Lactate dehydrogenase allozyme differentiation of species in the Anopheles culicifacies complex

Abstract Genetically controlled enzyme variation exists within and between four sibling species of the Anopheles culicifacies complex of malaria vectors in India. A study on electrophoretic variation of nine enzymes in An.culicifacies sibling species revealed that the lactate dehydrogenase ( Ldh ) locus has Fast (F) and Slow (S) allozymes distinguishing species A+D from species B+C with a probability of c . 95%.     

ITS-2 secondary structures and phylogeny of Anopheles culicifacies species

Background: Second internal transcribed spacer (ITS2) has proven to contain useful biological information at higher taxonomic levels. Objectives: This study was carried out to unravel the biological information in the ITS2 region of An. culicifacies and the internal relationships between the five species of Anopheles culicifacies. Methodology: In achieving these objectives, twenty two ITS2 sequences (~370bp) of An. culicifacies species were retrieved from GenBank and secondary structures were generated. For the refinement of the primary structures, i.e. nucleotide sequence of ITS2 sequences, generated secondary structures were used. The improved ITS2 primary structures sequences were then aligned and used for the construction of phylogenetic trees. Results and discussions: ITS2 secondary structures of culicifacies closely resembled near universal eukaryotes secondary structure and had three helices, and the structures of helix II and distal region of helix III of ITS2 of An. culicifacies were strikingly similar to those regions of other organisms strengthening possible involvement of these regions in rRNA biogenesis. Phylogenetic analysis of improved ITS2 sequences revealed two main clades one representing sibling B, C and E and A and D in the other. Conclusions: Near sequence identity of ITS2 regions of the members in a particular clade indicate that this region is undergoing parallel evolution to perform clade specific RNA biogenesis. The divergence of certain isolates of An. culicifacies from main clades in phylogenetic analyses suggests the possible existence of camouflaged sub-species within the complex of culicifacies. Using the fixed nucleotide differences, we estimate that these two clades have diverged nearly 3.3 million years ago, while the sibling species in clade 2 are under less evolutionary pressure, which may have evolved much later than the members in clade 1.     

Anopheles culicifacies in Baluchistan, Iran

Abstract. Anopheles culicifacies (probably species A) is the main vector of malaria in Baluchistan, southeastern Iran. Adult mosquitoes were collected during 1990-92 by five methods of sampling: knock-down pyrethrum space-spray indoors, human and animal bait (18.00–05.00 hours), pit shelters and CDC light traps, yielding 62%, 3%, 6%, 4% and 25% of specimens, respectively. Whereas spray-catches comprised c. 70% gravid and semi-gravid females, light trap catches were mostly ( c . 60%) unfed females, while females from pit shelters comprised all abdominal stages more equally (13–36%). An. culicifacies populations peaked in April-May and rose again during August-November. Densities of indoor-resting mosquitoes were consistently greater in an unsprayed village than in villages subjected to residual house-spraying with propoxur, malathion or pirimiphos-methyl. Monthly malaria incidence generally followed fluctuations of An. culicifacies density, usually with a peak in May-June.     

Presence of Anopheles culicifacies B in Cambodia established by the PCR-RFLP assay developed for the identification of Anopheles minimus species A and C and four related species

Abstract A polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) assay developed for identification of five species of the Anopheles minimus Theobald group and a related mosquito species of the Myzomyia Series (Diptera: Culicidae) was applied to morphologically identified adult female specimens collected in Ratanakiri Province, north‐eastern Cambodia. In addition to finding An. aconitus Dönitz, An. minimus species A and An. pampanai Büttiker & Beales, some specimens showed a new restriction banding pattern. Siblings of specimens that exhibited this new PCR‐RFLP pattern were morphologically identified as An. culicifacies James sensu lato. Based on nucleotide sequences of the ribonuclear DNA internal transcribed spacer 2 region (ITS2) and the mitochondrial cytochrome oxidase I gene (COI), these specimens were recognized as An. culicifacies species B (sensu Green & Miles, 1980 ), the first confirmed record of the An. culicifacies complex from Cambodia. This study shows that the PCR‐RFLP assay can detect species not included in the initial set‐up and is capable of identifying at least seven species of the Myzomyia Series, allowing better definition of those malaria vector and non‐vector anophelines in South‐east Asia.     

