Speciation and distribution of the members of the Anopheles punctulatus (Diptera : Culicidae) group in Papua New Guinea

Mosquito collections were made throughout the mainland of Papua New Guinea to identify the members of the Anopheles punctulatus group present and to determine their distribution. Identification was made using morphology, DNA hybridization, and polymerase chain reaction (PCR)-RFLP analysis. Nine members of the group were identified: An. farauti s.s. Laveran, An.farauti 2, An. koliensis Owen, and An. punctulatus Donitz, were common and widespread; An. farauti 4 was restricted to the north of the central ranges where it was common; An. farauti 6 was found only in the highlands above 1,000 m; and An. farauti 3, An. sp. near punctulatus and An. clowi Rozeboom & Knight were uncommon and had restricted distributions. Identification of An. koliensis and An. punctulatus using proboscis morphology was found to be unreliable wherever An. farauti 4 occurred. The distribution and dispersal of the members of the An. punctulatus group is discussed in regard to climate, larval habitats, distance from the coast, elevation, and proximity to human habitation.

Development and evaluation of a species diagnostic polymerase chain reaction-restriction fragment-length polymorphism procedure for cryptic members of the Culex sitiens (Diptera : Culicidae) subgroup in Australia and the southwest Pacific

Members of the Culex sitiens subgroup are important vectors of arboviruses, including Japanese encephalitis virus, Murray Valley encephalitis virus and Ross River virus. Of the eight described species, Cx. annulirostris Skuse, Cx. sitiens Wiedemann, and Cx. palpalis Taylor appear to be the most abundant and widespread throughout northern Australia and Papua New Guinea (PNG). Recent investigations using allozymes have shown this subgroup to contain cryptic species that possess overlapping adult morphology. We report the development of a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) procedure that reliably separates these three species. This procedure utilizes the sequence variation in the ribosomal DNA ITS1 and demonstrates species-specific PCR-RFLP profiles from both colony and field collected material. Assessment of the consistency of this procedure was undertaken on mosquitoes sampled from a wide geographic area including Australia, PNG, and the Solomon Islands. Overlapping adult morphology was observed for Cx. annulirostris and Cx. palpalis in both northern Queensland and PNG and for all three species at one site in northwest Queensland.

Distribution and evolution of the Anopheles punctulatus group (Diptera : Culicidae) in Australia and Papua New Guinea

The members of the Anopheles punctulatus group are major vectors of malaria and Bancroftian filariasis in the southwest Pacific region. The group is comprised of 12 cryptic species that require DNA-based tools for species identification. From 1984 to 1998 surveys were carried out in northern Australia, Papua New Guinea and on islands in the southwest Pacific to determine the distribution of the A. punctulatus group. The results of these surveys have now been completed and have generated distribution data from more than 1500 localities through this region. Within this region several climatic and geographical barriers were identified that restricted species distribution and gene flow between geographic populations. This information was further assessed in light of a molecular phylogeny derived from the ssrDNA (18S). Subsequently, hypotheses have been generated on the evolution and distribution of the group so that future field and laboratory studies may be approached more systematically. This study suggested that the ability for widespread dispersal was found to have appeared independently in species that show niche-specific habitat preference (Anopheles farauti s.s. and A. punctulatus) and conversely in species that showed diversity in their larval habitat (Anopheles farauti 2). Adaptation to the monsoonal climate of northern Australia and southwest Papua New Guinea was found to have appeared independently in A. farauti s.s., A. farauti 2 and Anopheles farauti 3. Shared or synapomorphic characters were identified as saltwater tolerance (A. farauti s.s. and Anopheles farauti 7) and elevational affinities above 1500 m (Anopheles farauti 5, Anopheles farauti 6 and A. farauti 2). (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Malaria vectors on Buka and Bougainville islands, Papua New Guinea

