Morphometric discrimination of females of five species of Anopheles of the subgenus Nyssorhynchus from Southern and Northwest Colombia

The most important vectors of human Plasmodium in the neotropics belong to the subgenus Nyssorhynchus. These species are generally sympatric in terms of their geographical distributions. Some are difficult to identify based solely on examination of adult females using the available morphological keys, in these cases examination of immature stages and male genitalia is required to make correct determinations. However, in epidemiological studies it is necessary to identify the species of adult females which are found near humans, i.e. in studies of malaria transmission or evaluation of control measures. The purpose of the present study was to evaluate the discrimination of adult females of different species of Nyssorhynchus isolated mainly from Southern Colombia (department of Putumayo), using morphometric analysis. Adult females were obtained after rearing larvae collected in natural breeding places and from the progeny of females collected on humans. The morphological characteristics of the immature stages allowed the identification of four species of the subgroup Oswaldoi from Southern Colombia: Anopheles rangeli Gabaldon, Cova Garcia & Lopez, An. oswaldoi (Peryassu), An. benarrochi Gabaldon, Cova Garcia & Lopez and An. triannulatus (Neiva & Pinto). The species An. nuneztovari (Gabaldon) from the Northwest of Colombia was included for comparison. Morphometric analysis allowed differentiation of the females of all species to a confidence level approaching 90% using principal components analysis of 10 wing and leg variables, followed by canonical variate analysis of the first four principal components. We conclude that morphometrics may represent a useful taxonomic tool for this group and that its use should be further studied.

Use of molecular methods in identification of Candida Species and evaluation of fluconazole resistance

The aim of this study was to evaluate the use of one of the molecular typing methods such as PCR (polymerase chain reaction) following by RFLP (restriction fragment length polymorphism) analysis in the identification of Candida species and then to differentiate the identified azole susceptible and resistant Candida albicans strains by using AP-PCR (arbitrarily primed-polymerase chain reaction). The identification of Candida species by PCR and RFLP analysis was based on the size and primary structural variation of rDNA intergenic spacer regions (ITS). Forty-four clinical Candida isolates comprising 5 species were included to the study. The amplification products were digested individually with 3 different restriction enzymes: HaeIII, DdeI, and BfaI. All the isolates tested yielded the expected band patterns by PCR and RFLP analysis. The results obtained from this study demonstrate that Candida species can be differentiated as C. albicans and non-C. albicans strains only by using HaeIII restriction enzyme and BfaI maintains the differentiation of these non-C. albicans species. After identification Candida species with RFLP analysis, C. albicans strains were included to the AP-PCR test. By using AP-PCR, fluconazole susceptible and resistant strains were differentiated. Nine fluconazole susceptible and 24 fluconazole resistant C. albicans were included to the study. Fluconazole resistant strains had more bands when evaluating with the agarose gel electrophoresis but there were no specific discriminatory band patterns to warrant the differentiation of the resistance. The identification of Candida species with the amplification of intergenic spacer region and RFLP analysis is a practical, short, and a reliable method when comparing to the conventional time-consuming Candida species identification methods. The fluconazole susceptibility testing with AP-PCR seems to be a promising method but further studies must be performed for more specific results.

Slime production and proteinase activity of Candida species isolated from blood samples and the comparison of these activities with minimum inhibitory concentration values of antifungal agents

Slime and proteinase activity of 54 strains consisting of 19 Candida parapsilosis and 35 C. albicans strains isolated from blood samples were investigated in this study. Ketoconazole, amphothericin B, and fluconazole susceptibility of Candida species were compared with slime production and proteinase activity of these species. For both Candida species, no correlation was detected between the slime activity and minimum inhibitory concentration (MIC) values of the three antifungal agents. For both Candida species no correlation was detected between the proteinase activity and the MIC values of amphothericin B, and fluconazole however, statistically significant difference, was determined between the proteinase activity and MIC values of ketoconazole (p = 0.007). Slime production was determined by using modified Christensen macrotube method and proteinase activity was measured by the method of Staib. Antifungal susceptibility was determined through the guidelines of National Committee for Laboratory Standards (NCCLS M27-A).