Four new mutations and a linkage map of species A of Anopheles culicifacies

Four new mutations, brown eye (bw), colorless eye (c), scarlet eye (sca), and abnormal eye (ae) have been mapped on chromosome 2 of species A of Anopheles culicifacies. The gene sequence is bw--c--sca--ae. A linkage map of the genetically analyzed loci in this species is presented.     

Responses of Anopheles culicifacies sibling species A and B to DDT and HCH in India: implications in malaria control

Differential responses of Anopheles culicifacies Giles sibling species A and B to DDT were evident from higher survival rate of species B in laboratory bioassays and greater proportions of species B in DDT-sprayed villages of northern India, compared with those under HCH pressure. Both species A and B have become almost completely resistant to HCH in this area due to regular house-spraying with HCH for about the last 10 years. Because species A predominates in northern India, where it has been incriminated as an important vector of malaria, and species A is more susceptible than species B to DDT, it is suggested that DDT would control malaria transmission more effectively than HCH in this situation. Monitoring of insecticide resistance in species A is therefore recommended as the basis for future choice of insecticides to be used by the National Malaria Eradication Programme.     

Anopheles culicifacies Y-chromosome dimorphism indicates sibling species (B and E) with different malaria vector potential in Sri Lanka

In Sri Lanka, malaria is transmitted mainly by Anopheles culicifacies Giles sensu lato (Diptera: Culicidae). In India, this nominal taxon comprises sibling species A, B, C, D and E, distinguished by their chromosome morphology. Species B (identified by polytene chromosome sequence Xab, 2g1 + h1) is not such an efficient vector of malaria as other members of the An. culicifacies complex in India. All specimens of An. culicifacies s.l. examined from Sri Lanka possess Xab, 2g1 + h1 polytenes, previously interpreted as species B, despite their important vector status. Recently, species E was described from Rameshwaram Island (Tamil Nadu, India) between Sri Lanka and the Indian mainland, where both species B and E are sympatric. Species B and E share polytene sequence Xab, 2g1 + h1 but differ by the mitotic Y-chromosome being acrocentric in species B, submetacentric in species E, the latter implicated as vector of vivax malaria. From May 1999 to January 2000, we surveyed Y-chromosomes of male progeny from An. culicifacies Xab, 2g1 + h1 females collected from cattle bait in diverse malarious districts of Sri Lanka: Badulla, Monaragala, Puttalam and Trincomalee. Karyotypes of readable quality were obtained from 42/83 families examined, with overall proportions 24% acrocentric and 76% submetacentric Y-chromosome carriers, both types being sympatric in at least 3/4 localities sampled. By analogy with the situation on Rameshwaram Island, we interpret these observations to demonstrate widespread presence of two members of the An. culicifacies complex in Sri Lanka, their karyotypes being compatible with species B and E, the latter predominant and having greater vector potential.     

The Anopheles culicifacies complex and control of malaria

Of the 50 or so species and varieties of anopheline mosquito in India, about 20 are implicated as vectors of human malaria. Of these, perhaps the most important and widespread is Anopheles culicifacies s.l. For the first 10 years of widespread DDT spraying, An. culicifacies remained susceptible to this insecticide - indeed, some thought it lacked resistance genes altogether. By 1960 however, resistance to DDT was apparent. DDT is a cheap insecticide, and its use is still favoured for mosquito control wherever it remains effective. But by the end of the late 1970s it appeared that DDT resistance in An. culicifacies (and other species) was a major barrier to effective vector control in several areas - particularly in parts of the northwestern states. Yet in other areas DDT still seemed to be effective. There was also increasing evidence from other studies suggesting differences in An. culicifacies found in different areas - particularly differences in seasonal prevalence and man-biting activity. We now know - as Sarala Subbarao discusses here - that An. culicifacies s.l. represents a complex of at least four sibling species. But in this case, one of the most important findings is that DDT resistance is mainly associated with species B which proves to be a very poor vector of malaria. Such findings, made possible by careful cytogenetic studies, have very important consequences for malaria epidemiology and control policies.     

A phosphoglucomutase polymorphism in the mosquito, Anopheles culicifacies

A survey of phosphoglucomutase (Pgm) among laboratory strains of Anopheles culicifacies has uncovered two electrophoretic variants. Detailed genetic analysis revealed that these variants are inherited as codominant alleles at a single locus. The Pgm locus has been assigned to linkage group III approximately 39 map units from Acph (acid phosphatase) and 8.5 map units from Dl (dieldrin resistance). The data indicate that the probable gene sequence is Acph-Dl-Pgm.     

