Systematics and Population Level Analysis of Anopheles darlingi

A new phylogenetic analysis of the Nyssorhynchus subgenus (Danoff-Burg and Conn, unpub. data) using six data sets {morphological (all life stages); scanning electron micrographs of eggs; nuclear ITS2 sequences; mitochondrial COII, ND2 and ND6 sequences} revealed different topologies when each data set was analyzed separately but no heterogeneity between the data sets using the arn test. Consequently, the most accurate estimate of the phylogeny was obtained when all the data were combined. This new phylogeny supports a monophyletic Nyssorhynchus subgenus but both previously recognized sections in the subgenus (Albimanus and Argyritarsis) were demonstrated to be paraphyletic relative to each other and four of the seven clades included species previously placed in both sections. One of these clades includes both Anopheles darlingi and An. albimanus, suggesting that the ability to vector malaria effectively may have originated once in this subgenus. Both a conserved (315 bp) and a variable (425 bp) region of the mitochondrial COI gene from 15 populations of An. darlingi from Belize, Bolivia, Brazil, French Guiana, Peru and Venezuela were used to examine the evolutionary history of this species and to test several analytical assumptions. Results demonstrated (1) parsimony analysis is equally informative compared to distance analysis using NJ; (2) clades or clusters are more strongly supported when these two regions are combined compared to either region separately; (3) evidence (in the form of remnants of older haplotype lineages) for two colonization events; and (4) significant genetic divergence within the population from Peixoto de Azevedo (State of Mato Grosso, Brazil). The oldest lineage includes populations from Peixoto, Boa Vista (State of Roraima) and Dourado (State of São Paulo).

Numerical Taxonomy of Old World Phlebotominae (Diptera: Psychodidae): 1. Considerations of Morphological Characters in the Genus Phlebotomus Rondani & Berté 1840

Numerical analyses (correspondence analysis, ascending hierarchical classification, cladistic approach) were applied to the morphological characters of the adults of the genus Phlebotomus Rondani & Berté 1840. They confirm the reliability of the classic classifications, and also redefine the taxonomic and phylogenetic position of certain taxa. Thus, Spelaeophlebotomus Theodor 1948, Idiophlebotomus Quate & Fairchild 1961 and Australophlebotomus Theodor 1948 deserve generic rank. Among the vectors of leishmaniasis, the subgenus Phlebotomus Rondani & Berté 1840 is probably ancient. The results attribute an intermediate taxonomic and phylogenetic position to the taxa Euphlebotomus Theodor 1948 and Anaphlebotomus Theodor 1948, and reveal the probable artificial nature of the latter. The comparatively large numbers of species of subgenera Paraphlebotomus Theodor 1948, Synphlebotomus Theodor 1948 and, above all, Larroussius Nitzulescu 1931 and Adlerius Nitzulescu 1931, suggest that they are relatively recent. The development of adult morphological characters, the validity of their use in taxonomy and proposals for further studies are discussed.

Numerical Taxonomy of Old World Phlebotominae (Diptera: Psychodidae): 2. Restatement of Classification upon Subgeneric Morphological Characters

Numerical analyses (correspondence analysis, ascending hierarchical classification, and cladistics) were done with morphological characters of adult phlebotomine sand flies. The resulting classification largely confirms that of classical taxonomy for supra-specific groups from the Old World, though the positions of some groups are adjusted. The taxa Spelaeophlebotomus Theodor 1948, Idiophlebotomus Quate & Fairchild 1961, Australophlebotomus Theodor 1948 and Chinius Leng 1987 are notably distinct from other Old World groups, particularly from the genus Phlebotomus Rondani & Berté 1840. Spelaeomyia Theodor 1948 and, in particular, Parvidens Theodor & Mesghali 1964 are clearly separate from Sergentomyia França & Parrot 1920.

