Implementation of a novel PCR based method for detecting malaria parasites from naturally infected mosquitoes in Papua New Guinea

<p>Abstract</p> <p>Background</p> <p>Detection of <it>Plasmodium species </it>in mosquitoes is important for designing vector control studies. However, most of the PCR-based detection methods show some potential limitations. The objective of this study was to introduce an effective PCR-based method for detecting <it>Plasmodium vivax </it>and <it>Plasmodium falciparum </it>from the field-caught mosquitoes of Papua New Guinea.</p> <p>Methods</p> <p>A method has been developed to concurrently detect mitochondrial cytochrome b (<it>Cyt b</it>) of four human <it>Plasmodium </it>species using PCR (<it>Cytb</it>-PCR). To particularly discriminate <it>P. falciparum </it>from <it>P. vivax</it>, <it>Plasmodium ovale </it>and <it>Plasmodium malariae</it>, a polymerase chain reaction-repeated fragment length polymorphism (PCR-RFLP) has further been developed to use with this method. However, due to limited samples number of <it>P. ovale </it>and <it>P. malariae</it>; this study was mainly confined to <it>P. vivax </it>and <it>P. falciparum</it>. The efficiency of <it>Cytb</it>-PCR was evaluated by comparing it with two 'gold standards' enzyme linked immunosorbent assay specific for circumsporozoite protein (CS-ELISA) using artificially infected mosquitoes; and nested PCR specific for small subunit ribosomal RNA (<it>SSUrRNA</it>) using field caught mosquitoes collected from three areas (Kaboibus, Wingei, and Jawia) of the East Sepic Province of Papua New Guinea.</p> <p>Results</p> <p>A total of 90 mosquitoes were artificially infected with three strains of <it>Plasmodium</it>: <it>P. vivax-</it>210 (<it>n </it>= 30), <it>P. vivax</it>-247 (<it>n </it>= 30) and <it>P. falciparum </it>(<it>n </it>= 30). These infected mosquitoes along with another 32 unfed mosquitoes were first checked for the presence of <it>Plasmodium </it>infection by CS-ELISA, and later the same samples were compared with the <it>Cytb</it>-PCR. CS-ELISA for <it>P. vivax</it>-210, <it>P. vivax</it>-247 and <it>P. falciparum </it>detected positive infection in 30, 19 and 18 mosquitoes respectively; whereas <it>Cytb</it>-PCR detected 27, 16 and 16 infections, respectively. The comparison revealed a close agreement between the two assays (κ = 0.862, 0.842 and 0.894, respectively for Pv-210, Pv-247 and <it>P. falciparum </it>groups). It was found that the eight CS-ELISA-positive mosquitoes detected negative by <it>Cytb</it>-PCR were false-positive results. The lowest detection limit of this <it>Cytb</it>-PCR was 10 sporozoites. A highly concordance result was also found between nested PCR and <it>Cytb</it>-PCR using 107 field caught mosquitoes, and both tests concordantly detected <it>P. falciparum </it>in an <it>Anopheles punctulatus </it>mosquito collected from Kaboibus. Both tests thus suggested an overall sporozoite rate of 0.9% (1/107) in the study areas. Subsequently, PCR-RFLP efficiently discriminated <it>P. falciparum </it>from <it>P. vivax </it>for all of the <it>Cytb</it>-PCR positive samples.</p> <p>Conclusion</p> <p>A single step PCR based method has been introduced here that is highly sensitive, efficient and reliable for identifying <it>P. vivax </it>and <it>P. falciparum </it>from mosquitoes. The reliability of the technique was confirmed by its ability to detect <it>Plasmodium </it>as efficiently as those of CS-ELISA and nested PCR. Application of the assay offers the opportunity to detect vector species of Papua New Guinea and may contribute for designing further vector control programmes.</p

Utility of Nd isotope ratio as a tracer of marine animals : regional variation in coastal seas and causal factors

