Allozyme analysis reveals six species within the Anopheles punctulatus complex of mosquitoes in Papua New Guinea

Abstract. Among samples collected from nineteen localities in Papua New Guinea, we have identified six species within the Anopheles punctulatus complex of mosquitoes, by means of cellulose acetate allozyme electrophoresis. An.punctulatus Dönitz sensu stricto was collected from seven villages in the Madang area and from Buksak, Sausi Mission and an area 18 km SW of Tari; An.koliensis Owen from eight villages in the Madang area, from Popondetta and Brown River near Karema; and An.farauti No. 1 from ten coastal areas including Madang, Lorengau, Popondetta, Port Moresby, Rabaul and Wewak. Three newly recognized species, reported here for the first time, are designated as An.farauti No. 4 from Gonoa and Hudini, Madang area; An.farauti No. 5 from Ketarabo near Goroka; and An.farauti No. 6 from Hiwanda near Tari. Three other known members of the complex, An.clowi Rozeboom & Knight, An.farauti No. 2 (Bryan, 1973) and An.farauti No. 3 (Mahon & Meithke, 1982) were not detected in Papua New Guinea. Problems arising with morphological characters for the identification of species in this group are discussed.     

Electrophoretic keys to identify members of the Anopheles punctulatus complex of vector mosquitoes in Papua New Guinea

Abstract. Electrophoretic keys are given for the six species of the Anopheles punctulatus complex (Diptera: Culicidae) known from Papua New Guinea plus An.farauti No. 2 and No. 3 from Australia. The categories ‘faster’, ‘standard’ and ‘slower’ are used in keys to relate allozyme band migration following cellulose acetate electrophoresis to the standard pattern. Alternative keys are given depending on the availability of different species for use as standards.     

Differential ecology of Anopheles punctulatus and three members of the Anopheles farauti complex of mosquitoes on Guadalcanal, Solomon Islands, identified by PCR-RFLP analysis

From a series of larval collections made across northern Guadalcanal during the dry season, October–November 1997, four members of the Anopheles punctulatus group of mosquitoes (Diptera: Culicidae) were identified using PCR‐RFLP analysis. Anopheline larvae were found in 54/57 (95%) of the sites sampled, comprising An. farauti Laveran sensu stricto (32 sites), An. farauti species no. 2 (39 sites), An. farauti no. 7 (36 sites) and An. punctulatus Dönitz (10 sites). Anopheles punctulatus occurred only on the coastal plain, where it was associated with the more transient sites. Anopheles farauti sensu lato was more widespread throughout the survey region, with similar proportions of all three sibling species in both transient and permanent sites. Two members of the An. farauti complex, An. farauti s.s. and species no. 2, were found in brackish water. All breeding sites of An. punctulatus were cohabited by An. farauti s.l., sometimes by all three sibling species. Anopheles farauti s.s. was the only species collected on human bait, with a much higher biting rate early in the evening (57 bites/human/hour at 18.30–20.00 hours) than later (0.8 bites/human/hour at 21.00–24.00 hours).     

Systematics of malaria vectors with particular reference to the Anopheles punctulatus group

The appearance of groups and complexes of closely related cryptic or sibling species in many of the anopheline taxa has impeded studies on malaria transmission and the evaluation of control strategies which have relied on morphological characters to identify the vector species involved. The advantages of morphological identification are low cost, speed and simplicity, which allow large numbers of specimens to be processed rapidly in the field. The need for accurate identification is crucial, as time and money may be wasted in studying and controlling species of no medical importance. Various techniques such as cross-mating, chromosome studies and allozyme analysis have been developed to resolve problems of identifying sibling species, though none, as yet, can match the speed and simplicity afforded by morphology markers. The latest of these identification methods comes from advances that have been made in DNA-based technology. Although costly and requiring fairly sophisticated laboratory support, methods such as DNA probe hybridisation and PCR are the quickest and most user-friendly to date. The use of DNA has other advantages in the study of intraspecific differences and in providing characters for phylogenetic studies. This review looks at the development of DNA-based techniques for taxonomic and systematic studies of anopheline mosquitoes. The Anopheles punctulatus group of the southwest Pacific is featured as an example of how this technology has been applied and how it has progressed.     

Morphological and chromosomal descriptions of new species in the Anopheles subpictus complex

Abstract. Anopheles subpictus Grassi is shown to comprise four reproductively distinct species, designated A, B, C and D, occurring sympatrically in villages of Pondicherry, southeast India.
Adult females were reared individually from wild larvae and examined for their morphological and chromosomal characters. Paracentric fixed inversions on the X-chromosome serve to distinguish the species cytogenetically, with no inversion heterozygotes (i.e. no interspecific hybrids) among totals of 717 species A (X+^a,+^b), 1863 species B (Xa, b), 869 species C (Xa,+^b) and 1365 species D (X +^a,b) identified.
Morphologically, diagnostic characters for each of the four species are seen in the egg float ridge number, larval mesothoracic seta 4, pupal seta 7-1 and the palpi of female adults. Species A. C and D immatures inhabit freshwater, whereas the malaria vector species B breeds in saltwater and was found only in coastal villages.     