Anopheles albitarsis Embryogenesis: Morphological Identification of Major Events

Anopheles albitarsis embryogenesis was analyzed through confocal microscopy of clarified eggs. Using Drosophila melanogaster as reference system, the major morphogenetic events (blastoderm, gastrulation, germ band extension, germ band retraction, dorsal closure) were identified. The kinetics of early events is proportionally similar in both systems, but late movements (from germ band retraction on) progress slower in An. albitarsis. Major differences in An. albitarsis related to D. melanogaster were: (1) pole cells do not protrude from the blastoderm; (2) the mosquito embryo undergoes a 180º rotation movement, along its longitudinal axis; (3) the head remains individualized throughout embryogenesis; (4) extraembryonary membranes surround the whole embryo. A novel kind of malaria control is under development and is based on the use of genetically modified mosquitoes. Phenotypic analysis of the embryonic development of mutants will be imposed as part of the evaluation of effectiveness and risk of employment of this strategy in the field. In order to accomplish this, knowledge of the wild type embryo is a prerequisite. Morphological studies will also serve as basis for subsequent development biology approaches.

Analysis of the first and second internal transcribed spacer sequences of the ribosomal DNA in Biomphalaria tenagophila complex (Mollusca: Planorbidae)

The first and second internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA of Biomphalaria tenagophila complex (B. tenagophila, B. occidentalis, and B. t. guaibensis) were sequenced and compared. The alignment lengths of these regions were about 655 bp and 481 bp, respectively. Phylogenetic relationships among the Biomphalaria species were inferred by Maximum Parsimony and Neighbor-joining methods. The phylogenetic trees produced, in most of the cases, were in accordance with morphological systematics and other molecular data previously obtained by polymerase chain reaction and restriction fragment length polymorphism analysis. The present results provide support for the proposal that B. tenagophila represents a complex comprising B. tenagophila, B. occidentalis and B. t. guaibensis.

Isoenzimatic analysis of four Anopheles (Kerteszia) cruzii (Diptera: Culicidae) populations of Brazil

Anopheles cruzii is a small sylvatic mosquito and primary human Plasmodium vector in Southern Brazil. The distribution of this bromeliad-breeding mosquito follows the Atlantic forest coastal distribution, where bromeliads are abundant. Morphological, genetic, and molecular polymorphisms among different populations have been reported and it has recently been suggested that An. cruzii is a complex of cryptic species. The aim of this work is to analyze the gene flow between different populations of An. cruzii collected in four localities within the geographic distribution range of the species, and to examine if An. cruzii is a complex of cryptic species. The genetic distances show that populations of the states of Santa Catarina, São Paulo, and Rio de Janeiro are genetically closer (0.032 to 0.083) than populations of Bahia (0.364 to 0.853) based on profiles from 10 distinct isoenzyme loci. The Fst was lower (0.077) when the Bahia population was excluded than when it was included (0.300) in the analyses. The inferred number of migrants per generation was 2.99 individuals among populations from the states of Santa Catarina, São Paulo, and Rio de Janeiro and 0.58 migrants per generation among all populations. Results suggest that An. cruzii is a complex of species and that the specimes of state of Bahia can be considered as belonging to a species that is distinct from other three closely-related populations studied.

Molecular and morphological characterization of heterorhabditid entomopathogenic nematodes from the tropical rainforest in Brazil

Despite massive losses of primary forest, the Amazonian rainforest remains an extremely rich source of biodiversity. In recent years, entomopathogenic nematodes (EPNs) have been isolated from soil in various parts of the world and used successfully as biological control agents against numerous insect pests. Therefore, a sampling in the rainforest of Monte Negro, Rondônia, Brazil was conducted with the aim of discovering new strains and/or species of EPNs for future development as biological control agents. From 156 soil samples taken at nine collecting sites, 19 isolates were obtained, all of them belonging to the genus Heterorhabditis. Four strains were subjected to detailed morphological and molecular evaluation. Based on morphometrics and internal transcribed spacer (ITS) sequence data, the strains LPP1, LPP2 and LPP4 were identified as Heterorhabditis indica, whereas LPP7 was considered Heterorhabditis baujardi. Comparative analysis of the ITS1 sequence of H. indica and H. baujardi isolates showed a polymorphic site for the restriction enzyme Tth 111 that could be used to distinguish the two species. Consequently, strains LPP1, LPP2, LPP3, LPP4, and LPP9 were identified as H. indica, whereas LPP5, LPP7, LPP8 and LPP10 were identified as H. baujardi.

