After infection with Leishmania infantum, Golden Hamsters (Mesocricetus auratus) become more attractive to female sand flies (Lutzomyia longipalpis).

In Brazil, human and canine visceral leishmaniasis is caused by infection with Leishmania infantum, a Protist parasite transmitted by blood-feeding female Lutzomyia longipalpis sand flies. The objective of this study was to determine if the odour of hamsters, infected with Le. infantum, was more attractive than the odour of the same hamsters, before they were infected. The attractiveness of odour collected from individual hamsters (n = 13), before they were infected, was compared in a longitudinal study, with the attractiveness of the odour of the same hamster in a Y-tube olfactometer bioassay, at a late stage of infection. The odour of six of the golden hamsters was significantly more attractive to 50% of the female sand flies at the end of infection compared to before infection and the odour of four of the golden hamsters was significantly more attractive to 75% of the female sand flies at the end of infection. These results strongly indicate that hamsters infected with Le. infantum become significantly more attractive to a greater proportion of female sand flies as the infection progresses.

Courtship behaviour of Phlebotomus papatasi the sand fly vector of cutaneous leishmaniasis.

BACKGROUND: The sand fly Phlebotomus papatasi is an Old World vector of Leishmania major, the etiologic agent of zoonotic cutaneous leishmaniasis. This study describes the courtship behaviour of P. papatasi and compares it with that of Lutzomyia longipalpis, the New World vector of visceral leishmaniasis. Understanding the details of courtship behaviour in P. papatasi may help us to understand the role of sex pheromones in this important vector. RESULTS: P. papatasi courtship was found to start with the female touching the male, leading him to begin abdomen bending and wing flapping. Following a period of leg rubbing and facing, the male flaps his wings while approaching the female. The female then briefly flaps her wings in response, to indicate that she is willing to mate, thereby signaling the male to begin copulation. Male P. papatasi did not engage in parading behaviour, which is performed by male L. longipalpis to mark out individual territories during lekking (the establishment and maintenance of mating aggregations), or wing-flap during copulation, believed to function in the production of audio signals important to mate recognition. In P. papatasi the only predictor of mating success for males was previous copulation attempts and for females stationary wing-flapping. By contrast, male L. longipalpis mating success is predicted by male approach-flapping and semi-circling behaviour and for females stationary wing-flapping. CONCLUSIONS: The results show that there are important differences between the mating behaviours of P. papatasi and L. longipalpis. Abdomen bending, which does not occur in L. longipalpis, may act in the release of sex pheromone from an as yet unidentified site in the male abdomen. In male L. longipalpis wing-flapping is believed to be associated with distribution of male pheromone. These different behaviours are likely to signify significant differences in how pheromone is used, an observation that is consistent with field and laboratory observations.

Enhancing a cancer prevention and control curriculum through interactive group discussions.

The Principles and Practice of Cancer Prevention and Control course (Principles course) is offered annually by the National Cancer Institute Cancer Prevention Fellowship Program. This 4-week postgraduate course covers the spectrum of cancer prevention and control research (e.g., epidemiology, laboratory, clinical, social, and behavioral sciences) and is open to attendees from medical, academic, government, and related institutions across the world. In this report, we describe a new addition to the Principles course syllabus, which was exclusively a lecture-based format for over 20 years. In 2011, cancer prevention fellows and staff designed and implemented small group discussion sessions as part of the curriculum. The goals of these sessions were to foster an interactive environment, discuss concepts presented during the Principles course, exchange ideas, and enhance networking among the course participants and provide a teaching and leadership opportunity to current cancer prevention fellows. Overall, both the participants and facilitators who returned the evaluation forms (n=61/87 and 8/10, respectively) reported a high satisfaction with the experience for providing both an opportunity to explore course concepts in a greater detail and to network with colleagues. Participants (93%) and facilitators (100%) stated that they would like to see this component remain a part of the Principles course curriculum, and both groups provided recommendations for the 2012 program. The design, implementation, and evaluation of this initial discussion group component of the Principles course are described herein. The findings in this report will not only inform future discussion group sessions in the Principles course but may also be useful to others planning to incorporate group learning into large primarily lecture-based courses.

Behavioral evidence for the presence of a sex pheromone in male Phlebotomus papatasi scopoli (Diptera: Psychodidae).