Isotopic compositions of animal tissue are an intrinsic marker commonly used to trace animal origins and migrations; however, few isotopes are effective for this purpose in marine environments, especially on a local scale. The isotope ratio of the lanthanoid element neodymium (Nd) is a promising tracer for coastal animal migrations. Neodymium derives from the same geologic materials as strontium, well known as an isotopic tracer (87Sr/86Sr) for terrestrial and anadromous animals. The advantage of the Nd isotope ratio (143Nd/144Nd, expressed as εNd) is that it varies greatly in the ocean according to the geology of the neighboring continents, whereas oceanic 87Sr/86Sr is highly uniform. This study explored the utility of the Nd isotope ratio as a marine tracer by investigating the variation of εNd preserved in tissues of coastal species, and the causes of that variation, in a region of northeastern Japan where the bedrock geology is highly variable. We measured εNd and 87Sr/86Sr in seawater, river water, and soft tissues of sedentary suspension feeders: the mussels Mytilus galloprovincialis and Mytilus coruscus and the oyster Crassostrea gigas. We also measured concentrations of three lanthanoids (La, Ce, and Pr) in shellfish bodies to determine whether the Nd in shellfish tissue was derived from solution in seawater or from suspended particulates. The εNd values in shellfish tissue varied regionally (−6 to +1), matching the ambient seawater, whereas all 87Sr/86Sr values were homogeneous and typical of seawater (0.7091–0.7092). The seawater εNd values were in turn correlated with those in the adjacent rivers, linking shellfish εNd to the geology of river catchments. The depletion of Ce compared to La and Pr (negative Ce anomaly) suggested that the Nd in shellfish was derived from the dissolved phase in seawater. Our results indicate that the distinct Nd isotope ratio derived from local geology is imprinted, through seawater, on the soft tissues of shellfish. This result underscores the potential of εNd as a tracer of coastal marine animals

Obliquity pacing of the western Pacific Intertropical Convergence Zone over the past 282,000 years

The Intertropical Convergence Zone (ITCZ) encompasses the heaviest rain belt on the Earth. Few direct long-term records, especially in the Pacific, limit our understanding of long-term natural variability for predicting future ITCZ migration. Here we present a tropical precipitation record from the Southern Hemisphere covering the past 282,000 years, inferred from a marine sedimentary sequence collected off the eastern coast of Papua New Guinea. Unlike the precession paradigm expressed in its East Asian counterpart, our record shows that the western Pacific ITCZ migration was influenced by combined precession and obliquity changes. The obliquity forcing could be primarily delivered by a cross-hemispherical thermal/pressure contrast, resulting from the asymmetric continental configuration between Asia and Australia in a coupled East Asian-Australian circulation system. Our finding suggests that the obliquity forcing may play a more important role in global hydroclimate cycles than previously thought

Missing western half of the Pacific Plate: Geochemical nature of the Izanagi-Pacific Ridge interaction with a stationary boundary between the Indian and Pacific mantles

The source mantle of the basaltic ocean crust on the western half of the Pacific Plate was examined using Pb–Nd–Hf isotopes. The results showed that the subducted Izanagi–Pacific Ridge (IPR) formed from both Pacific (180–∼80 Ma) and Indian (∼80–70 Ma) mantles. The western Pacific Plate becomes younger westward and is thought to have formed from the IPR. The ridge was subducted along the Kurile–Japan–Nankai–Ryukyu (KJNR) Trench at 60–55 Ma and leading edge of the Pacific Plate is currently stagnated in the mantle transition zone. Conversely, the entire eastern half of the Pacific Plate, formed from isotopically distinct Pacific mantle along the East Pacific Rise and the Juan de Fuca Ridge, largely remains on the seafloor. The subducted IPR is inaccessible; therefore, questions regarding which mantle might be responsible for the formation of the western half of the Pacific Plate remain controversial. Knowing the source of the IPR basalts provides insight into the Indian–Pacific mantle boundary before the Cenozoic. Isotopic compositions of the basalts from borehole cores (165–130 Ma) in the western Pacific show that the surface oceanic crust is of Pacific mantle origin. However, the accreted ocean floor basalts (∼80–70 Ma) in the accretionary prism along the KJNR Trench have Indian mantle signatures. This indicates the younger western Pacific Plate of IPR origin formed partly from Indian mantle and that the Indian–Pacific mantle boundary has been stationary in the western Pacific at least since the Cretaceous