Morphological and molecular description of Blastocrithidia cyrtomeni sp. nov. (Kinetoplastea: Trypanosomatidae) associated with Cyrtomenus bergi Froeschner (Hemiptera: Cydnidae) from Colombia

A new trypanosomatid species, Blastocrithidia cyrtomeni, is herein described using morphological and molecular data. It was found parasitising the alimentary tract of the insect host Cyrtomenus bergi, a polyphagous pest. The morphology of B. cyrtomeni was investigated using light and transmission microscopy and molecular phylogeny was inferred from the sequences of spliced leader RNA (SL rRNA) - 5S rRNA gene repeats and the 18S small subunit (SSU) rRNA gene. Epimastigotes of variable size with straphanger cysts adhering to the middle of the flagellum were observed in the intestinal tract, hemolymph and Malpighian tubules. Kinetoplasts were always observed anterior to the nucleus. The ultrastructure of longitudinal sections of epimastigotes showed the flagellum arising laterally from a relatively shallow flagellar pocket near the kinetoplast. SL RNA and 5S rRNA gene repeats were positive in all cases, producing a 0.8-kb band. The amplicons were 797-803 bp long with > 98.5% identity, indicating that they originated from the same organism. According to the sequence analysis of the SL-5S rRNA gene repeats and the 18S SSU rRNA gene, B. cyrtomeni is different from all other known species or isolates of Trypanosomatidae. Both analyses indicate that among known species, it is most closely related to Blastocrithidia triatomae.

Genetic and morphological characterisation of a new species of the genus Hysterothylacium (Nematoda) from Paralichthys isosceles Jordan, 1890 (Pisces: Teleostei) of the Neotropical Region, state of Rio de Janeiro, Brazil

Taking into account the difficulties of taxonomic identification of larval anisakid nematodes based on morphological characters, genetic analyses were performed, together with those usually applied, in order to identify anisakid larvae found in the flounder Paralichthys isosceles from the littoral of the state of Rio de Janeiro, Brazil. The analysis of 1,820 larvae revealed a new species, similar to Hysterothylacium MD, Hysterothylacium 2, Hysterothylacium KB and Hysterothylacium sp regarding the absence of the larval tooth, an excretory pore situated below the nerve ring level, and slender lateral alae. Moreover, the new species differs from Hysterothylacium fortalezae and Hysterothylacium reliquens with regard to the number and size of spines present on the tail end and from Hysterothylacium patagonicus by the absence of interlabia. The maximum parsimony and neighbour joining tree topologies based on the 18S ribosomal DNA gene, complete internal transcribed spacer region and cytochrome oxidase 2 (COII) gene demonstrated that the Brazilian larvae belong to Raphidascarididae and represent a unique genetic entity, confirmed as a new Hysterothylacium species. Furthermore, the new species presents COII genetic signatures and shares polymorphisms with Raphidascarididae members. This is the first description of a new anisakid species from Brazil through the integration of morphological and molecular taxonomy data.

Potential sources of Triatoma infestans reinfesting peridomiciles identified by morphological characterization in Los Llanos, La Rioja, Argentina

The presence of Triatoma infestans in habitats treated with insecticides constitutes a frequent problem in endemic areas. Basing our study on the hypothesis that descendants of a residual population should be more similar to the pre-treatment population than to any other, we compared the indications of two quantitative morphological approaches. This study seeks to find the origin of 247 T. infestans from three populations found in two chicken coops and a goat corral after treatment with insecticides. The results obtained by quantitative morphology suggest that the T. infestans found between three-34 months after the application of insecticides formed mixed populations with insects derived from residual foci and neighbouring habitats. Our analyses also showed the presence of a phenotype which does not resemble neither the pre-treatment phenotype nor the one from neighbouring populations, suggesting the presence of a particular post-treatment phenotype. The heads size showed some variations in males from different populations and remained unchanged in females, which reinforces the hypothesis of an intraspecific competition for food with priority for females. This article presents, for the first time, the combined analysis of geometric morphometry of heads and antennal phenotypes to identify the composition of reinfesting populations.