Susceptibility of species A, B, and C of Anopheles culicifacies complex to Plasmodium yoelii yoelii and Plasmodium vinckei petteri infections

The comparative susceptibilities of colonized species A, B, and C of Anopheles culicifacies complex and Anopheles stephensi were determined for 2 rodent malaria parasites Plasmodium vinckei petteri and Plasmodium yoelii yoelii. All the 3 members of the complex were found to support complete sporogony with varying success. Controls, A. stephensi, become readily infected, with >70% developing oocysts. Of the test groups, species A had the highest percentage of mosquitoes with oocysts (>25%) and sporozoites (>15%). Anopheles culicifacies species B were least susceptible; less than 10% had oocysts and sporozoites in the salivary glands. The results demonstrate that A. culicifacies species A is most susceptible and species B is least susceptible to infections with both the parasites.     

Anopheles culicifacies sibling species B and E in Sri Lanka differ in longevity and in their susceptibility to malaria parasite infection and common insecticides

Members of the Anopheles culicifacies Giles complex (Diptera: Culicidae) are well established as the predominant vectors of malaria in Sri Lanka. Until recently, only sibling species B was reported to be present in Sri Lanka, which was surprising as species B is a poor vector of malaria in India. This was clarified by the identification through Y-chromosome morphology that what was reported as B on the island is really a mixture of B and E. The fecundity, longevity and insecticide resistance of B and E are of relevance to malaria transmission and its control and are reported in this study. The mean egg production of these two sibling species did not differ significantly. The mean age of wild mosquitoes was assessed by the Polovodova technique of observing ovarian dilatations. More of species E than B had three or more dilatations, i.e. had reached an age at which sporozoites could have developed to maturity, although the difference between the species was of borderline significance. Following feeding on Plasmodium vivax or Plasmodium falciparum infected blood, some females of species E developed oocysts but none of species B did so. Both sibling species were found fully susceptible in laboratory tests to lambdacyhalothrin and deltamethrin, but resistant to DDT and partially resistant to malathion.     

