Reproductive isolation between sympatric and allopatric Brazilian populations of Lutzomyia longipalpis s.l. (Diptera: Psychodidae)

Lutzomyia longipalpis s.l., the main vector of Leishmania chagasi in Latin America, is a species complex although the exact number of siblings is yet unknown. In Brazil, the siblings differ in male copulatory courtship songs and pheromones that most certainly act as pre-zygotic reproductive barriers. Here we analysed the reproductive isolation between three allopatric and two sympatric populations of Lu. longipalpis s.l. from Brazil. The results indicate a strong copulatory and pre-mating isolation between the three allopatric populations. In addition, the results also indicate a stronger pre-mating isolation between the two sympatric siblings than between the three allopatric ones, suggesting a role for reinforcement in the speciation of the Lu. longipalpis s.l. complex.

Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil

The molecular types of 443 Brazilian isolates of Cryptococcus neoformans and Cryptococcus gattii were analyzed to determine their geographic distribution within Brazil and their underlying host conditions. The following data, imported from previous epidemiological studies as well as two culture collections, were analyzed for: place of isolation, source (clinical or environmental), host risk factors, species, serotype, mating type, and molecular type. Molecular typing by PCR-fingerprinting using primers for the minisatellite-specific core sequence of the wild-type phage M13 or microsatellites [(GACA)4, (GTG)5], restriction fragment length polymorphism of URA5 gene analysis, and/or amplified fragment length polymorphism (AFLP) identified eight major genotypes: VNI/AFLP1, VNII/AFLP1A, VNIII/AFLP2, and VNIV/AFLP3 for C. neoformans, and VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5, and VGIV/AFLP7 for C. gattii. The most common molecular type found in Brazil was VNI (64%), followed by VGII (21%), VNII (5%), VGIII (4%), VGI and VNIV (3% each), and VNIII (< 1%). Primary cryptococcosis caused by the molecular type VGII (serotype B, MAT) prevails in immunocompetent hosts in the North and Northeast regions, disclosing an endemic regional pattern for this specific molecular type in the Northern Brazil.

The Pneumocystis life cycle

First recognised as "schizonts" of Trypanosoma cruzi, Pneumocystis organisms are now considered as part of an early-diverging lineage of Ascomycetes. As no robust long-term culture model is available, most data on the Pneumocystis cell cycle have stemmed from ultrastructural images of infected mammalian lungs. Although most fungi developing in animals do not complete a sexual cycle in vivo, Pneumocystis species constitute one of a few exceptions. Recently, the molecular identification of several key players in the fungal mating pathway has provided further evidence for the existence of conjugation and meiosis in Pneumocystisorganisms. Dynamic follow-up of stage-to-stage transition as well as studies of stage-specific proteins and/or genes would provide a better understanding of the still hypothetical Pneumocystislife cycle. Although difficult to achieve, stage purification seems a reasonable way forward in the absence of efficient culture systems. This mini-review provides a comprehensive overview of the historical milestones leading to the current knowledge available on the Pneumocystis life cycle.

Genotyping of the MTL loci and susceptibility to two antifungal agents of Candida glabrata clinical isolates

The opportunistic fungal pathogen Candida glabrata is the second most common isolate from bloodstream infections worldwide and is naturally less susceptible to the antifungal drug fluconazole than other Candida species. C. glabrata is a haploid yeast that contains three mating-type like loci (MTL), although no sexual cycle has been described. Strains containing both types of mating information at the MTL1 locus are found in clinical isolates, but it is thought that strains containing type a information are more common. Here we investigated if a particular combination of mating type information at each MTLlocus is more prevalent in clinical isolates from hospitalized patients in Mexico and if there is a correlation between mating information and resistance to fluconazole and 5-fluorocytosine. We found that while both types of information at MTL1 are equally represented in a collection of 64 clinical isolates, the vast majority of isolates contain a-type information at MTL2 and α-type at MTL3. We also found no correlation of the particular combination of mating type information at the three MTL loci and resistance to fluconazole.

Cryptococcus neoformans var. grubii - Pathogenicity of environmental isolates correlated to virulence factors, susceptibility to fluconazole and molecular profile

The pathogenicity of Cryptococcus neoformans is heterogeneous and is associated with the expression of virulence factors. This study aimed to correlate the pathogenicity of C. neoformans var. grubii in BALB/c mice with in vitro virulence factors, fluconazole minimal inhibitory concentrations (MICs) and molecular profiles, before and after animal passage. Ten environmental isolates and one ATCC strain of C. neoformans var. grubii mating type α were evaluated. Most isolates (91%) killed 50% or more of the infected animals by day 24 postinfection and were recovered from the lungs and brains of surviving animals on days 7 and 14 postinfection. The burden of yeast in the lungs was more variable than that in the brain. The differences in the expression of virulence factors (growth at 37ºC, presence and size of the capsule and production of melanin, urease, proteinase and phospholipase) by most isolates pre and postpassage in animals were not statistically significant. The fluconazole MICs in postpassaged lines differed by a one-dilution from the MIC of the corresponding prepassaged line for six isolates. Using molecular typing [polymerase chain reaction-fingerprinting with (GACA)4 and M13], eight isolates were identified as VNI and three as VNII. We concluded that different isolates with the same molecular and phenotypic profiles, including isolates that are markedly hypervirulent, span a wide range of virulence and there were no changes in virulence factors in the postpassaged lines when compared with the corresponding nonpassaged lines.

