Resistance to deltamethrin in Triatoma infestans (Hemiptera: Reduviidae): Does it influence the phenotype of antennae, wings, and heads?

In vector control terms, insecticide resistance is the development of the capacity, of an insect population, to tolerate doses of an insecticide that are lethal to most individuals in a typical population of the same species. The genetic changes that determine resistance may have adaptive costs in the resistant phenotype or, conversely, may result in an adaptive advantage when compared to susceptible insects in the environment without insecticides. Triatoma infestans is one of the main vectors of Trypanosoma cruzi in the southern cone of South America. High insecticide resistance in T. infestans was detected in Argentina in Salta and Chaco provinces. The objective of this study was to determine the possible morphometric changes in wings, heads, and the antennal phenotype of deltamethrin-resistant T. infestans (RR) males and females compared to susceptible insects (SS), evaluating its implication in adaptive processes such as olfactory capacity, dispersion, and probability of colonizing new habitats, among others. Nine type I landmarks were marked on wings, 5 type II landmarks on heads, and 10 antennal sensilla were counted on 106 adults of both sexes (resistant and susceptible from first and second laboratory generations). Morphological divergence was observed between the two groups (RR and SS). The RR insects showed smaller sizes of wings and heads and shape compatible with lower dispersal potential and different active dispersal behaviors. Antennae also revealed sensory simplification in RR and divergence between RR and SS, although more marked in females. This study characterizes for the first time T. infestans RR and SS through wings, heads, and antennae. The results suggest a lower dispersive potential in resistant insects and the differences described lay the foundations for the identification of a resistance biomarker in triatomines.

Shape relatedness between geographic populations of Culex tritaeniorhynchus, the primary vector of Japanese encephalitis virus: A landmark study.

BACKGROUND: Japanese encephalitis is a severe disease of acute encephalitis, with children and the elderly primarily affected, and with mortality rates reaching over 25%. The virus is transmitted mainly by species of the Culex (Culex) vishnui subgroup, primarily the widely spread Cx. tritaeniorhynchus Giles. The latter is known as a highly migratory mosquito which moves with airflow over large distances. We explored the geometric variation of the wing venation among distant areas of its geographic distribution. Our working hypothesis was that shape variation across geography could reveal known past and present migratory routes. MATERIALS METHODS: We compared the wing venation geometry of 236 female Culex tritaeniorhynchus from different locations in the Madagascan (La Reunion), Oriental (Thailand, Vietnam) and Paleartic (Japan) regions. To ascertain the taxonomic signal of the wing venation we also used two species as relative outgroups, Cx. whitmorei and Cx. brevipalpis. RESULTS: In spite of an increasing morphometric variation as expected with larger geographic dispersion, our Cx. tritaeniorhynchus samples were clustered as a single species when considered relative to other Culex species. The relationships between geographic sites of Cx. tritaeniorhynchus globally conformed with an isolation by distance model. The shape homogeneity of our Palearctic samples (Japan) contrasted with some heterogeneity observed in the Oriental region (Thailand, Vietnam), and could be related to the different regimes of wind trajectories in these regions. CONCLUSION: The average shape variation of Culex tritaeniorhynchus disclosed a separation between Madagascan, Oriental and Palearctic regions in accordance with geography. The wing venation not only could reflect geography, it also contained a clear taxonomic signal separating three Culex species. Within Cx. tritaeniorhynchus, a contrasting pattern of shape variation between the Palearctic and the Oriental regions is tentatively explained by the influence of wind trajectories.

Geometric morphometrics to distinguish the cryptic species Anopheles minimus and An. harrisoni in malaria hot spot villages, western Thailand.