Phlebotomus papatasi (Diptera: Psychodidae) is the Old World sand fly vector of zoonotic cutaneous leishmaniasis caused by Leishmania major (Trypanosomatidae: Kinetoplastida), a debilitating and disfiguring protist parasitic disease prevalent throughout southern Mediterranean countries, the Middle East, as well as southern and eastern European countries, where it is regarded as a serious public health problem. Little is known of the mating ecology of P. papatasi, and, in particular, the role (if any) of pheromones is not known. In this laboratory- and field-based study, we have shown that a male-produced sex pheromone exists in P. papatasi. Young female P. papatasi are attracted to the headspace volatiles of small groups of males, males and females together, but not females alone. Males were not attracted to males, females, or mixed groups of males and females in the laboratory. Larger groups of males or males and females together were repellent in the laboratory study. Field experiments showed that Centers for Disease Control (CDC) light traps baited with small groups of males and females together were attractive to females, but not males. CDC traps baited with large groups of males and females together caught significantly fewer females and males than the control traps; however, the proportion of females caught compared with males overall was much higher than with CDC traps baited with small numbers of males and females. These results suggest that females may be attracted in preference to males to the vicinity of the baited traps and are highly sensitive to the concentration of male pheromone. It also suggests that P. papatasi mating behavior is fundamentally different from that of Lutzomyia longipalpis, where large mating aggregations of males and females occur.

Synthetic sex pheromone attracts the leishmaniasis vector Lutzomyia longipalpis (Diptera: Psychodidae) to traps in the field.

Improving vector control remains a key goal in reducing the world's burden of infectious diseases. More cost-effective approaches to vector control are urgently needed, particularly because vaccines are unavailable and treatment is prohibitively expensive. The causative agent of American visceral leishmaniasis (AVL), Leishmania chagasi, Cunha and Chagas (Kinetoplastida: Trypanosomatidae), is transmitted between animal and human hosts by blood-feeding female sand flies attracted to mating aggregations formed on or above host animals by male-produced sex pheromones. Our results show the potential of using synthetic pheromones to control populations of Lutzomyia longipalpis Lutz and Neiva (Diptera: Psychodidae), the sand fly vector of one of the world's most important neglected diseases, AVL. We showed that a synthetic pheromone, (+/-)-9-methylgermacrene-B, produced from a low-cost plant intermediate, attracted females in the laboratory. By formulating dispensers that released this pheromone at a rate similar to that released by aggregating males, we were able to attract flies of both sexes to traps in the field. These dispensers worked equally well when deployed with mechanical light traps and inexpensive sticky traps. If deployed effectively, pheromone-based traps could be used to decrease AVL transmission rates through specific targeting and reduction of L. longipalpis populations. This is the first study to show attraction of a human disease-transmitting insect to a synthetic pheromone in the field, showing the general applicability of this novel approach for developing new tools for use in vector control.

Sandfly pheromones. Their biology and potential for use in control programs.

Lutzomyia longipalpis (Diptera: Psychodidae) is the vector of Leishmania chagasi the causative agent of visceral leishmaniasis (VL) in South and Central America, particularly Brazil, where the greatest incidence occurs. The disease is fatal if untreated. Although huge efforts have been made to control VL the incidence is increasing. Vector control remains an important element of disease control but residual spraying and other strategies have failed to make any lasting impact. Manipulation of sandfly chemical communication offers the opportunity to add new techniques and tools to reduce sandfly populations and thereby reduce Leishmania transmission. This paper reports the current understanding of several areas of sandfly chemical ecology and their prospects for application.

Courtship behaviour in the sandfly Lutzomyia longipalpis, the New World vector of visceral leishmaniasis.

Courtship behaviour in Lutzomyia longipalpis Lutz and Neiva (Diptera: Psychodidae) was examined to determine the sequence of behaviours that occur prior to copulation. Courtship consisted of a series of male and female touching and wing-flapping behaviours, with males performing a greater variety of wing-flapping behaviours than previously described. Occurrence of male approach-flapping, semi-circling and female stationary-flapping were all predictors of eventual copulation, and may coincide with the dispersal of pheromones or the production of auditory signals important to courtship. Touching occurred in the majority of observations, with contact most often made with the tips of the legs and antennae. This behaviour, not previously described in sandflies, was initiated by males and females, and may indicate the use of contact pheromones, a form of communication previously overlooked in L. longipalpis. Future studies are required to separate the auditory and chemical signals associated with wing-flapping, and to confirm whether L. longipalpis possesses cuticular hydrocarbons capable of inducing behavioural responses. The identification of signals that inhibit sexual behaviour during courtship may be particularly relevant to developing mating disruption techniques against L. longipalpis.

Host odor synergizes attraction of virgin female Lutzomyia longipalpis (Diptera: Psychodidae).

