Genetic evidence for the existence of cryptic species in theAnopheles albitarsis complex in Brazil: Allozymes and mitochondrial DNA restriction fragment length polymorphisms

Allozyme and mitochondrial DNA (mtDNA) restriction studies were undertaken to determine the extent of genetic divergence among field populations ofAnopheles albitarsis in Brazil. Two sympatric species,An. deaneorum andAn. marajoara, were identified in collections from Costa Marques (CM), Rondonia. Genetic evidence includes (1) the presence of two types of individuals, each with diagnostic allelic clusters (forHad-1, Pgi-1, Pep-1, Mpi-1, andIdh-1), (2) a deficiency of heterozygotes, and (3) characteristic mtDNA haplotypes. In addition, two allopatric cryptic species ofAn. marajoara were identified, one from Iguape (An. marajoara form IG), Sao Paulo state, and the other from the Island of Marajo (An. marajoara form MA). Though form IG and form-MA resemble form CM in wing spot morphology, they differ from it in diagnostic allozymes and mtDNA haplotypes.An. marajoara form CM had a higher variability (mean heterozygosity,H=0.22, and percentage of polymorphic loci,P=66.7) than did form IG and form MA (H=0.08 in both, andP=25.0 and 33.3, respectively). Form MA and form IG are genetically more similar to each other than both are to form CM. Based on wing morphology, estimates ofF statistics, and genetic similarities, we propose thatAn. albitarsis in Brazil is a species complex. It comprises at least two morphologically distinguishable species: (1)An. deaneorum (currently one taxon) and (2) theAn. marajoara species complex, which further consists of at least three cryptic forms,marajoara form MA,marajoara form IG, andmarajoara form CM. The views of the authors do not purport to reflect the position of the Department of the Army or the Department of Defense of the United States. This research was conducted when the senior author was on the staff of the USDA-ARS Laboratory in Gainesville, Florida.     

Abundance, biting behaviour and parous rate of anopheline mosquito species in relation to malaria incidence in gold-mining areas of southern Venezuela

Abstract A longitudinal entomological and epidemiological study was conducted in five localities of southern Venezuela between January 1999 and April 2000 to determine the abundance, biting behaviour and parity of anopheline mosquitoes (Diptera: Culicidae) in relation to climate variables and malaria incidence. A total of 3685 female anopheline mosquitoes, representing six species, were collected. The most abundant species were Anopheles marajoara Galvão & Damasceno (60.7%) and Anopheles darlingi Root (35.1%), which together represented 95.8% of the total anophelines collected. Abundance and species distribution varied by locality. Malaria prevalence varied from 12.5 to 21.4 cases per 1000 population. Transmission occurred throughout the year; the annual parasite index (API) for the study period was 813.0 cases per 1000 population, with a range of 71.6?2492 per 1000 population, depending on locality. Plasmodium vivax (Grassi & Feletti) (Coccidia: Plasmodiidae) accounted for 78.6% of cases, Plasmodium falciparum (Welch) for 21.4% and mixed infections (Pv+Pf) for < 0.1%. Anopheles marajoara and An. darlingi were more abundant during the rainy season (April–September). There was no significant correlation (P > 0.05) between mosquito abundance and rainfall. Correlations between malaria incidence by parasite species and mosquito abundance were not significant (P > 0.05). Monthly parous rates were similar for An. marajoara and An. darlingi throughout the year, with two peaks that coincided with the dry?rainy transition period and the period of less rain. Peaks in the incidence of malaria cases were observed 1 month after major peaks in biting rates of parous anophelines. Anopheles darlingi engages in biting activity throughout the night, with two minor peaks at 23.00–00.00 hours and 03.00–04.00 hours. Anopheles marajoara has a different pattern, with a biting peak at 19.00?21.00 hours and 76.6% of biting occurring before midnight. Although both vectors bite indoors and outdoors, they showed a highly significant (P < 0.01) degree of exophagic behaviour. The present study constitutes the first effort to characterize the bionomics of anophelines in malaria endemic foci in different ecological situations in relation to malaria transmission in southern Venezuela and to provide relevant information to be considered when planning and implementing vector control programmes.     