Studies Towards the Synthesis of Medermycin via Dötz Benzannulation

The C‐arylglycosides are available in enantiomerically pure form via the Dötz benzannulation reaction between Fischer alkenyl chromium carbene complexes and alkynes; it also could be converted to a precursor of medermycin by O‐carbamate directed ipso bromination and nitrile substitution in good overall yields. Chirality 27:18–22, 2015. © 2014 Wiley Periodicals, Inc.     

The Anopheles punctulatus complex: DNA probes for identifying the Australian species using isotopic, chromogenic, and chemiluminescence detection systems

Isotopic and enzyme-labeled species-specific DNA probes were made for the three known members of the Anopheles punctulatus complex of mosquitoes in Australia (Anopheles farauti Nos. 1, 2, and 3). Species-specific probes were selected by screening total genomic libraries made from the DNA of individual species with 32P-labeled DNA of homologous and heterologous mosquito species. The 32P-labeled probes for A. farauti Nos. 1 and 2 can detect less than 0.2 ng of DNA while the 32P-labeled probe for A. farauti No. 3 has a sensitivity of 1.25 ng of DNA. Probes were then enzyme labeled for chromogenic and chemiluminescence detection and compared to isotopic detection using 32P-labeled probes. Sequences of the probe repeat regions are presented. Species identifications can be made from dot blots or squashes of freshly killed mosquitoes or mosquitoes stored frozen, dried, and held at room temperature or fixed in isopropanol or ethanol with isotopic, chromogenic, or chemiluminescence detection systems. The use of nonisotopic detection systems will enable laboratories with minimal facilities to identify important regional vectors.     

The Anopheles punctulatus group of mosquitoes in the Solomon Islands and Vanuatu surveyed by allozyme electrophoresis

Abstract. Four species within the Anopheles punctulatus group of mosquitoes (Diptera: Culicidae) were identified by allozyme analysis of samples collected from thirty-three localities in Guadalcanal, Makira, Malaita, Temotu and Western Provinces in the Solomon Islands and six localities on Efate, Espiritu Santo, Maewo and Malekula Islands in Vanuatu. Three of these species are members of the An. farauti complex. A key is given to identify five species of the An. punctulatus group known to occur in the Solomon Islands using their isoenzyme characteristics.
An. farauti No. 1 was widespread in coastal areas of the Solomon Islands and was the only species detected in Vanuatu, including Efate Island (where Faureville is the type locality of An. farauti Laveran sensu stricto). An. farauti No. 2 and An. punctulatus were common in the Solomon Islands in more inland areas. An. farauti No. 7, reported here for the first time, was found as larvae in freshwater at six localities on north Guadalcanal. Three other members of the An. punctulatus group which have been reported previously from the Solomon Islands: An. koliensis, An. renellensis and an electrophoretic variant of An. farauti sensu lato, were not found in our samples.
Previously recognized vectors of malaria and bancroftian filariasis in the Solomon Islands are An. farauti No. 1 (i.e. An. farauti s.s. ), An. koliensis and An. punctulatus s. s. Adult females of An. farauti No. 2 and An. farauti No. 7 were not attracted to human bait in areas where their larvae occurred, indicating that these two species are not anthropophilic and therefore unlikely to transmit human pathogens.     

Morphological studies on the genusClematis Linn

The basic pattern of the vascular supply to stamens and carpels in the flowers ofClematis is discussed on the basis of serial sections. The bundles of the receptacular stele show fairly regular fusions and divisions in relation to the origin of the vascular supply, giving off a single trace for each stamen or carpel. In many cases the trace arises by the trifurcation of the “fused bundle” and the subsequent departure of the median strand. This is the pattern basic to the structure of the receptacular stele of the genus. Although the basic pattern involves a variety of modifications, each of the diverging traces fundamentally leaves a single independent gap in the stele, contrary to the conclusion of previous authors. Similarities and differences between a group of stamens and carpels and that of sepals and foliage leaves are also discussed based on the results of the present and previous studies on the vascular anatomy of the floral receptacle and the inflorescence axis.     

Morphological studies on the genusClematis linn

In four-sepaled flowers ofClematis the sepal is supplied by three main traces. The basic pattern of the vascular supply to sepals is found inC. alpina var.ochotensis which invariably has six-bundled pedicels. It is as follows: the median traces to the first pair of opposite sepals, as well as all the lateral traces, arise directly from pedicel bundles, while those to the second pair are formed secondarily, after fusion and subsequent division of two adjacent pedicel bundles. As to the manner of origin of the median traces, the pattern is similar to that of the vascular supply to foliage leaves. This gives further evidence for the generally accepted view that the sepals ofClematis, like foliage leaves, are decussately arranged. In most other species such asC. apiifolia, C. stans, etc. the number of pedicel bundles tends to be reduced from six to four so as to coincide with that of the sepals, so patterns are much simplified and specialized: all the traces arise directly from pedicel bundles. InC. japonica an iconsistent pattern is observed, since the number of pedicel bundles from which sepal traces arise is much higher and varied.     