The sand fly Lutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae) is the principle vector of Leishmania chagasi/infantum Cunha and Chagas, the causative agent of visceral leishmaniasis. The disease is transmitted by blood-feeding females, which seek aggregations of males above potential hosts both to mate and blood-feed. Pheromones produced by male sand flies could potentially be used as lures in L. longipalpis control programs. We investigated whether attraction of male and female sand flies to pheromone could be increased by addition of host odor. Pheromone was attractive to females in the absence of host odor, although a 10-fold increase in concentration did not increase numbers attracted or reduce the proportion of flies not responding during trials. Odors from Syrian hamsters, Mesocricetus auratus, were more attractive to females than air when presented without sex pheromone, but this effect was masked by hexane, suggesting attraction to host odor alone may be relatively weak. Addition of hamster odor both increased the number of virgin female L. longipalpis attracted to sex pheromone (relative to a solvent control) and reduced the number of nonresponders, indicating that host odor may have both a synergistic and activating effect. Male sand flies were not attracted to pheromone with or without host odor, although addition of pheromone did counteract an apparent avoidance of host odor combined with a hexane control. These results indicate L. longipalpis pheromones function primarily to attract females and that their efficacy as lures may be increased through addition of host odor, or by deploying traps in the vicinity of host animals.

Identification of sex pheromones of Lutzomyia longipalpis (Lutz & Neiva, 1912) populations from the state of São Paulo, Brazil.

In Brazil, four populations of Lutzomyia longipalpis each producing different sex pheromones are recognised. It has been suggested that these chemotype populations represent true sibling species. In this study we present the results of an analysis, by coupled gas chromatography-mass spectrometry, of the pheromones of males L. longipalpis from two different municipalities of the state of São Paulo. Our study showed that L. longipalpis from these two municipalities produced different sex pheromones from each other. This coupled with the remarkable difference between the epidemiological situation in Araçatuba and Espírito Santo do Pinhal, suggests that the (S)-9-methylgermacrene-B and cembrene-1 populations may have different vectorial capacities.

Identification of sex pheromones of Lutzomyia longipalpis (Lutz & Neiva, 1912) populations from the state of São Paulo, Brazil

In Brazil, four populations of Lutzomyia longipalpis each producing different sex pheromones are recognised. It has been suggested that these chemotype populations represent true sibling species. In this study we present the results of an analysis, by coupled gas cromotography - mass spectrometry, of the pheromones of males L. longipalpis from two different municipalities of the state of São Paulo. Our study showed that L. longipalpis from these two municipalities produced different sex pheromones from each other. This coupled with the remarkable difference between the epidemiological situation in Araçatuba and Espírito Santo do Pinhal, suggests that the (S)-9-methylgermacrene-B and cembrene-1 populations may have different vectorial capacities.

Analysis of the sex pheromone extract of individual male Lutzomyia longipalpis sandflies from six regions in Brazil.

Although the phlebotomine sandfly Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae) is generally accepted to be a species complex, it is unclear how many members there are, how they are related and which are the main vectors of leishmaniasis. The vectorial capacity of each sibling species is likely to differ, thus a means of identifying the most important vector species is of critical importance to the epidemiology and control of this debilitating disease in South and Central America. In Brazil four chemotypes have been distinguished by sex pheromone analysis. In this study the sex pheromone extracts of L. longipalpis from six regions of Brazil were analysed in detail. Samples included the sympatric 1-spot, 2-spot and intermediate spot morphotypes from Sobral, Ceará State. The results strongly suggest that members of the complex that produce different sex pheromones are reproductively isolated, thus strengthening the argument that the different chemotypes represent true sibling species. The study also found significant differences in morphology and the amounts of sex pheromone produced by members of each chemotype from different parts of Brazil, which suggests population substructuring that has not previously been recognized. Evidence of a fifth chemotype in Brazil is also presented.

A fourth chemotype of Lutzomyia longipalpis (Diptera: Psychodidae) from Jaibas, Minas Gerais State, Brazil.