Anatomical studies of sibling species within neotropical lymnaeids, snail intermediate hosts of fascioliasis

Several anatomical parameters of the reproductive system have been used to distinguish Lymnaea cubensis from L. viatrix, the snail hosts of fascioliasis in South America and the Caribbean area. Three samples have been collected in the type localities of L. cubensis (Cuba), L. viatrix var. A ventricosa (Argentina, Río Negro Lower Valley), and L. viatrix var. B elongata (Peru, Lima), respectively. Only one parameter, the relative lengths of the penis sheath and preputium, showed significant differences between L. viatrix var. ventricosa and the two other taxa. None of the studied parameters separated L. cubensis from L. viatrix var. elongata.

Biology of three species of the Meccus phyllosomus complex (Hemiptera: Reduviidae: Triatominae) fed on blood of hens and rabbits

Aspects related to hatching, life time, number of blood meals to molt, mortality, feeding time and postfeed defecation delay for each instar of Meccus phyllosomus, M. mazzottii, and M. bassolsae, life-cycle were evaluated and compared in two cohorts of each of those three species, fed on hens or rabbits. No significant (p > 0.05) differences were recorded among cohorts fed on hens respect to cohorts fed on rabbits in M. phyllosomus and M. mazzottii and the average time of hatching was 21.5 days for cohorts fed on hens and 22.5 for cohorts fed on rabbits. Average egg-to-adult development times were no significant (p > 0.05) different between both cohorts of M. phyllosomus and M. mazzotti, independent of the blood meal source. The average span in days for each instar fed on hens was not significantly different to the average span for each instar fed on rabbits, when comparisons were made by species. The number of blood meals at each nymphal instar varied from 1 to 6 in both cohorts of each species. The mortality rates were higher on older nymphs, in both cohorts of M. phyllosomus and M. bassolsae, whereas they were higher on first instar nymphs on M. mazzottii. Mean feeding time was no significant (p > 0.05) different in triatomines fed on hens or fed on rabbits, when each species were compared separately. A similar number of nymphs of each cohort, completed the cycle. Defecation delay was no significant (p > 0.05) different when cohorts fed on hens and fed on rabbits were compared by species. Most of the studied parameters showed no significant (p > 0.05) differences among those cohorts fed on hens and for fed on rabbits, which could mean a high degree of association of those species with birds as much as mammals, under wild conditions, increasing their capacity to colonize human dwellings.

Biology of three species of North American Triatominae (Hemiptera: Reduviidae: Triatominae) fed on rabbits

Aspects related to hatching, lifetime, number of blood meals for molting, mortality, feeding time, and postfeeding defecation delay were evaluated and compared in each instar of three North American Triatominae: Triatoma gerstaeckeri, Triatoma lecticularia and Triatoma protracta, all of them fed on rabbits. No significant differences (p > 0.05) were found among the three species regarding mean hatching rate, which was close to 20 days. Egg-to-adult development times were significantly shorter (p < 0.05) for T. lecticularia. Number of blood meals for molting to next instar ranged from one to five for T. protracta, and from one to six for T. gerstaeckeri and T. lecticularia. Mortality rates were higher in younger nymphs of T. lecticularia and T. protracta, while rates in T. gerstaeckeri were higher in fifth-instar nymphs. Mean feeding time was longest in T. gerstaeckeri, followed by T. lecticularia. More than twice the number of T. gerstaeckeri nymphs completed the development process, if compared to the nymphs from the other two species. Defecation delay was less than 10 min for T. lecticularia, T. protracta and the youngest nymphs of T. gerstaeckeri. Results point out that these three species may be important potential vectors of Trypanosoma cruzi for human populations, in areas of Mexico where these species are currently present.

Characterization of Aspergillus species based on fatty acid profiles

Cellular fatty acid (FA) composition was utilized as a taxonomic tool to discriminate between different Aspergillus species. Several of the tested species had the same FA composition and different relative FA concentrations. The most important FAs were palmitic acid (C16:0), estearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2), which represented 95% of Aspergillus FAs. Multivariate data analysis demonstrated that FA analysis is a useful tool for differentiating species belonging to genus Aspergillus. All the species analyzed showed significantly FA acid profiles (p < 0.001). Furthermore, it will be possible to distinguish among Aspergillus spp. in the Flavi Section. FA composition can serve as a useful tool for the identification of filamentous fungi.