Seasonal abundance of anopheline mosquitoes and their association with rainfall and malaria along the Matapí River,Amapí, Brazil

Three communities separated by 1.5–7.0 km, along the Matapí River, Amapá State, Brazil, were sampled monthly from April 2003 to November 2005 to determine relationships between seasonal abundance of host‐seeking anophelines, rainfall and malaria cases. Out of the 759 821 adult female anophelines collected, Anopheles darlingi Root (Diptera: Culicidae) was the most abundant (56.2%) followed by An. marajoara Galvão & Damasceno (24.6%), An. nuneztovari Gabaldón (12.4%), An. intermedius (Chagas) (4.4%) and An. triannulatus (Neiva and Pinto) (2.3%). Vector abundance, as measured by human landing catches, fluctuated during the course of the study and varied in species‐specific ways with seasonal patterns of rainfall. Anopheles darlingi and An. triannulatus were more abundant during the wet‐dry transition period in June to August, whereas An. marajoara began to increase in abundance in February in two villages, and during the wet‐dry transition in the other village. Anopheles nuneztovari and An. intermedius increased in abundance shortly after the rains began in January to February. A generalized linear mixed model (GLMM) analysis of 32 consecutive months of collections showed significant differences in abundance for each species by village and date (P < 0.0001). Correlations between lagged rainfall and abundances also differed among species. A strong positive correlation of An. darlingi abundance with rainfall lagged by 4 and 5 months (Pearson's r = 0.472–0.676) was consistent among villages and suggests that rainfall may predict vector abundance. Significant correlations were detected between numbers of malaria cases and abundances of suspected vector species. The present study shows how long‐term field research may connect entomological and climatological correlates with malaria incidence.     

Anopheles (Anopheles) neomaculipalpus: a new malaria vector in the Amazon basin?

Abstract. Anopheles (Anopheles) neomaculipalpus Curry (Diptera: Culicidae) collected by human landing catches and light traps in southern Venezuela were assayed by enzyme-linked immunosorbent assay (ELISA) for detection of Plasmodium circumsporozoite (CS) protein. A total of 356 An. neomaculipalpus were collected, of which three (0.84%) were positive for P. vivax, two for the variant 247 and one for the variant 210. The overall sporozoite rate in An. neomaculipalpus was similar to that for the principal vector An. (Nyssorhynchus) darlingi Root (0.82%) and higher than in An. (Nys.) marajoara Galvão & Damasceno (0.27%). This is the first report of An. neomaculipalpus naturally infected with Plasmodium parasites in Venezuela.     

Chemical Variation in Piper aduncum and Biological Properties of Its Dillapiole‐Rich Essential Oil

The essential oils of the specimens of Piper aduncum that occur in deforested areas of Brazilian Amazon, North Brazil, are rich in dillapiole (35–90%), a derivative of phenylpropene, to which are attributed biological properties. On the other hand, the oils of the specimens with occurrence in the Atlantic Forest, and Northeastern and Southeastern Brazil, do not contain dillapiole, but only terpene compounds such as (E)‐nerolidol and linalool. One specimen existing in the Amazon was hydrodistilled. The obtained oil was fractioned on a silica chromatographic column, resulting in fractions rich in dillapiole (95.0–98.9%) utilized for analyses by GC and GC/MS, structural characterization by NMR, confirmation of their biological properties, and to obtain the isomer isodillapiole. Dillapiole showed a fungicide action against the fungus Clinipellis perniciosa (witches' broom) by inhibition of its basidiospores, in concentrations ranging from 0.6 to 1.0 ppm. The larvicide and insecticide actions of dillapiole were tested against the larvae and the adult insects of Anopheles marajoara and Aedes aegypti (malaria and dengue mosquitoes), resulting in mortality of the larvae (48 h, 100%) at a concentration of 100 ppm, and mortality of the insects (30 min, 100%) at a concentration of 600 ppm. The isomeric isodillapiole showed no significant activity in the same biological tests.     

Development of simple sequence repeat markers for the plant pathogenic rust fungus,Puccinia graminis

Twenty‐four dinucleotide simple sequence repeat markers were developed for the phytopathogenic fungus, Puccinia graminis. The identified loci were polymorphic, with allelic diversity ranging from two to 11 alleles. Observed and expected levels of heterozygosity ranged from 0.000 to 0.960 and from 0.113 to 0.846, respectively. Fourteen of the loci deviated significantly from Hardy–Weinberg equilibrium. Null alleles were observed for 10 of the 24 loci with a frequency of 4–16%. A preliminary screen of other Puccinia cereal rust fungi (P. coronata, P. striiformis and P. triticina) indicated that these primer pairs are specific to P. graminis.     