Morphological and morphometrical assessment of spermathecae of Aedes aegypti females

The vectorial capacity of Aedes aegypti is directly influenced by its high reproductive output. Nevertheless, females are restricted to a single mating event, sufficient to acquire enough sperm to fertilize a lifetime supply of eggs. How Ae. aegypti is able to maintain viable spermatozoa remains a mystery. Male spermatozoa are stored within either of two spermathecae that in Ae. aegypti consist of one large and two smaller organs each. In addition, each organ is divided into reservoir, duct and glandular portions. Many aspects of the morphology of the spermatheca in virgin and inseminated Ae. aegypti were investigated here using a combination of light, confocal, electron and scanning microscopes, as well as histochemistry. The abundance of mitochondria and microvilli in spermathecal gland cells is suggestive of a secretory role and results obtained from periodic acid Schiff assays of cell apexes and lumens indicate that gland cells produce and secrete neutral polysaccharides probably related to maintenance of spermatozoa. These new data contribute to our understanding of gamete maintenance in the spermathecae of Ae. aegypti and to an improved general understanding of mosquito reproductive biology.

Male accessory gland substances from Aedes albopictus affect the locomotor activity of Aedes aegypti females

Dengue is one of the world’s most important mosquito-borne diseases and is usually transmitted by one of two vector species: Aedes aegypti or Aedes albopictus . These two diurnal mosquitoes are frequently found coexisting in similar habitats, enabling interactions between adults, such as cross-mating. The objective of this study was to assess cross-mating between Ae. aegypti females and Ae. albopictus males under artificial conditions and evaluate the locomotor activity of Ae. aegypti virgin females injected with male accessory gland (MAG) homogenates to infer the physiological and behavioural responses to interspecific mating. After seven days of exposure, 3.3-16% of Ae. aegypti females mated with Ae. albopictus males. Virgin Ae. aegypti females injected with conspecific and heterospecific MAGs showed a general decrease in locomotor activity compared to controls and were refractory to mating with conspecific males. The reduction in diurnal locomotor activity induced by injections of conspecific or heterospecific MAGs is consistent with regulation of female reproductive activities by male substances, which are capable of sterilising female Ae. aegypti through satyrisation by Ae. albopictus .

Acoustic communication in insect disease vectors

Acoustic signalling has been extensively studied in insect species, which has led to a better understanding of sexual communication, sexual selection and modes of speciation. The significance of acoustic signals for a blood-sucking insect was first reported in the XIX century by Christopher Johnston, studying the hearing organs of mosquitoes, but has received relatively little attention in other disease vectors until recently. Acoustic signals are often associated with mating behaviour and sexual selection and changes in signalling can lead to rapid evolutionary divergence and may ultimately contribute to the process of speciation. Songs can also have implications for the success of novel methods of disease control such as determining the mating competitiveness of modified insects used for mass-release control programs. Species-specific sound “signatures” may help identify incipient species within species complexes that may be of epidemiological significance, e.g. of higher vectorial capacity, thereby enabling the application of more focussed control measures to optimise the reduction of pathogen transmission. Although the study of acoustic communication in insect vectors has been relatively limited, this review of research demonstrates their value as models for understanding both the functional and evolutionary significance of acoustic communication in insects.

Circadian clock of Aedes aegypti: effects of blood-feeding, insemination and RNA interference

Mosquitoes are the culprits of some of the most important vector borne diseases. A species’ potential as a vector is directly dependent on their pattern of behaviour, which is known to change according to the female’s physiological status such as whether the female is virgin/mated and unfed/blood-fed. However, the molecular mechanism triggered by and/or responsible for such modulations in behaviour is poorly understood. Clock genes are known to be responsible for the control of circadian behaviour in several species. Here we investigate the impact mating and blood-feeding have upon the expression of these genes in the mosquito Aedes aegypti . We show that blood intake, but not insemination, is responsible for the down-regulation of clock genes. Using RNA interference, we observe a slight reduction in the evening activity peak in the fourth day after dstim injection. These data suggest that, as in Drosophila , clock gene expression, circadian behaviour and environmental light regimens are interconnected in Ae. aegypti .

Molecular analysis of an odorant-binding protein gene in two sympatric species of Lutzomyia longipalpis s.l.

Lutzomyia longipalpis s.l. is the main vector of American visceral leishmaniasis (AVL) and occurs as a species complex. DNA samples from two Brazilian sympatric species that differ in pheromone and courtship song production were used to analyse molecular polymorphisms in an odorant-binding protein ( obp29 ) gene. OBPs are proteins related to olfaction and are involved in activities fundamental to survival, such as foraging, mating and choice of oviposition site. In this study, the marker obp29 was found to be highly polymorphic in Lu. longipalpis s.l. , with no fixed differences observed between the two species. A pairwise fixation index test indicated a moderate level of genetic differentiation between the samples analysed.