Lutzomyia longipalpis (Lutz and Neiva) is a species complex of Lutzomyia pseudolongipalpis (Arrivillaga and Feliciangeli) and at least three other as yet undefined siblings. Isozyme and mitochondrial studies of allopatric populations across Central and South America have suggested the presence of four "clades" that have been hypothesized to have arisen mainly because of geographical isolation mechanisms. Parallel studies of sexual behavior as well as cross-mating and genetic analysis, of both allopatric and sympatric populations, suggest at least four sibling species that do not seem to correspond to the defined four "clades." In an effort to understand this apparent discrepancy, sympatric populations of L. longipalpis from a single South American country, Brazil, are being studied. In Brazil, three putative species can be identified by their male-produced sex pheromones: (S)-9-methylgermacrene-B, 3-methyl-a-himachalene, and a cembrene. We report here that analysis by coupled gas chromatography-mass spectrometry shows that L. longipalpis from Jaibas, Minas Gerais State, Brazil, occurs as two sympatric sex pheromone chemotypes. One chemotype is the cembrene type previously recorded in a L. longipalpis population from Sobral, Ceará State, Brazil, and the other is a new cembrene isomer not previously observed in L. longipalpis. The finding of this new chemotype strongly suggests that the L. longipalpis species complex in Brazil consists of four members rather than the three previously recognized and confirms previous analysis of genetic variation that had suggested the presence of a complex in Brazil.

Navigation of Lutzomyia longipalpis (Diptera: Psychodidae) under dusk or starlight conditions.

The responses of male and female Lutzomyia longipalpis (Lutz & Neiva) to different wavelengths of light was tested by presenting the sandflies with two light sources simultaneously, a series of test wavelengths between 350-670 nm and a 400 nm control. To test whether L. longipalpis could discriminate between the test and control, three sets of experiments were carried out in which the test wavelengths were presented at higher, equivalent or lower intensity than the control. In all three experiments, ultra-violet (350 nm) and blue-green-yellow (490-546 nm) light was more attractive to L. longipalpis than the control wavelength. However, at low intensity, UV was less attractive, than equivalent or higher intensity UV light. At intensities equivalent to or higher than the control wavelength, ultra-violet light was more attractive than blue-green. Furthermore, at low intensity, green-yellow (546 nm) light was more attractive to males whereas blue-green (490 nm) was more attractive to females. Blue-violet (400 nm) and orange-red (600-670 nm) light were least attractive in all three sets of experiments. Response function experiments indicated that the responses were dependent on both intensity and wavelength and that therefore more than one photoreceptor must be involved in the response. The results indicated that L. longipalpis can discriminate between different wavelengths at different intensities and thus have true colour vision. It also suggests that L. longipalpis may be able to navigate at dusk or under moonlight or starlight conditions using light in the blue-green-yellow part of the spectrum. The difference in response of males and females to light in this region is interesting and may indicate the different ecology of the sexes at night. Overall, these results may have important implications for sandfly trap design.

Genetic identification of two sibling species of Lutzomyia longipalpis (Diptera: Psychodidae) that produce distinct male sex pheromones in Sobral, Ceará State, Brazil.

Lutzomyia longipalpis, the main sandfly vector for New World visceral leishmaniasis is a complex of an as yet undefined number of sibling species. At present, there is no consensus on the status (single species vs. species complex) of Brazilian populations. We applied five microsatellite loci to test the hypothesis that L. longipalpis occurs as two sympatric cryptic species in Sobral, Ceará State, Brazil as predicted by male sex pheromone chemotypes described previously for field specimens from this site [S-9-methyl-germacrene-B (9MGB) and a cembrene compound]. Abdominal spot morphology corresponds with pheromone type at this locality (9MGB in '1 spot' males and cembrene in '2 spot' males). Genotype data from 190 wild-caught L. longipalpis specimens collected in October 1999 and April 2001 were used to estimate genetic differentiation between the two sex pheromone populations and sampling dates. No significant (P > 0.05) genetic differences were found between the 1999 and 2001 9MGB samples (theta = 0.018; RST = -0.005), and genetic differentiation was low between the cembrene collections (theta = 0.037, P < 0.05; RST = -0.043, P > 0.05). By contrast, highly divergent allelic frequencies (largely at two microsatellite loci) corresponded to significant (P > 0.05) genetic differentiation (theta = 0.221; RST = 0.215) for all comparisons between samples with different pheromones. When pheromone samples were pooled across sample date, genetic differentiation was high (theta = 0.229; P < 0.001; Nem = 0.84). The allele frequency distribution at each of the five microsatellite loci was similar for males and females from the two collection years. Two of these loci showed highly divergent allele frequencies in the two sex pheromone populations. This was reflected in the highly significant genetic differentiation obtained from the male genotypes, between populations producing different pheromones (theta = 0.229-0.268; P < 0.0001 for the 2001 and theta = 0.254-0.558; P < 0.0001 for the 1999 collections, respectively). Similar results were obtained when the females, assigned to a pheromone type, were included in the analysis. Both a Bayesian analysis of the data set and a population assignment test provided strong evidence for two distinct populations corresponding to pheromone type. Given its genotype, the probability of assigning a 9MGB male to the original 9MGB population was 100% once the two years' collections were pooled. For cembrene-producing '2 spot' males this probability although still high, was lower than for 9MGB males, at 86%. This microsatellite data together with previously reported reproductive isolation between the two Sobral populations confirm that premating barriers are important in speciation of L. longipalpis.