Silver nanoparticle production by the fungus Fusarium oxysporum: nanoparticle characterisation and analysis of antifungal activity against pathogenic yeasts

The microbial synthesis of nanoparticles is a green chemistry approach that combines nanotechnology and microbial biotechnology. The aim of this study was to obtain silver nanoparticles (SNPs) using aqueous extract from the filamentous fungus Fusarium oxysporum as an alternative to chemical procedures and to evaluate its antifungal activity. SNPs production increased in a concentration-dependent way up to 1 mM silver nitrate until 30 days of reaction. Monodispersed and spherical SNPs were predominantly produced. After 60 days, it was possible to observe degenerated SNPs with in additional needle morphology. The SNPs showed a high antifungal activity against Candida and Cryptococcus , with minimum inhibitory concentration values ≤ 1.68 µg/mL for both genera. Morphological alterations of Cryptococcus neoformans treated with SNPs were observed such as disruption of the cell wall and cytoplasmic membrane and lost of the cytoplasm content. This work revealed that SNPs can be easily produced by F. oxysporum aqueous extracts and may be a feasible, low-cost, environmentally friendly method for generating stable and uniformly sized SNPs. Finally, we have demonstrated that these SNPs are active against pathogenic fungi, such as Candida and Cryptococcus .

Phylogenetic relationships among four species of the guarani group of Drosophila (Diptera, Drosophilidae) as inferred by molecular and morphological analyses

Traditionally, the Drosophila guarani species group has been divided into two subgroups: the guarani and the guaramunu subgroups. Two, out of the four species included in this research, are members of the guarani subgroup (D. ornatifrons Duda, 1927 and D. subbadia Paterson & Mainland, 1943) and two are included in the guaramunu subgroup (D. maculifrons Duda, 1927 and D. griseolineata Duda, 1927). However, some authors have suggested that D. maculifrons and D. griseolineata are much closer to some species of the Drosophila tripunctata group than to some of the species of the guarani group. To add new data to the matter under dispute, Polyacrylamide Gel Eletrophoresis (PAGE-SDS) was used for the analysis and comparison of protein composition and Random Amplified Polymorphic DNA (RAPD) analysis to find differences in genomic DNA, in addition to the analysis of quantitative morphological characters previously described. Analysis of PAGE-SDS results in a dendrogram that pointed out D. subbadia as being the most distant within the Drosophila guarani group. However, these results were not supported either by RAPD analysis or by the analysis of continuous morphological characters, which supplied the clustering of D. subbadia with D. ornatifrons. Although our data give strong support to the clustering of D. subbadia and D. ornatifrons, none of the dendrograms provided a clade comprising D. maculifrons and D. griseolineata. Thus, this research does not support the traditional subdivision of the D. guarani group into those two subgroups.

Description and phylogenetic analysis of the Calycopidina (Lepidoptera, Lycaenidae, Theclinae, Eumaeini): a subtribe of detritivores