Characterization of the external female genitalia of six species of Triatominae (Hemiptera: Reduviidade) by scanning electron microscopy

By macroscopic and microscopic dorsal side observation, it was noted that the IX and X segments of two species each of Panstrongylus and Triatoma terminate in an elongated way, whereas they terminate abruptly in the two species of Rhodnius. Scanning observation of the dorsal, ventral, lateral and posterior sides of the female genitalia of Panstrongylus herreri, Panstrongylus megistus, Rhodnius colombiensis, Rhodnius prolixus, Triatoma infestans and Triatoma vitticeps revealed that these six species are generally and specifically distinguished based on these elements. We describe several components that distinguish P. herreri from P. megistus: four on the dorsal side: the VII, VIII, IX and X segments, on the ventral view, three: VII sternite, VIII gonocoxite and VIII gonapophyse, on the lateral view one character, VIII gonocoxite and on the posterior view three characters: VIII and IX gonocoxite and XI gonopophyse. Comparing R. colombiensis and R. prolixus, there were three distinct characters on the dorsal side: the VII, VIII and X segments, on the ventral view three characters: the IX and X segments and VIII gonocoxite and on the posterior view four characters: the VIII, IX, X segments and VIII gonapophyse that distinguish the two species. T. infestans and T. vitticeps have four different characters on the dorsal side: the VII, VIII, IX and X segments, on the ventral view four characters: the VII and X segments, VIII gonocoxite and VIII gonapophyse, on the lateral view two characters, IX and X segments and on the posterior view four characters: the IX and X segments, VIII gonocoxite and VIII gonapophyse that distinguish the two species. Examination of the external female genitalia of six triatomine species by scanning suggests that these components are useful for taxonomical studies.

Alternative PCR protocol using a single primer set for assessing DNA quality in several tissues from a large variety of mammalian species living in areas endemic for leishmaniasis

The aim of this work was to establish a modified pre-diagnostic polymerase chain reaction (PCR) protocol using a single primer set that enables successful amplification of a highly conserved mammalian sequence in order to determine overall sample DNA quality for multiple mammalian species that inhabit areas endemic for leishmaniasis. The gene encoding interphotoreceptor retinoid-binding protein (IRBP), but not other conserved genes, was efficiently amplified in DNA samples from tail skin, ear skin, bone marrow, liver and spleen from all of the species tested. In tissue samples that were PCR-positive for Leishmania, we found that DNA from 100%, 55% and 22% of the samples tested resulted in a positive PCR reaction for the IRBP, beta-actin and beta-globin genes, respectively. Nucleotide sequencing of an IRBP amplicon resolved any questions regarding the taxonomical classification of a rodent, which was previously based simply on the morphological features of the animal. Therefore, PCR amplification and analysis of the IRBP amplicon are suitable for pre-diagnostically assessing DNA quality and identifying mammalian species living in areas endemic to leishmaniasis and other diseases.

Distribution of sibling species of Anopheles culicifacies s.l. and Anopheles fluviatilis s.l. and their vectorial capacity in eight different malaria endemic districts of Orissa, India

The study was undertaken in eight endemic districts of Orissa, India, to find the members of the species complexes of Anopheles culicifacies and Anopheles fluviatilis and their distribution patterns. The study area included six forested districts (Keonjhar, Angul, Dhenkanal, Ganjam, Nayagarh and Khurda) and two non-forested coastal districts (Puri and Jagatsingpur) studied over a period of two years (June 2007-May 2009). An. culicifacies A, B, C and D and An. fluviatilis S and T sibling species were reported. The prevalence of An. culicifacies A ranged from 4.2-8.41%, B from 54.96-76.92%, C from 23.08-33.62% and D from 1.85-5.94% (D was reported for the first time in Orissa, except for occurrences in the Khurda and Nayagarh districts). The anthropophilic indices (AI) were 3.2-4.8%, 0.5-1.7%, 0.7-1.37% and 0.91-1.35% for A, B, C and D, respectively, whereas the sporozoite rates (SR) were 0.49-0.54%, 0%, 0.28-0.37% and 0.41-0.46% for A, B, C and D, respectively. An. fluviatilis showed a similarly varied distribution pattern in which S was predominant (84.3% overall); its AI and SR values ranged from 60.7-90.4% and 1.2-2.32%, respectively. The study observed that the co-existence of potential vector sibling species of An. culicifacies (A, C and D) and An. fluviatilis S (> 50%) was responsible for the high endemicity of malaria in forested districts such as Dhenkanal, Keonjhar, Angul, Ganjam, Nayagarh and Khurda (> 5% slide positivity rate). Thus, the epidemiological scenario for malaria is dependent on the distribution of the vector sibling species and their vectorial capacity.