Anopheles minimus Theobald 1901 and An. harrisoni Harbach & Manguin 2007 belong to the same species complex. They are morphologically similar and can exist in sympatry but have blood host preferences. The most accurate method for their identification is based on molecular techniques. Here, we measure the level of interspecific discrimination by geometric morphometry. Sixty-seven An. minimus and 22 An. harrisoni specimens were selected based on their morphological integrity and confirmed by identification polymerase chain reaction of internal transcribed spacer 2. These samples were used as reference data allowing for a morphometric identification based on geometric shape. Despite size overlap between the two species, there was a significant shape divergence allowing for differentiation of An. minimus and An. harrisoni with 90% accuracy. An intraspecific study of An. minimus showed a summer period associated to the reducing of wing size, which did not influence the shape-based differentiation of An. harrisoni. Wing venation geometry can be used to distinguish between these cryptic species mainly based on shaped divergence. This study suggests that geometric morphometrics represent a convenient low-cost method to complement morphological identification, especially concerning damaged specimens, i.e., insects having accidentally lost the anatomical features allowing a reliable morphological identification.

Geometric morphometric analysis of the effect of temperature on wing size and shape in Aedes albopictus.

Wing geometry helps to identify mosquito species, even cryptic ones. On the other hand, temperature has a well-known effect on insect metric properties. Can such effects blur the taxonomic signal embedded in the wing? Two strains of Aedes albopictus (laboratory and field strain) were examined under three different rearing temperatures (26, 30 and 33 °C) using landmark- and outline-based morphometric approaches. The wings of each experimental line were compared with Aedes aegypti. Both approaches indicated similar associations between wing size and temperature. For the laboratory strain, the wing size significantly decreased as the temperature increased. For the field strain, the largest wings were observed at the intermediate temperature. The two morphometric approaches describing shape showed different sensibilities to temperature. For both strains and sexes, the landmark-based approach disclosed significant wing shape changes with temperature changes. The outline-based approach showed lesser effects, detecting significant changes only in laboratory females and in field males. Despite the size and shape changes induced by temperature, the two strains of Ae. albopictus were always distinguished from Ae. aegypti. The present study confirms the lability of size. However, it also suggests that, despite environmentally-induced variation, the architecture of the wing still provides a strong taxonomic signal.

The wing venation patterns to identify single tsetse flies.

This is the first study to explore the potential of various geometric morphometrics methods to help the morphological diagnostic of tsetse species, vectors of human and animal trypanosomiases in sub-Saharan Africa. We compared landmarks, semilandmarks and outlines techniques on male and female samples of species, and suggested adapted strategies according to the countries and their own Glossina fauna. We could compare up to 7 taxa belonging to the three main subgenera of the Glossina genus: Nemorhina (5 species), Glossina (1 species) and Austenina (1 species). Our sample included the major vectors of sleeping sickness: G. palpalis palpalis, G. p. gambiensis, G. fuscipes fuscipes and G. f. quanzensis, as well as two important vectors of African animal trypanosomoses: G. tachinoides and Glossina morsitans submorsitans. The average level of correct species recognition by the wing shape was satisfactory, and slightly higher for females than for males. The best scores of correct assignment, in both sexes, were obtained by the contour technique (96% of correct attribution in females, 92% in males), slightly higher than for semilandmarks (95% and 91%) or landmarks (94% and 89%) techniques. We made our images of wings freely available to be used as reference images (http://mome-clic.com), and we describe the conditions and the analytical steps to be followed to identify unknown specimens using external reference images. Under adequate conditions, such use of reference images obtained from a free access server could help species identification of new samples anywhere in Africa.

Geometric morphometrics for the taxonomy of 11 species of Anopheles (Nyssorhynchus) mosquitoes.

The subgenus Anopheles (Nyssorhynchus) (Diptera: Culicidae) includes the primary vectors of Plasmodium spp. in Colombia. Most adult females of this subgenus are difficult to identify in the field using the available keys. With the objective of further investigating the discriminatory power of modern morphometrics, both landmark-based and outline-based approaches were explored using the wing venation geometry of 11 Anopheles (Nyssorhynchus) species. Wing shape was able to separate the closest species of the subgenus. When the 11 species were analysed together, validated classification scores on average 5.3-8.6 times higher than those expected by chance were observed. These scores computed from the total sample of 11 species were not satisfactory for the recognition of Anopheles benarrochi B, Anopheles oswaldoi s.l. and Anopheles strodei. These sibling species were captured in sympatry. To improve the identification power of the morphometric tool, it was necessary to analyse these species separately from the remaining species. The best classification scores were obtained using a combination of 12 landmarks collected not only on the intersections of wing veins, but also on spots. An outline approach also gave excellent reclassification scores. Another pair of sibling species, collected in allopatry, Anopheles nuneztovari and Anopheles rangeli, also showed high classification scores.