Pigeon liver malic enzyme

Summary Malic enzyme of pigeon liver is a tetrameric molecule with identical, or nearly-identical subunits. It catalyzes, in addition to oxidative decarboxylation of L-malate, the following metal activated component reactions: Oxalacetate decarboxylase; reductase with broad specificity on -ketocarboxylic acids; a NADP⁺-dependent dismutation of L-malate to L-lactate; and proton exchange between pyruvate and medium water. The kinetic mechanism of oxidative decarboxylase is sequential and ordered, with NADP⁺ adding first to the metal enzyme, followed by L-malate, and by the release of products CO₂, pyruvate, and NADPH. NADPH release, or a conformation change preceeding it, is rate-limiting in the overall reaction.Chemical modification studies indicate the presence of histidyl and lysyl residues at the nucleotide site, and tyrosyl residues at the carboxylic acid site. The involvement of protonated histidine(s) in NADPH binding is implicated by results of direct titration experiments, which also suggest a role of this residue as a proton sink in the catalytic reaction.A cysteinyl SH group is located near (but not at) each of the substrate-sites on the enzyme tetramer. Reaction of these groups with SH reagents causes selective loss of activities involving decarboxylation (i.e., oxidative decarboxylase, reductive carboxylase, and oxalacetate decarboxylase), owing to blockage of the reversible carbon-carbon cleavage step by the bulky substituent. All-of-the-sites reactivity is observed for non-specific thiol reagents such as 5,5 dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide, iodoacetate, and iodoacetamide. While bromopyruvate, which is reduced by the enzyme to L-bromolactate under catalytic conditions, alkylates these groups in an active-site directed manner with half-of-the-sites stoichiometry. The remaining two SH groups are reactive toward non-specific reagents, but at rates 2.4 - 3.6 fold lower than do the same groups on the unalkylated enzyme. This behavior is interpreted in terms of the ligand-induced negative cooperativity concept of Koshland, et al. (Biochemistry 5: 365–385, 1966): Reaction of bromopyruvate induces a conformation change on the alkylated subunit which is transmitted to the unoccupied subunit neighbor, turning off its catalytic site for reaction with L-malate, as well as converting the initial fast SH groups into slow, or unreactive SH groups.In equilibrium binding experiments, all-of-the-sites reactivity is seen with nucleotide cofactors NADP⁺ and NADPH. Binding of Mn²⁺, or L-malate in the presence of Mn²⁺ and NADPH is biphasic, showing two tight sites with dissociation constants in the micromolar range, and two weak sites with 10–100 fold lower affinities. The presence of tight and weak L-malate sites is confirmed by fluorescence titration experiments which also yields similar affinities for the substrate molecule. In kinetic studies, two types of non-equivalent, and functionally distinct sites are detected. At saturating NADP⁺, and Mn²⁺ and L-malate levels corresponding to binding at tight sites, typical Michaelian behavior is observed. The reaction is inhibited uncompentitively by L-malate at higher concentrations corresponding to occupancy at all of the L-malate sites. Occupancy of Mn²⁺ at weak metal sites as well has no effect at low L-malate, but prevents substrate inhibition at high L-malate.A tentative half-of-the-sites model consistent with results of chemical modification, binding, and kinetic experiments is proposed for this enzyme. This model implicates involvement of subunit cooperativity in the catalytic process. Malic enzyme is depicted as a tetramer composed of inititally identical subunits, each containing an active-site capable of binding all reactants. Mn²⁺ and L-malate bind anticooperatively to the tight and weak sites, in contrast to NADP⁺ which binds equivalently to all sites. On the fully active enzymes, only half (or the tight) of the subunits are simultaneously undergoing catalysis. Binding of L-malate (but not Mn²⁺) at the adjacent weak subunits causes a slow isomerization of the enzyme, and inhibition of NADPH dissociation from the catalytic subunits. Binding of Mn²⁺ at the same sites prevents this change and thereby relieving substrate inhibition. This model is further supported by results of active-site titration experiments, such as the half-size burst of enzyme-bound NADPH in the transient state, and half-of-the-sites reactivity of oxalate, an analog for the transition state intermediate of the reaction.Abbreviations DTNB 5,5 dithiobis-(2-nitrobenzoic acid) - NEM N-ethylmaleimide - BP bromopyruvate - DTT dithiothreitol     

Mitochondrial malic enzyme in human leukocytes

Leukocyte samples from 316 unrelated blood donors were screened for malic enzyme (MEM). The frequency of the common allele in this investigation was MEM1 = 0.63. There is evidence for the existence of a rare fourth allele MEM4.     

Crossing-over in the long arm of the X and Y chromosomes in Anopheles culicifacies

Genetic and cytogenetic investigations of two unstable Y-linked translocations in Anopheles culicifacies indicate that crossing over can occur between the X and Y chromosomes in this species. Moreover, these observations suggest that the long arms of the X and Y chromosomes are homologous and that the sex determining mechanism(s) is located in the short arms.     

High malic enzyme activity in tumor cells and its cross-reaction with antipigeon liver malic enzyme serum

Summary Rabbit antibodies against pigeon liver malic enzyme (EC 1.1.1.40) were prepared. The antiserum gave single precipitation line with crude pigeon liver extract. Cross reaction was observed with partially purified malic enzyme or crude extract from chicken liver. Positive cross reaction was also observed with the concentrated cytosolic fraction of two human carcinoma cell lines which were demonstrated to contain high malic enzyme activity. All other proteins examined did not react with the antibodies. When purified pigeon liver malic enzyme was mixed with the antiserumin vitro, a time-dependent inactivation of the enzyme activity was observed. Protection of the enzyme activity against antiserum inactivation was afforded by NADP⁺ orL-malate. Metal Mn²⁺ gave little protection.     

Isolation and characterization of microsatellite markers from malaria vector,Anopheles culicifacies

Anopheles culicifacies, an important vector in the Indian subcontinent is a complex of five sibling species of which four are vectors. We describe the isolation of 31 microsatellite markers from the recently recognized isomorphic species A of which 13 were characterized in sympatric populations of Anopheles culicifacies isomorphic species A and B. The allele frequencies ranges from two to 12 in species A and two to seven in species B. Species A being a vector, and that these markers can be used in closely related species, makes the isolation of these markers important to study population structure of all sibling species in this complex.