Anthropogenic influence on the distribution, abundance and diversity of sandfly species (Diptera: Phlebotominae: Psychodidae), vectors of cutaneous leishmaniasis in Panama

In Panama, species of the genus Lutzomyia are vectors of American cutaneous leishmaniasis (ACL). There is no recent ecological information that may be used to develop tools for the control of this disease. Thus, the goal of this study was to determine the composition, distribution and diversity of Lutzomyia species that serve as vectors of ACL. Sandfly sampling was conducted in forests, fragmented forests and rural environments, in locations with records of ACL. Lutzomyia gomezi, Lutzomyia panamensis and Lutzomyia trapidoi were the most widely distributed and prevalent species. Analysis of each sampling point showed that the species abundance and diversity were greatest at points located in the fragmented forest landscape. However, when the samples were grouped according to the landscape characteristics of the locations, there was a greater diversity of species in the rural environment locations. The Kruskal Wallis analysis of species abundance found that Lu. gomezi and Lu. trapidoi were associated with fragmented environments, while Lu. panamensis, Lutzomyia olmeca bicolor and Lutzomyia ylephiletor were associated with forested environments. Therefore, we suggest that human activity influences the distribution, composition and diversity of the vector species responsible for leishmaniasis in Panama.

Molecular characterisation of Bartonella species in cats from São Luís, state of Maranhão, north-eastern Brazil

Bartonella species are fastidious bacteria that predominantly infect mammalian erythrocytes and endothelial cells and cause long-lasting bacteraemia in their reservoir hosts. Reports that describe the epidemiology of bartonellosis in Brazil are limited. This study aimed to detect and characterise Bartonella spp DNA from cat blood samples in São Luís, Maranhão, north-eastern Brazil. Among 200 cats tested for multiple genes, nine (4.5%) were positive for Bartonella spp: six cats for Bartonella henselae and three for Bartonella clarridgeiae. Based on the phylogenetic analysis of four genes, the B. henselae strain matched strains previously observed in Brazil and was positioned in the same clade as B. henselae isolates from the United States of America. Moreover, sequence alignment demonstrated that the B. clarridgeiae strain detected in the present study was the same as the one recently detected in cats from southern Brazil.

Molecular identification of Sporothrix species involved in the first familial outbreak of sporotrichosis in the state of Espírito Santo, southeastern Brazil

Sporotrichosis is a widespread subcutaneous mycosis caused by the dimorphic fungi now known as the Sporothrix schenckii complex. This complex is comprised of at least six species, including Sporothrix albicans, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei, Sporothrix mexicana and S. schenckii. Cases of sporotrichosis have significantly increased in Brazil over the past decade, especially in the state of Rio de Janeiro (RJ), where an epidemic among cat owners has been observed. The zoonotic transmission from cats to humans suggests a common source of infection and indicates that animals can act as vectors. We performed a molecular characterisation of samples collected during the first outbreak of familial sporotrichosis caused by S. brasiliensis in the state of Espírito Santo, Brazil. These results represent the first description of such an outbreak outside the endemic area of zoonotic sporotrichosis in RJ.

Preliminary data on the geographical distribution of Drosophila species within morphoclimatic domains of Brazil. III. The cardini group

A reanalysis, based on museum specimens, of our previously published data on the geographical distribution of the species of Drosophila belonging to the cardini group in Brazil is presented and discussed. As previously recorded in several papers, including ours, the following four species were recognized: D. cardini, D. cardinoides, D. neocardini, and D. polymorpha. However, it was realized that most of the flies we have previously identified as Drosophila cardinoides belong in fact to Drosophila cardini. To facilitate the proper identification of these four near-sibling species, their holotypes were analyzed and their terminalia were described and illustrated. A key to the four species is also provided.

Record of two species of Orius Wolff (Hemiptera, Anthocoridae) in Brazil

The genus Orius Wolff, 1811 comprises predatory species, with approximately 70 known species. Informations about the genus in Brazil are scarce. Therefore, the aim of this investigation was to identify Orius species present in four localities in the southeastern Brazil. Samples were taken from several plants, and the material screened in laboratory. The genitalia of both sexes were studied and illustrated. Two species were identified, Orius insidiosus (Say, 1832) and Orius thyestes Herring, 1966. O. insidiosus, the most common species, was collected in all of the localities sampled [Lavras (MG), Holambra, Pindorama and Campinas (SP)]. O. thyestes, registered for the first time in Brazil, occurred only in Lavras (MG) and Pindorama (SP). Some morphologic aspects of these two species are also presented.