Microgeographic spatial structuring of Triatoma infestans (Hemiptera: Reduviidae) populations using wing geometric morphometry in the Argentine Chaco.

We investigated the occurrence of spatial structuring in Triatoma infestans (KLug) (Hemiptera: Reduviidae) populations 12 yr after the last community-wide insecticide spraying campaign in rural Pampa del Indio, in the Gran Chaco of northeastern Argentina. In total, 172 male and 149 female right wings collected at 16 georeferenced sites with at least 10 individuals of the same sex were analyzed using geometric morphometry. Mean female body length and wing centroid size (CS) were significantly larger than for males. Log-transformed CS and length were significantly and positively correlated both for males and females. Males collected in domiciles had significantly smaller CS than those collected in peridomestic structures both closed (kitchens or storerooms) or open (chicken coops), in agreement with our previous results elsewhere in the dry Argentine Chaco. Female wing CS was not significantly different between ecotopes. Wing shape analyses showed the occurrence of significant geographic structuring in males and females combined and in males only. Male wings showed a strong association between Mahalanobis distance and geographic distance. In general, Mahalanobis distances were significantly different between collection sites located > 4 km apart. For collection sites located < 4 km apart, the greater the geographic distance the larger the difference in wing shape variables. Among females, only a partial correspondence between geographic groups and Mahalanobis distances was recorded. The strong spatial structuring found in T. infestans populations may be useful for the identification of putative reinfestation sources after vector control interventions.

Deciphering morphology in Triatominae: the evolutionary signals.

Many species of Triatominae show evidence for morphological plasticity. Frequent taxonomic questions arose from this variability leading to disputes about describing new subspecies, species or even genera. We suggest this phenotypic flexibility is primarily an intraspecific feature, but with potential for evolutionary changes. We present arguments for a selection regime leading to the separation of species having low developmental canalization into morphologically distinct ecotypes. We suggest that these ecotypes, or morphs, or forms, may have evolutionary importance even if gene flow still exists between them. Thus, although we consider the morphological plasticity of Triatominae as an intraspecific trait, we defend the idea that it might represent a common evolutionary route to new species. Speciation processes in Triatominae could result from disruptive selection regimes combined with weak developmental canalization. Added to this basic pattern, accidental events could hasten evolutionary change. We suggest the heterosis as one of them.

Influence of larval density or food variation on the geometry of the wing of Aedes (Stegomyia) aegypti.

BACKGROUND AND METHOD: Variation in wing length among natural populations of Aedes (Stegomyia) aegypti (L.) (Diptera: Culicidae) is associated with different vectorial capacities. Geometric morphometrics allowed us to use a more powerful estimator of wing size ('centroid size'), as well as to visualize the variation of wing shape, to describe the effects of density or food variation at larval stage on 20 anatomical landmarks of the wing of A. aegypti. RESULTS: Almost perfect correlations between (centroid) size and larval density or size and larval food were observed in both sexes: a negative correlation with increasing density and a positive one with increasing amount of food. The allometric component of shape change was always highly significant, with stronger contribution of size to shape under food effects. Within each experiment, either food or density effects, and excluding extreme conditions, allometric trends were similar among replicates and sexes. However, they differed between the two experiments, suggesting different axes of wing growth. CONCLUSION: Aedes aegypti size is highly sensible to food concentration or population density acting at larval stages. As larger individuals could be better vectors, and because of the stronger effect of food concentration on size, vector control activities should pay more attention in eliminating containers with rich organic matter. Furthermore, as a simple reduction in larval density could significantly increase the size of the survivors, turning them into potentially better vectors, the control activities should try to obtain a complete elimination of the domestic populations.