Description and phylogenetic analysis of the Calycopidina (Lepidoptera, Lycaenidae, Theclinae, Eumaeini): a subtribe of detritivores. The purpose of this paper is to establish a phylogenetic basis for a new Eumaeini subtribe that includes those lycaenid genera in which detritivory has been recorded. Morphological characters were coded for 82 species of the previously proposed "Lamprospilus Section" of the Eumaeini (19 of these had coding identical to another species), and a phylogenetic analysis was performed using the 63 distinct ingroup terminal taxa and six outgroups belonging to four genera. Taxonomic results include the description in the Eumaeini of Calycopidina Duarte & Robbins new subtribe (type genus Calycopis Scudder, 1876), which contains Lamprospilus Geyer, Badecla Duarte & Robbins new genus (type species Thecla badaca Hewitson), Arzecla Duarte & Robbins new genus (type species Thecla arza Hewitson), Arumecla Robbins & Duarte, Camissecla Robbins & Duarte, Electrostrymon Clench, Rubroserrata K. Johnson & Kroenlein revalidated status, Ziegleria K. Johnson, Kisutam K. Johnson & Kroenlein revalidated status, and Calycopis. Previous "infratribe" names Angulopina K. Johnson & Kroenlein, 1993, and Calycopina K. Johnson & Kroenlein, 1993, are nomenclaturally unavailable and polyphyletic as proposed. New combinations include Badecla badaca (Hewitson), Badecla picentia (Hewitson), Badecla quadramacula (Austin & K. Johnson), Badecla lanckena (Schaus), Badecla argentinensis (K. Johnson & Kroenlein), Badecla clarissa (Draudt), Arzecla arza (Hewitson), Arzecla tarpa (Godman & Salvin), Arzecla canacha (Hewitson), Arzecla calatia (Hewitson), Arzecla tucumanensis (K. Johnson & Kroenlein), Arzecla sethon (Godman & Salvin), Arzecla nubilum (H. H. Druce), Arzecla paralus (Godman & Salvin), Arzecla taminella (Schaus), Arzecla albolineata (Lathy), Electrostrymon denarius (Butler & H.Druce), Electrostrymon guzanta (Schaus), Electrostrymon perisus (H. H. Druce), Rubroserrata mathewi (Hewitson), Rubroserrata ecbatana (Hewitson), Kisutam micandriana (K. Johnson), and Kisutam syllis (Godman & Salvin). The structure of the male genitalia lateral window, labides, and brush organs are described and discussed, as are the female genitalia signa of the corpus bursae and 8th abdominal tergum. Widespread wing pattern sexual dimorphism in the Calycopidina is noted and illustrated, and the presence of alternating dark and light bands on the ventral wings of both sexes is discussed. The evidence for detritivory in Lamprospilus, Badecla, Arzecla, Arumecla, Camissecla, Electrostrymon, Ziegleria, Kisutam, and Calycopis is summarized using the new classification.

Morphological and physiological variation between queens and workers of Protonectarina sylveirae (de Saussure) (Hymenoptera, Vespidae, Epiponini)

Morphological and physiological variation between queens and workers of Protonectarina sylveirae (de Saussure) (Hymenoptera, Vespidae, Epiponini). The Neotropical swarm-founding wasps, Epiponini, range from the absence of morphological differentiation between castes to highly distinct castes. We measured eight body parts of females of two colonies of Protonectarina sylveirae (de Saussure, 1854). ANOVA and Discriminant Analysis evidenced significant differences between castes, as previously observed by other authors for other species of Epiponini. However, some females previously categorized as queens, were actually workers, supported by our statistic analyses. These individuals showed intermediate morphological features between queens and workers, having distinct patterns of hairs and clypeal spots. The castes of P. sylveirae are distinct, however intermediate individuals may be found in colonies promoting social flexibility.

Toxicological and ultrastructural analysis of the impact of pesticides used in temperate fruit crops on two populations of Chrysoperla externa (Neuroptera, Chrysopidae)

This study aimed to evaluate the effects of (g a.i. L-1) abamectin (0.02), carbaryl (1.73), sulphur (4.8), fenitrothion (0.75), methidathion (0.4), and trichlorfon (1.5) on the survival of larvae and pupae, on the oviposition of adults and hatching of eggs from treated Chrysoperla externa third-instar larvae from two different populations (Bento Gonçalves and Vacaria, Rio Grande do Sul State, Brazil). Morphological changes caused by abamectin to eggs laid by C. externa from Vacaria population were evaluated by mean of ultrastructural analysis. The pesticides were applied on glass plates. Distilled water was used as control. For the evaluation of larvae mortality, a fully randomized experimental design in a 2 x 7 (two populations x seven treatments) factorial scheme was used, whereas for the effects of the compounds on oviposition capacity and egg viability, a 2 x 4 factorial scheme was used. Carbaryl, fenitrothion, and methidathion caused 100% mortality of larvae. Abamectin reduced the hatching of eggs from treated third-instar larvae of both populations; however, this pesticide presented highest toxicity on insects from Vacaria. The ultrastructural analysis showed that abamectin caused malformations in micropyle and in chorion external surface of C. externa eggs. Based in the total effect (E), carbaryl, fenitrothion, and methidathion are harmful to C. externa; trichlorfon is harmless to third-instar larvae, while abamectin and sulphur are harmless and slightly harmful to third-instar larvae from Bento Gonçalves and Vacaria, respectively.