Fine-root respiration in a loblolly pine and sweetgum forest growing in elevated CO2.

• The loss of carbon below-ground through respiration of fine roots may be modified by global change. Here we tested the hypothesis that a reduction in N concentration of tree fine-roots grown in an elevated atmospheric CO2 concentration would reduce maintenance respiration and that more energy would be used for root growth and N uptake. We partitioned total fine-root respiration (RT ) between maintenance (RM ), growth (RG ), and N uptake respiration (RN ) for loblolly pine (Pinus taeda) and sweetgum (Liquidambar styraciflua) forests exposed to elevated CO2 . • A substantial increase in fine-root production contributed to a 151% increase in RG for loblolly pine in elevated CO2 . Root specific RM for pine was 24% lower under elevated CO2 but when extrapolated to the entire forest, no treatment effect could be detected. • R G (< 10%) and RN (< 3%) were small components of RM in both forests. Maintenance respiration was the vast majority of RT , and contributed 92% and 86% of these totals at the pine and sweetgum forests, respectively. • The hypothesis was rejected because the majority of fine-root respiration was used for maintenance and was not reduced by changes in root N concentration in elevated CO2 . Because of its large contribution to RT and total soil CO2 efflux, changes in RM caused by warming may greatly alter carbon losses from forests to the atmosphere.

Isolation and identification of 9-methylgermacrene-B as the putative sex pheromone of Lutzomyia cruzi (Mangabeira, 1938) (Diptera: Psychodidae).

Lutzomyia (Lutzomyia) cruzi has been named as a probable vector of Leishmania chagasi in Corumbá, Mato Grosso do Sul, Brazil. Taxonomically L. cruzi is closely related to the L. longipalpis species complex. Females of L. cruzi and L. longipalpis are morphologically indistinguishable and associated males must be examined carefully to confirm identifications. Chemical analysis hexane extracts of male L. cruzi has revealed the presence of a 9-methylgermacrene-B (C16), a homosesquiterpene (mw 218) previously shown to be the sex pheromone of one of the members of the L. longipalpis species complex.

Enhanced sandfly attraction to Leishmania-infected hosts.

We report that hamsters infected with Leishmania infantum are more attractive to female sandflies in bioassays. Entrained odours from infected animals were shown by gas chromatography to contain peaks absent from uninfected individuals. Implications of enhanced transmission, potential for developing novel diagnoses and the significance to epidemiological models are discussed.

Distribution of putative male sex pheromones among Lutzomyia sandflies (Diptera: Psychodidae).

Male Lutzomyia longipalpis produce terpene sex pheromones in glandular tissue underlying the cuticle. The pheromones are transmitted to the surface via cuticle-lined ducts (measuring 0.25 microm in diameter), each of which reaches the surface in the centre of a papule (measuring 3-3.5 microm in diameter). Similar papules, in a range of shapes but all characterized by the presence of a central pore and absence of macroserae, occur in some other species of sandfly. The aim of the present study was to determine the distribution of sex pheromones in sandflies of the genus Lutzomyia that do and do not have the papules. The results indicate that sex pheromones are not widely distributed amongst male Lutzomyia spp. Male members of the genus can be subdivided into three groups: those that produce terpenes and have cuticular papules; those that do not produce terpenes but still have the associated papules; and those that have neither terpenes nor papules. The papules seen in the species that do not synthesise sex pheromones are presumably vestigial, non-functional structures. Such species may have stopped producing pheromone as the result of changes in the way in which the females found and selected mates or changing feeding preferences. A similar event has occurred in the Lepidoptera, where vestigial pheromone-secreting structures remain in some species which no longer produce pheromone. Lutzomyia lenti collected in southern Brazil produced a novel diterpene whereas male L. lenti from north-eastern Brazil did not, supporting suggestions by others that L. lenti is, like L. longipalpis, a species complex.

Copulation songs in three siblings of Lutzomyia longipalpis (Diptera: Psychodidae).

We present the results of recording male courtship songs of the sandfly Lutzomyia longipalpis. The striking differences in the songs from 3 Brazilian populations of this sandfly with 3 distinct male pheromones support the 3 sibling species previously proposed based on this characteristic.