Evolutionary relationships based on genetic and phenetic characters between Triatoma maculata, Triatoma pseudomaculata and morphologically related species (Reduviidae: Triatominae).

The maculata group currently comprises two species of Triatominae, Triatoma maculata and Triatoma pseudomaculata, which share morphologic and chromatic characteristics. In order to clarify the systematic status of these two vectors of Trypanosoma cruzi and to infer their evolutionary relationships, we performed an enzymatic, morphometric and cytogenetic comparison of them, also taking into account two sister species not included in the group (T. arthurneivai and T. wygodzinskyi). According to our results, T. maculata and T. pseudomaculata belong to distinct evolutionary lineages. Similarly, T. arthurneivai topotypes from Minas Gerais form an independent isolated group by morphometrics. Our results also support the specific status of the Triatoma population from São Paulo State (formerly referred to T. arthurneivai), and suggest the possibility that it is T. wygodzinskyi. Finally, we suggest that only the arboricolous T. pseudomaculata from northeast Brazil and the rupicolous sister species originated from São Paulo State should be classified together in the same group.

The tsetse fly Glossina palpalis palpalis is composed of several genetically differentiated small populations in the sleeping sickness focus of Bonon, Côte d'Ivoire.

Glossina palpalis is the main vector of human African trypanosomosis (HAT, or sleeping sickness) that dramatically affects human health in sub-Saharan Africa. Because of the implications of genetic structuring of vector populations for the design and efficacy of control campaigns, G. palpalis palpalis in the most active focus of sleeping sickness in Côte d'Ivoire was studied to determine whether this taxon is genetically structured. High and statistically significant levels of within population heterozygote deficiencies were found at each of the five microsatellite loci in two temporally separated samples. Neither null alleles, short allele dominance, nor trap locations could fully explain these deviations from random mating, but a clustering within each of the two samples into different genetic sub-populations (Wahlund effect) was strongly suggested. These different genetic groups, which could display differences in infection rates and trypanosome identity, were composed of small numbers of individuals that were captured together, leading to the observed Wahlund effect. Implications of this population structure on tsetse control are discussed.

Origin and phylogeography of the Chagas disease main vector Triatoma infestans based on nuclear rDNA sequences and genome size.

For about half of all Chagas disease cases T. infestans has been the responsible vector. Contributing to its genetic knowledge will increase our understanding of the capacity of geographic expansion and domiciliation of triatomines. Populations of all infestans subcomplex species, T. infestans, T. delpontei, T. platensis and T. melanosoma and the so-called T. infestans "dark morph", from many South American countries were studied. A total of 10 and 7 different ITS-2 and ITS-1 haplotypes, respectively, were found. The total intraspecific ITS-2 nucleotide variability detected in T. infestans is the highest hitherto known in triatomines. ITS-1 minisatellites, detected for the first time in triatomines, proved to be homologous and thus become useful markers. Calculations show that ITS-1 evolves 1.12-2.60 times faster than ITS-2. Despite all species analyzed presenting the same n=22 chromosome number, a large variation of the haploid DNA content was found, including a strikingly high DNA content difference between Andean and non-Andean specimens of T. infestans (mean reduction of 30%, with a maximum of up to 40%) and a correlation between presence/absence of minisatellites and larger/smaller genome size. Population genetics analysis of the eight composite haplotypes of T. infestans and net differences corroborate that there are clear differences between western and eastern populations (60%), and little genetic variation among populations (1.3%) and within populations (40%) within these two groups with migration rates larger than one individual per generation corresponding only to pairs of populations one from each of these groups. These values are indicative either of a large enough gene flow to prevent population differentiation by drift within each geographic area or a very recent spread, the latter hypothesis fitting available data better. Phylogenetic trees support a common ancestor for T. infestans and T. platensis, an origin of T. infestans in Bolivian highlands and two different dispersal lines, one throughout Andean regions of Bolivia and Peru and another in non-Andean lowlands of Chile, Paraguay, Argentina, Uruguay and Brazil.

