and diacylglycerides. Radiotracer studies indicated that E. roborator microsomes and cytosol did not contain noncompetitive or uncompetitive inhibitors of glycerophosphate acyltransferase. E. roborator microsomes acylated exogenous phosphatidic acid but not dihydroxyacetone phosphate or glycerol. The maximum rate of glycerophosphate acylation was less than 0.1 nmole/min/mg microsomal protein after 15 min incubation. The incorporation was subject to rapid lipolysis on further incubation. The addition of bovine serum albumin (BSA) reduced the ability of E. roborator microsomes to acylate mono- and diacylglycerides with endogenous acyl groups. In the absence of BSA, palmitoyl-CoA was a more effective substrate than stearoyl-CoA for both mono- and diacylglyceride acyltransferases.     

Trichomonas vaginalis: NADH oxidase activity.

NADH oxidase activity was detected in the 105,000g supernatant (“soluble”) fraction of Trichomonas vaginalis and the enzyme was purified 50-fold by centrifugation, ammonium sulfate precipitation, Sephadex G-200, and DEAE-Sephadex A-25 chromatography. The ratio of oxygen uptake to NADH oxidation was approximately one-half. Addition of catalase did not affect the rate of oxygen uptake elicited by NADH. Since the purified fraction was free from interfering enzymes, the postulated reaction is as follows: $\text{NADH}\text{+}\text{H}{}^{\mathrm{+}}\text{+}\text{1}\text{2}\text{= NAD}{}^{\mathrm{+}}\text{+ H}{}_2\text{O}$. Among numerous substances tested, only NADH was a functional substrate, whereas NADPH was not oxidized. The purified enzyme had a V_max of 16.5 μmole of oxygen consumed/min/mg protein, and the apparent K_m for NADH was 7.4 μM. Substrate inhibition was observed at 3.7 mM NADH. The purified NADH oxidase was competitively inhibited by NAD⁺ as well as by NADP⁺ with 50% inhibition at 1 and 5 mM, respectively. The enzyme was also markedly inhibited by p-chloromercuribenzoate, hydrogen peroxide, and transient metal-chelators such as bathophenanthroline or o-phenanthroline. A flavoprotein antagonist, atebrin was slightly less inhibitory. Various quinones, flavin nucleotides and artificial dyes, except for p-benzoquinone, ferricyanide and cytochrome c, did not function in accepting electrons from NADH oxidase. These three compounds, however, were still poor electron acceptors in the enzymatic reaction suggesting that the trichomonad NADH oxidase has little diaphorase activity. All of these findings indicate that T. vaginalis has an unique NADH oxidizing enzyme in that H₂O seems to be the prdouct of oxygen reduction. This NADH oxidase appears important in the aerobic metabolism of this parasite.