Anophelines were sampled from 82 locations oil Buka and Bougainville islands in Papua New Guinea by larval collections, carbon dioxide-baited Mosquito traps, and human biting catches. Anopheles farauti s.s. was collected in larval Surveys but infrequently in mosquito traps on both islands; on Buka Island this species was readily collected in human biting catches. Anopheles faraunti 2 was commonly collected in larval surveys on both islands however. it was not collected in either mosquito traps or human biting catches. Anopheles punctulatus was found only on Buka Island, where it was commonly collected as larvae, but rarely in human biting catches and mosquito traps. Anopheles lungae was collected Lis larvae from only I site on Bougainville. Anopheles farauti s.s. led consistently throughout the night (1900-0600 h): small peaks at midnight and dawn were not statistically significant. Of 1,156 An. farauti s.s. specimens examined by enzyme-linked immunosorbent assay for malaria sporozoites. 20 were found to be positive: 12 were positive for Plasmodium falciparum and 8 were positive for P. vivax (247 variant = 5: 210 variant = 3). Anopheles farauti s.s. seems to be the major malaria vector on these islands, whereas An. punctulatus may play a minor role on Buka Island. Anophele farauti 2 is unlikely to be involved in malaria transmission on Buka or Bougainville islands.

Neural control of the heart: developmental changes in ionic conductances in mammalian intrinsic cardiac neurons

The expression and properties of ionic channels were investigated in dissociated neurons from neonatal and adult rat intracardiac ganglia. Changes in the hyperpolarization-activated and ATP-sensitive K+ conductances during postnatal development and their role in neuronal excitability were examined. The hyperpolarization-activated nonselective cation current, I-h, was observed in all neurons studied and displayed slow time-dependent rectification. An inwardly rectifying K+ current, I-K(I), was present in a population of neurons from adult but not neonatal rats and was sensitive to block by extracellular Ba2+. Using the perforated-patch recording configuration, an ATP-sensitive K+ (K-ATP) conductance was identified in greater than or equal to 50% of intracardiac neurons from adult rats. Levcromakalim evoked membrane hyperpolarization, which was inhibited by the sulphonylurea drugs. glibenclamide and tolbutamide. Exposure to hypoxic conditions also activated a membrane current similar to that induced by levcromakalim and was inhibited by glibenclamide. Changes in the complement of ion channels during postnatal development may underlie observed differences in the function of intracardiac ganglion neurons during maturation. Furthermore, activation of hyperpolarization-activated and KATP channels in mammalian intracardiac neurons may play a role in neural regulation of the mature heart and cardiac function during ischaemia-reperfusion. (C) 2002 Elsevier Science B.V All rights reserved.

The synthesis and structure of an n-terminal dodecanoic acid conjugate of a-conotoxin MII

The alpha-conotoxin MII is a 16 amino acid long peptide toxin isolated from the marine snail, Conus magus. This toxin has been found to be a highly selective and potent inhibitor of neuronal nicotinic acetylcholine receptors of the subtype alpha3beta2. To improve the bioavailability of this peptide, we have coupled to the N-terminus of conotoxin MII, 2-amino-D,L-dodecanoic acid (Laa) creating a lipidic linear peptide which was then successfully oxidised to produce the correctly folded conotoxin MII construct.

A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NAIR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

Basal nonselective cation permeability in rat cardiac microvascular endothelial cells

The presence of a basal nonselective cation permeability was mainly investigated in primary cultures of rat cardiac microvascular endothelial cells (CMEC) by applying both the patch-clamp technique and Fura-2 microfluorimetry. With low EGTA in the pipette solution, the resting membrane potential of CMEC was -21.2 +/- 1.1 mV, and a Ca2+-activated Cl- conductance was present. When the intracellular Ca2+ was buffered with high EGTA, the membrane potential decreased to 5.5 +/- 1.2 mV. In this condition, full or partial substitution of external Na+ by NMDG(+) proportionally reduced the inward component of the basal I-V relationship. This current was dependent on extracellular monovalent cations with a permeability sequence of K+ > Cs+ > Na+ > Li+ and was inhibited by Ca2+, La3+, Gd3+, and amiloride. The K+/Na+ permeability ratio, determined using the Goldman-Hodgkin-Katz equation, was 2.01. The outward component of the basal I-V relationship was reduced when intracellular K+ was replaced by NMDG(+), but was not sensitive to substitution by Cs+. Finally, microfluorimetric experiments indicated the existence of a basal Ca2+ entry pathway, inhibited by La3+ and Gd3+. The basal nonselective cation permeability in CMEC could be involved both in the control of myocardial ionic homeostasis, according to the model of the blood-heart barrier, and in the modulation of Ca2+ -dependent processes. (C) 2002 Elsevier Science (USA).

Factorizations of the complete graph into C-5-factors and 1-factors

In this paper, we show that K-10n can be factored into alpha C-5-factors and beta 1-factors for all non-negative integers alpha and beta satisfying 2alpha + beta = 10(n) - 1.