Four New Flavonoid C-Glycosides Isolated from Achyranthes aspera and Their Nitric Oxide Production Inhibitory Activities

A chemical study of the methanol extract of the aerial parts of Achyranthes aspera led to the isolation of four new flavonoid C-glycosides ( 1 – 4 ) along with eight known analogs ( 5 – 12 ). Their structures were elucidated by a combination of spectroscopic data analysis, HR-ESI-MS, 1D and 2D NMR spectra. All the isolates were evaluated their NO production inhibitory activity in LPS-activated RAW264.7 cells. Compounds 2 , 4 , and 8 – 11 showed significant inhibition with IC₅₀ values ranging from 25.06 to 45.25 μM, compared to that of the positive control compound, L-NMMA, IC₅₀ value of 32.24 μM, whereas the remaining compounds were weak inhibitory activity with IC₅₀ values over 100 μM. This is the first report of 7 from Amaranthaceae family, and 11 from the genus Achyranthes.     

Development and characterization of microsatellite markers for an endangered shrub, <Emphasis Type="Italic">Ammopiptanthus mongolicus</Emphasis> (Leguminosae) and cross-species amplification in <Emphasis Type="Italic">Ammopiptanthus nanus</Emphasis>

We developed 11 polymorphic microsatellite markers for an endangered shrub Ammopiptanthus mongolicus using the Fast Isolation by AFLP of Sequences Containing repeats protocol. Polymorphism of each locus was assessed in 24 individuals from five natural populations of A. mongolicus. The average allele number of these markers was 4.2, ranging from 2 to 7. The observed (H _O) and expected (H _E) heterozygosities were 0.044–0.956 and 0.044–0.827, respectively. Further assessment in the congeneric species A. nanus revealed that nine of eleven loci were successfully amplified, but only two loci showed polymorphism. These markers provide powerful tools for the conservation genetics studies of A. mongolicus.     

Geographic distribution,evolution, and disease importance of species within the Neotropical Anopheles albitarsis Group (Diptera,Culicidae)

The Anopheles albitarsis group of mosquitoes comprises eight recognized species and one mitochondrial lineage. Our knowledge of malaria vectorial importance and the distribution and evolution of these taxa is incomplete. We constructed ecological niche models (ENMs) for these taxa and used hypothesized phylogenetic relationships and ENMs to investigate environmental and ecological divergence associated with speciation events. Two major clades were identified, one north (Clade 1) and one south (Clade 2) of the Amazon River that likely is or was a barrier to mosquito movement. Clade 1 species occur more often in higher average temperature locations than Clade 2 species, and taxon splits within Clade 1 corresponded with a greater divergence of variables related to precipitation than was the case within Clade 2. Comparison of the ecological profiles of sympatric species and sister species support the idea that phylogenetic proximity is related to ecological similarity. Anopheles albitarsis I, An. janconnae, and An. marajoara ENMs had the highest percentage of their predicted suitable habitat overlapping distribution models of Plasmodium falciparum and P. vivax, and warrant additional studies of the transmission potential of these species. Phylogenetic proximity may be related to malaria vectorial importance within the Albitarsis Group.     

Electrospun Ultrafine Fibers from Black Bean Protein Concentrates and Polyvinyl Alcohol

In this study, ultrafine fibers were produced from black bean protein concentrates (BPCs) and polyvinyl alcohol (PVA) by electrospinning. The BPC was denatured under acidic (pH 2) or basic (pH 11) conditions. Polymer solutions containing different PVA concentrations (11% or 21%, w/v) and different BPC: PVA ratios (50:50 or 75:25, v/v) were used for fiber production. The electrical conductivity and rheological properties of the fiber-forming solutions were evaluated, as well as the morphology, size distribution, infrared spectrum, and thermal properties of the electrospun fibers. The fibers showed a homogeneous morphology and diameters ranging from 115 to 541 nm. Fibers from the solution containing BPC denatured at pH 11, 11% PVA, and 75:25 (v/v) BPC: PVA presented the lowest diameter, and those from BPC denatured at pH 2 had less beads than the fibers obtained from BPC denatured at pH 11. The solution formulation affected the thermal properties of the fibers, with weight loss increases ranging from 39.0% to 60.9%. The polymeric solutions containing PVA and BPC (whether denatured under basic or acidic conditions) resulted in ultrafine electrospun fibers with highly favorable characteristics that could potentially be used for the encapsulation of bioactive compounds and food applications.