Morphological studies on neuroglia

Immunohistochemical studies with the use of the peroxidase-antiperoxidase (PAP) method revealed that "amoeboid microglial cells", in the brains of neonatal rats and "brain macrophages" in lesioned brains of adult rats react positively to an antiserum raised against macrophages. In brains of neonatal rats, "amoeboid microglial cells" stained by means of the PAP-method were observed in the corpus callosum, internal capsule, dorso-lateral region of the thalamus, subventricular zone of the lateral ventricle, and the subependymal layer of the ventricular system. These cellular elements were not detected in brains of rats aged 21 days or older. Resting microglial cells displaying a typical ramified structure were not specifically stained. Cells reacting positively to the macrophage antiserum appeared (i) in the cerebral cortex of adult rats following placement of a stab wound, or (ii) in the hippocampal formation after kainic acid-induced lesions; in the damaged areas immunoreactive cells exhibited the typical features of "brain macrophages". "Brain macrophages" and "amoeboid microglial cells" are considered to belong to the class of exudate macrophages derived from blood monocytes. Thus, elements of hematogenous origin do exist in the intact brain parenchyma of neonatal rats and in lesioned brains of adult rats. The relationship between brain macrophages and resting microglial cells is discussed.     

Morphological studies on the genusClematis Linn

While the axes of branched axillary inflorescences seen in most species ofClematis show fundamentally the same features in nodal vasculature as the vegetative stems, those of simple axillary inflorescences, having only a pair of opposite sterile bracts at specific positions, exhibit an exclusive feature in nodal vasculature because they have entirely lost lateral branches or buds in the bract axils. Noticeably, at the nodal level of the axis of the simple inflorescence ofC. japonica andC. Williamsii, trace bundles to the missing lateral branches are formed and extend unaltered beyond the node. Thus, in these two species stelar bundles in the inflorescence axis generally increase in number upwardly from six to eight or more through the node with sterile bracts. On the basis of both anatomical and morphological data, a probable evolutionary trend in floral shoots with simple axillary inflorescences is proposed. The type of floral shoot ofC. tosaensis bearing simple, scale-subtended and basally fascicled inflorescences is the most primitive and constitutes an initial phase of evolution, and progressive changes in a kind of inflorescence-subtending leaves as well as in the shape and position of the sterile bracts resulting in the type of floral shoot ofC. japonica and further in that ofC. obvallata. A floral shoot ofC. Williamsii follows another line of evolution from an ancestral type similar to that ofC. tosaensis.     

Morphological studies on neuroglia

The postnatal development of microglial cells was investigated in the neonatal rat brain by use of light- and electron microscopy, including enzyme-histochemical techniques. Microglial cells were selectively stained by demonstration of their nucleoside diphosphatase (NDPase) activity and classified into three types: 1) In the early postnatal period "primitive microglial cells" showing scantily ramified processes were found in the cerebral cortex, the hippocampal formation, and the hypothalamus. During the course of the first postnatal week the processes of this cell type developed gradually and the cells were transformed into typical ramified microglial cells, called "resting microglial cells". 2) "Amoeboid microglial cells "showing typical features of macrophages were characteristic of the cerebral white matter. 3) "Round microglial cells" possessing a round soma and few pseudopodia but no characteristic processes occurred in large numbers in the subventricular zone of the lateral ventricle and as single elements in the vicinity of blood vessels. Histochemically, thiamine pyrophosphatase (TPPase) was demonstrated only in the fully developed, ramified microglial cells ("resting microglial cells"), which could be readily observed in the central nervous tissue from the age of 14 day. "Round and amoeboid microglial cells" did not show TPPase activity and disappeared after 14 days of postnatal life. By use of electron microscopy, in neonatal rats NDPase activity was apparent in the plasma membrane of the three types of microglial cells ("primitive, round, and amoeboid" types). They showed basically similar submicroscopic characteristics, i.e., well-developed Golgi apparatus, long strands of rough-surfaced endoplasmic reticulum, single dense bodies and vacuoles, and numerous ribosomes. "Amoeboid microglial cells" were characterized by their well-developed cytoplasmic vacuoles and phagocytic inclusion bodies. The present study strongly suggests a mesodermal origin for these microglial elements.     

Morphological studies on neuroglia

Summary Autoradiographic studies showed that in the rat hippocampus microglia-like reactive cells (MRC) and astrocytes are capable of proliferation in response to kainic acid (KA)-induced lesions. A marked increase in the number of labeled MRC was observed during the first four days after the induction of the KA-lesion. A proliferative response of astrocytes occurred at two days after the KA-lesion. After the induction of a KA-lesion brain macrophages and oligodendrocytes were only slightly labeled with ³H-thymidine. It appears likely that MRC is the main cellular element responding to this type of lesion.This work was supported by a grant (No. 437002) from the Ministry of Education, Science and Culture, Japan     

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).