Large indoor cage study of the suppression of stable Aedes aegypti populations by the release of thiotepa-sterilised males

The sterile insect technique (SIT) is a promising pest control method in terms of efficacy and environmental compatibility. In this study, we determined the efficacy of thiotepa-sterilised males in reducing the target Aedes aegypti populations. Treated male pupae were released weekly into large laboratory cages at a constant ratio of either 5:1 or 2:1 sterile-to-fertile males. A two-to-one release ratio reduced the hatch rate of eggs laid in the cage by approximately a third and reduced the adult catch rate by approximately a quarter, but a 5:1 release drove the population to elimination after 15 weeks of release. These results indicate that thiotepa exposure is an effective means of sterilising Ae. aegypti and males thus treated are able to reduce the reproductive capacity of a stable population under laboratory conditions. Further testing of the method in semi-field enclosures is required to evaluate the mating competitiveness of sterile males when exposed to natural environmental conditions. If proven effective, SIT using thiotepa-sterilised males may be incorporated into an integrated programme of vector control to combat dengue in Cuba.

Elytra colour polymorphism and randomness of matings in Chauliognathus fallax Germar 1824 from southern Brazil (Coleoptera, Cantharidae)

Beetles of the species Chauliognathus fallax Germar 1824 are polymorphic for elytron colouration with six morphs distinguished on the basis of black pigmentation on a yellow background. We investigated samples of C. fallax taken in eight consecutive weeks aiming to determine the frequency of the morphs which were grouped, for statistical analysis, into three classes according to elytra pigmentation as well as the possible occurrence of assortative mating. Our results showed a variation in the frequency of the classes throughout the season, both in males and females, with the maximum frequency of each class at the fourth and fifth week. The three classes (A, B, C) showed the same pattern of variation, and class B was always the more frequent. To test randomness of matings two approaches were taken: in the first, we compared the frequency of each class in copulating and non-copulating insects. In the second, the frequency of each class in the whole sample was taken as the probability of occurrence of the respective class; then, using the criterion of the probability of independent events we calculated the expected proportion of copulating insects for each pair of events. Both methods gave non-significant differences, suggesting that the matings were random.

Territórios de machos, acasalamento, distribuição e relação com plantas em Protomeliturga turnerae (Ducke, 1907) (Hymenoptera, Andrenidae)

Protomeliturga turnerae (Ducke, 1907) represents the monotypic tribe Protomeliturgini (Andrenidae, Panurginae). The species is oligolectic on flowers of Turnera L. (Turneraceae). A survey of bees on flowers of Turneraceae and of material in entomological collections showed that P. turnerae is common and endemic in Northeastern Brazil, occurring from the State of Maranhão to Alagoas. In João Pessoa, Paraíba, we studied the reproductive biology and mating behavior of P. turnerae and its relations to plants. At the study site, the species was univoltine with males emerging 5-8 days before the females. Soon after emergence the males established territories on flowers of Turnera subulata Smith which they occupied during several days. Parts of each territory overlapped with those of 1 to 3 other males. On the average, a territory comprised 124 flowers, 59 being shared with other males. Males showed two mating strategies: patrolling the flowers of T. subulata in which females collected pollen or waiting in a specific flower inside the territory for arriving females. P. turnerae showed multiple mating. On the average, a male mated 7 times a day, each copula lasting 3 to 25 sec. We observed 2 to 3 males attempting to copulate with the same female. At the end of anthesis of T. subulata the males stopped flying activity and remained inside flowers until their closure.

Sexual associations for two species of mutillid wasps (Hymenoptera, Mutillidae), with the description of a new species of Anomophotopsis

The sexes of Pseudometocha melanocephala (Perty, 1833) and of a new species of Anomophotopsis are associated based on mating pairs found in the field and also in laboratory mating trials. The previously unknown male of Pseudomethoca melanocephala (Perty, 1833) and both sexes of Anomophotopsis quinteroi Cambra, sp. nov., are described. We present the first distribution record of P. melanocephala from Argentina. Anecdotal data on their mating behavior are also discussed.

Learning of courtship components in Drosophila mercatorum (Paterson & Wheller) (Diptera, Drosophilidae)

In Drosophila, courtship is an elaborate sequence of behavioural patterns that enables the flies to identify conspecific mates from those of closely related species. This is important because drosophilids usually gather in feeding sites, where males of various species court females vigorously. We investigated the effects of previous experience on D. mercatorum courtship, by testing if virgin males learn to improve their courtship by observing other flies (social learning), or by adjusting their pre-existent behaviour based on previous experiences (facilitation). Behaviours recorded in a controlled environment were courtship latency, courtship (orientation, tapping and wing vibration), mating and other behaviours not related to sexual activities. This study demonstrated that males of D. mercatorum were capable of improving their mating ability based on prior experiences, but they had no social learning on the development of courtship.