     

Mycotoxin production by Fusarium species isolated from New Zealand maize fields

Forty Fusarium isolates obtained from maize fields were screened for moniliformin production on maize kernels. Twelve isolates, including seven of F. subglutinans, were found to produce moniliformin at levels ranging from 0.4 to 64 ppm. Twenty six isolates were also screened for production of deoxynivalenol, diacetoxyscirpenol, T-2 toxin and zearalenone. Of these, 22, including all 11 isolates of F. graminearum, produced zearalenone at levels ranging from 0.1 to 96.0 ppm, while 13 produced T-2 toxin at low levels, (<1.1 ppm). Deoxynivalenol and diacetoxyscirpenol were each produced by six isolates, also at low levels (<1.0 ppm). Three isolates of F. graminearum and one of F. sambucinum produced four toxins simultaneously.     

Development and characterization of eight polymorphic microsatellite markers for Daphnia atkinsoni (Crustacea: Ctenodaphnia)

A microsatellite‐enriched genomic library was developed for the water flea Daphnia atkinsoni Baird, 1859, a dominant species of intermittent wetlands in Europe. Eight polymorphic microsatellite markers were successfully optimized. Characterization of 77 individuals from Belgium and Spain showed moderate (in the former) to high (in the latter) levels of polymorphism with two to 11 alleles per locus and an observed heterozygosity ranging from 0 to 0.87. Some of these microsatellite markers were successfully amplified in three other Daphnia species (D. magna n = 4, D. similis n = 6; D. obtusa n = 6).     

Purification of a diagnostic, secreted cysteine protease-like protein from the hookworm Ancylostoma caninum

The enteric infection of humans with the canine hookworm Ancylostoma caninum varies in its clinical presentation, ranging from asymptomatic to eosinophilic gastroenteritis requiring surgical intervention. Infections are not patent, but can be diagnosed immunologically by detecting antibodies to an immunodominant secreted hookworm protein termed Ac68. To characterise Ac68, we purified the native protein from A. caninum excretory/secretory products using size exclusion followed by anion exchange chromatography. The epitopes in the purified protein recognised by human infection sera were shown to be proteins and not carbohydrates. The N-terminal amino acid sequence of the purified Ac68 was determined and six of the 11 residues obtained were shared with a previously characterised cysteine protease of A. caninum, AcCP1.     

Expression of trypsin-like serine peptidases in pre-imaginal stages of Aedes aegypti (Diptera: Culicidae)

This study reports the biochemical characterization and comparative analyses of highly active serine proteases in the larval and pupal developmental stages of Aedes aegypti (Linnaeus) using substrate‐SDS‐PAGE. Zymographic analysis of larval stadia detected proteolytic activity in 6–8 bands with apparent molecular masses ranging from 20 to 250 kDa, with activity observed from pH 5.5 to 10.0. The pupal stage showed a complex proteolytic activity in at least 11 bands with apparent Mr ranging from 25 to 250 kDa, and pH optimum at 10.0. The proteolytic activities of both larval and pupal stages were strongly inhibited by phenyl‐methyl sulfonyl‐fluoride and N‐α‐Tosyl‐L ‐lysine chloromethyl ketone hydrochloride, indicating that the main proteases expressed by these developmental stages are trypsin‐like serine proteases. The enzymes were active at temperatures ranging from 4 to 85°C, with optimal activity between 37 and 60°C, and low activity at 85°C. Comparative analysis between the proteolytic enzymes expressed by larvae and pupae showed that substantial changes in the expression of active trypsin‐like serine proteases occur during the developmental cycle of A. aegypti. © 2011 Wiley Periodicals, Inc.     

Isolation and characterization of chloroplast microsatellite markers in four mangrove species,Aegiceras corniculatum,Avicennia marina,Acanthus ilicifolius and Lumnitzera racemosa

One, three, seven, and six of polymorphic chloroplast microsatellite (cpSSR) markers were developed from four mangrove species, Acanthus ilicifolius, Aegiceras corniculatum, Avicennia marina, and Lumnitzera racemosa, respectively. Characterization of 229, 509, 369, and 216 individuals of A. ilicifolius, A. corniculatum, A. marina, and L. racemosa, collected from different natural mangrove populations (A. ilicifolius, 6; A. corniculatum, 14; A. marina, 10; L. racemosa, 6) in the southern coastline of China showed that these loci provide cpSSR markers with polymorphisms ranging from two to four alleles per locus and gene diversity between 0.005 and 0.675. Combining the polymorphic cpSSR loci of each species, 3, 5, 11, and 4 of cpSSR haplotypes were separately detected in populations of A. ilicifolius, A. corniculatum, A. marina, and L. racemosa in the southern coastline of China. These cpSSR markers will be useful for analyzing the maternal lineage distributions and population genetic structures of these four species.     