Is Lutzomyia serrana (Diptera, Psychodidae, Phlebotominae) present in Ecuador?

We describe a multivariate metric comparison of three sandfly species showing strong differences in size, which questions the geographical distribution of one of them. Two species are represented by a single population (L. robusta and L. guilvardae) and one by two populations (L. serrana). All of them belong to the series serrana (Diptera, Psychodidae, Phlebotominae). The morphometric data confirm that L. guilvardae is a distinct species. However, they suggest that L. robusta and L. serrana in Ecuador are the same taxon, and that it is distinguishable from the population of L. serrana in Bolivia. A multilocus enzyme electrophoresis analysis comparing L. serrana in Bolivia and L. robusta in Ecuador adds further evidence that these two populations are distinct species. Thus, our data seem to indicate that, in Ecuador, the population previously identified as L. serrana is actually the same species as the allopatric population previously identified as L. robusta. Accepting L. serrana in Ecuador as small-sized L. robusta, the resulting geographic distribution of this latter becomes in closer agreement with ecology and epidemiology.

Domiciliation process of Rhodnius stali (Hemiptera: Reduviidae) in Alto Beni, La Paz, Bolivia.

We report a systematic collection of Triatominae inside houses and in the peridomestic environment of Alto Beni, department of La Paz, Bolivia. This area is free of Triatoma infestans and although we detected previously seropositivity for Trypanosoma cruzi, the Alto Beni region is not officially considered as endemic for Chagas disease. From 11 houses of five localities, we collected adults, nymphs and eggs of a Rhodnius species, which was confirmed by morphological and morphometric analysis as Rhodnius stali. This little-known species has long been confused with R. pictipes, and was originally described from museum specimens labelled as R. pictipes. Our data show that R. stali is able to establish colonies in domestic and peridomestic habitats in Bolivia, and it is probably the vector responsible for Chagas disease seropositivity observed in the indigenous population of Alto Beni.

Wing shape divergence between Rhodnius prolixus from Cojedes (Venezuela) and Rhodnius robustus from Mérida (Venezuela).

The existence of Rhodnius robustus as a species distinct from Rhodnius prolixus has long been the main epidemiological question about Chagas disease transmission in Venezuela and surrounding countries. These two taxa are morphologically and genetically very similar, but only R. prolixus is assumed to colonize houses and transmit Chagas disease to humans. R. robustus is assumed to be an exclusively sylvatic species, restricted to palm trees. If robustus and prolixus are actually the same species, the theoretical possibility exists of sylvatic specimens invading houses, even after insecticide application, and a control strategy similar to that of the successful Southern Cone Initiative against Triatoma infestans would be difficult to consider. Since no valid alternative control strategy exists, the answer to this biological question could be decisive about the future of vector control in this region. Although we believe genetic techniques are best suited to define species boundaries, we present here an example of the relevance of modern morphometrics in dealing with such an issue. Using both traditional and geometric morphometrics, we compared the wing size and shape in both sexes of these two taxa reared in the same laboratory for one generation. R. robustus specimens were collected from palm trees in the state of Mérida (Venezuela), and R. prolixus were collected from houses in the state of Cojedes (Venezuela). Our study provided no argument to question their specific status. Even after one generation of living in the same laboratory conditions, the two lines showed clear size differences, divergent allometric trends, and significant allometry-free differences in shape. These results suggest that R. robustus (Mérida, Venezuela) and R. prolixus (Cojedes, Venezuela) are distinct evolutionary units. Due to the epidemiological importance of this question, further studies in other geographic areas of Venezuela are required to accurately define the relationships of R. robustus and R. prolixus.

Nuclear rDNA ITS-2 sequences reveal polyphyly of Panstrongylus species (Hemiptera: Reduviidae: Triatominae), vectors of Trypanosoma cruzi.

Panstrongylus species are widely distributed throughout the Americas, where they act as vectors of Trypanosoma cruzi, agent of Chagas disease. Their intraspecific relationships, taxonomic position and phylogeny in relation to other Triatomini were explored using ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS-2) sequence polymorphisms and maximum parsimony, distance and maximum likelihood analyses of 10 populations representing six species of the genus (P. megistus, P. geniculatus, P. rufotuberculatus, P. lignarius, P. herreri and P. chinai). At the subspecific level, P. megistus appeared more homogeneous than P. rufotuberculatus and P. geniculatus (both with broader distribution). Several dinucleotide microsatellites were detected in the sequences of given species. Many of these microsatellites (GC, TA, GT and AT) showed different number of repeats in different populations and thus, may be very useful for population differentiation and dynamics analyses in future studies. The sequences of P. lignarius (considered sylvatic) and P. herreri (a major disease vector in Peru) were identical, suggesting that these species should be synonymised. Intrageneric analysis showed a clear separation of P. rufotuberculatus, with closest relationships between P. geniculatus and P. chinai, and P. megistus occupying a separate branch. Genetic distances between Panstrongylus species (0.11585-0.22131) were higher than those between Panstrongylus and other Triatomini (16 species from central and North America and South America) (0.08617-0.11039). The distance between P. megistus and P. lignarius/herreri (0.22131) was the largest so far recorded in the tribe. The pronounced differences in length and nucleotide composition suggest a relatively old divergence of Panstrongylus species. P. rufotuberculatus was closer to Mesoamerican Triatoma, Meccus and Dipetalogaster species than to other Panstrongylus. All Panstrongylus clustered with the Mesoamerican clade; P. rufotuberculatus clustered with the phyllosoma complex and T. dimidiata, with D. maxima and T. barberi in a basal position. The rest of Panstrongylus appeared paraphyletically in the tree. This is evidence suggesting polyphyly within the genus Panstrongylus, whose species may be related to the ancestors giving rise to central and North American Triatomini.

Adult and nymphs of Microtriatoma trinidadensis (Lent, 1951) (Hemiptera: Reduviidae) caught from peridomestic environment in Bolivia.

This is the first report of adult and nymphs (20 nymphs of all stages and 4 adults) of Microtriatoma trinidadensis (Lent 1951) (Hemiptera: Reduviidae: Triatominae) collected in peridomestic environment, in the department of La Paz, Bolivia. These specimens were associated to Rhodnius stali Lent, Jurberg & Galvão 1993. The exceptional finding of M. trinidadensis in peridomestic environment, illustrates the general tendency of triatominae to adapt to human dwellings and dependences.

Rhodnius robustus in Bolivia identified by its wings.

Wings of a Rhodnius specimen from Alto Beni (Bolivia) was examined for identification and compared with R. stali, R. robustus, (certified Bolivian species), R. pictipes and R. prolixus (suspected Bolivian species). A projection of the unidentified wings as supplementary data into a discriminant analysis of shape revealed clear cut differences with R. stali and R. pictipes, less differences with R. prolixus, and none with R. robustus. Combining global size and shape of the wings, the unknown specimen was identified as R. robustus. Thus, this study confirmed the presence of R. robustus in Bolivia. It also highlighted the possibility of morphometrics to taxonomically interpret one individual, or even one piece of an individual, when related species data are available for comparison.

Genetic studies of Psammolestes tertius (hemiptera: reduviidae: triatominae) using male genital morphology, morphometry, isoenzymes, and random amplified polymorphic DNA.

Two Brazilian populations of Psammolestes tertius (Ceará and Minas Gerais) collected from thornbird nests (Furnariidae) were compared by male genital morphology, morphometry, isoenzymes, and random amplified polymorphic DNA (RAPD). Male genitalia showed no difference between the populations. In contrast, morphometry, isoenzyme, and RAPD clearly distinguished the two populations. Possible mechanisms of dispersal and the origin of Psammolestes are discussed.