Eleven polymorphic microsatellite loci for the salmonid trematode Plagioporus shawi

We isolated and characterized 11 microsatellite loci from Plagioporus shawi, a parasitic trematode of salmonid fishes (Oncorhynchus spp.). Extensive polymorphism (expected heterozygosities ranging from 0.476 to 0.981 and number of alleles from 10 to 55) was found in a sample of 94 trematodes from 24 infected cutthroat trout (Oncorhynchus clarki) individuals. Nine of the 11 loci will be useful for future genetic studies on within population dynamics of P. shawi.     

Aflatoxin B1 producing potential of isolates of Aspergillus flavus Link ex Fries from cotton,maize and wheat

Twenty-one isolates ofAspergillus flavus Link ex Fries obtained from cotton, maize and wheat were screened for their ability to produce aflatoxins on two liquid media. Of these, sixteen isolates were toxigenic and produced only aflatoxin B₁ as assessed by bioassay on okra seedlings and TLC method. For screening isolates ofA. flavus for aflatoxin formation, 0.7 % YES+ Salt medium was found to be good as also for obtaining higher yields of the toxin. Isolates ofA. flavus produced aflatoxin B₁ ranging from 0.85 to 17.2 mg/50 ml. Maximum yield of aflatoxin was obtained when rice was used as the substrate in case of toxigenic isolates L-27 and C-9, and on maize in isolate M-11.     

A luxCDABE-based bioluminescent bioreporter for the detection of phenol

A bioluminescent reporter strain, Acinetobacter sp. DF4-8, was constructed for the detection of phenol by inserting a mopR-like promoter upstream of the Vibrio fischeri bioluminescent luxCDABE gene cassette in a modified mini-Tn5 construct. When introduced into the chromosome of Acinetobacter sp. DF4, the bioreporter produced a sensitive bioluminescent response to phenol at concentrations ranging from 2.5 to 100 ppm. This response was linear (R ²=0.986) in the range from 20 to 90 ppm. A significant bioluminescent response was also recorded when strain DF4-8 was incubated with slurries from aged, phenol-contaminated soil. Received 13 February 2002/ Accepted in revised form 11 July 2002     

New microsatellite loci for estimating genetic diversity and structure in Octopus hubbsorum from Nayarit,México

Background

Octopus hubbsorum Berry, 1953 is the most important species for commercial fishing in the Mexican Pacific. However, there is a lack of information regarding population structure that could have important management implications. We tested 44 microsatellite loci in O. hubbsorum by cross-amplification from O. bimaculatus.

Methods and results

Genetic diversity and structure was tested over 30 octopus sampled from Santa Cruz de Miramar (Nayarit, México). A total of 11 loci were successfully amplified. All loci were polymorphic with the number of effective alleles ranging from 2.13 to 23.14, while three loci significantly deviated from Hardy-Weinberg equilibrium. No significant LD was observed between pairs of loci (P?≥?0.05). The application of the new markers in a O. hubbsorum population from Santa Cruz de Miramar Nayarit, México, did not showed Wahlund or isolate breaking effects due to the mixing of distinct populations.

Conclusions

The loci were useful to estimate levels of pairwise relatedness and to discard the presence of recent demographic bottlenecks in the population. We consider that eight microsatellites are adequate from the 11 amplified loci

     

Isolation and characterization of 18 microsatellites in the Peking duck (Anas platyrhynchos) and their application in other waterfowl species

We have isolated and characterized 18 microsatellite loci in the Peking duck (Anas platyrhynchos). The average number of alleles per locus was 3.5, ranging from one to six in domestic Peking ducks (n = 40). All of the markers were polymorphic in a sample of five mallards (A. platyrhynchos; two to eight alleles). Seventeen of the 18 markers amplified in Muscovy ducks (Cairina moschata) with 11 being polymorphic in our sample (n = 14). Amplification of the markers in different species comprising the subfamilies Anatinae and Anserinae indicates their potential value for population genetic applications in a wide range of waterfowl species.