LINCOLN SUESDEK ROCHA
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- Wing Morphometry and Genetic Variability Between Culex coronator and Culex usquatus (Diptera: Culicidae), Two Sibling Species of the Coronator Group(2017) DEMARI-SILVA, Bruna; MULTINI, Laura Cristina; SUESDEK, Lincoln; OLIVEIRA, Tatiane M. P.; SALLUM, Maria Anice Mureb; MARRELLI, Mauro ToledoCulex coronator Dyar and Knab and Culex usquatus Dyar belong to the Coronator Group of the subgenus Culex. Culex coronator and Cx. usquatus are widespread and sympatric throughout their distribution range, which includes Brazil. Morphological identification of these species is based primarily on the characteristics of the male genitalia; females are indistinguishable using the qualitative characteristics employed in identification keys. The primary objective of this study was to distinguish females of Cx. coronator from those of Cx. usquatus employing both wing geometric morphometrics, and DNA sequences (NADH5, COI, Hunchback, and CAD). Additionally, we employed the isolation with migration model (IMa) to evaluate: 1) the migration rates and 2) the divergence time, between Cx. coronator and Cx. usquatus. Specimens were captured in Pariquera-Acu and Cananeia south-eastern Sao Paulo, Ribeira Valley, Brazil. Canonical variate analysis (CVA) demonstrated two groups in the morphospace. The accuracy of species recognition was moderate (82.6%) for Cx. coronator and low (60.8%) for Cx. usquatus. Bayesian analyses of concatenated gene sequences recovered from specimens of Cx. coronator separated the species into three lineages (herein referred to as Culex coronator A, B, and C), whereas Cx. usquatus specimens clustered into a single lineage. Lineages A and B of Cx. coronator intermixed with specimens of Cx. usquatus, and one specimen of Cx. coronator clustered into the Cx. usquatus lineage. The IMa analysis indicated that the divergence of Cx. coronator and Cx. usquatus is a slow process, with some degree of gene flow between the two species.
- Altitudinal population structure and microevolution of the malaria vector Anopheles cruzii (Diptera: Culicidae)(2014) LORENZ, Camila; MARQUES, Tatiani Cristina; SALLUM, Maria Anice Mureb; SUESDEK, LincolnBackground: In Brazil, the autochthonous transmission of extra-Amazonian malaria occurs mainly in areas of the southeastern coastal Atlantic Forest, where Anopheles cruzii is the primary vector. In these locations, the population density of the mosquito varies with altitude (5-263 m above sea level), prompting us to hypothesise that gene flow is also unevenly distributed. Describing the micro-geographical and temporal biological variability of this species may be a key to understanding the dispersion of malaria in the region. We explored the homogeneity of the An. cruzii population across its altitudinal range of distribution using wing shape and mtDNA gene analysis. We also assessed the stability of wing geometry over time. Methods: Larvae were sampled from lowland (5-20 m) and hilltop (81-263 m) areas in a primary Atlantic Forest region, in the municipality of Cananeia (State of Sao Paulo, Brazil). The right wings of males and females were analysed by standard geometric morphometrics. Eighteen landmarks were digitised for each individual and a discriminant analysis was used to compare samples from the hilltop and lowland. A 400-bp DNA fragment of the mitochondrial cytochrome oxidase gene subunit I (CO-I) was PCR-amplified and sequenced. Results: Wing shapes were distinct between lowland and hilltop population samples. Results of cross-validated tests based on Mahalanobis distances showed that the individuals from both micro-environments were correctly reclassified in a range of 54-96%. The wings of hilltop individuals were larger. The CO-I gene was highly polymorphic (haplotypic diversity = 0.98) and altitudinally structured (Phi st = 0.085 and Jaccard = 0.033). We found 60 different haplotypes but only two were shared by the lowland and hilltop populations. Wing shape changed over the brief study period (2009-2013). Conclusions: Wing geometry and CO-I gene analysis indicated that An. cruzii is vertically structured. Wing shape varied rapidly, but altitude structure was maintained. Future investigations should identify the biotic/abiotic causes of these patterns and their implications in the local epidemiology of malaria.
- Polymorphism in male genitalia of Aedes (Ochlerotatus) scapularis Rondani, 1848(2018) PETERSEN, V.; VIRGINIO, F.; SUESDEK, L.Morphology of male genitalia of culicids is generally species-specific and often used as a taxonomic marker. However, some characters of the male genitalia vary intraspecifically and are not taxonomically diagnostic. This might be the case of Aedes scapularis, a Neotropical culicid with vector competence for arboviruses and filarial worms. Males of this species may or not present a retrorse process (RP) in the genitalic claspette filaments, which led authors to suspect that this variance might be indicative of population divergence or incipient speciation process. This suspicion has not been investigated hitherto and it is not known if there are variable patterns of RPs. We hypothesized that the presence of the RP varies intraspecifically in Ae. scapularis and then we statistically evaluated the variability of this character in a single population. To this study the genitalia of 73 males of Ae. scapularis were prepared, and their RPs were meristically quantified and categorized according to the phenotypes observed. We noted that the presence or RPs is a polymorphic character because it varied inter and intra-individually. The presence of a single RP on each claspette filament was the predominant pattern (77%), but absent or multiple RPs in each filament were also found either in bilateral symmetry or asymmetry. Thus, we conclude that the presence of RPs owing to its high variability is not indicative of populational divergence or diagnostic of species complex within Ae. scapularis.
- Spatial and temporal epidemiology of malaria in extra-Amazonian regions of Brazil(2015) LORENZ, Camila; VIRGINIO, Flavia; AGUIAR, Breno S.; SUESDEK, Lincoln; CHIARAVALLOTI-NETO, FranciscoBackground: Mosquitoes, Plasmodium parasites, and humans live in sympatry in some extra-Amazonian regions of Brazil. Recent migrations of people from Amazonia and other countries to extra-Amazonian regions have led to many malaria outbreaks. Lack of relevant expertise among health professionals in non-endemic areas can lead to a neglect of the disease, which can be dangerous given its high fatality rate. Therefore, understanding the spatial and temporal epidemiology of malaria is essential for developing strategies for disease control and elimination. This study aimed to characterize imported (IMP) and autochthonous/introduced (AU/IN) cases in the extra-Amazonian regions and identify risk areas and groups. Methods: Epidemiological data collected between 2007 and 2014 were obtained from the Notifiable Diseases Information System of the Ministry of Health (SINAN) and from the Department of the Unified Health System (DATA-SUS). High malaria risk areas were determined using the Local Indicator of Spatial Association. IMP and AU/IN malaria incidence rates were corrected by Local Empirical Bayesian rates. Results: A total of 6092 malaria cases (IMP: 5416, 88.9 %; AU/IN: 676, 11.1 %) was recorded in the extra-Amazonian regions in 2007-2014. The highest numbers of IMP and AU/IN cases were registered in 2007 (n = 862) and 2010 (n = 149), respectively. IMP cases were more frequent than AU/IN cases in all states except for Espirito Santo. Piaui, Espirito Santo, and Parana states had high incidences of AU/IN malaria. The majority of infections were by Plasmodium falciparum in northeast and southeast regions, while Plasmodium vivax was the predominant species in the south and mid-west showed cases of dual infection. AU/IN malaria cases were concentrated in the coastal region of Brazil, which contains the Atlantic Forest and hosts the Anopheles transmitters. Several malaria clusters were also associated with the Brazilian Pantanal biome and regions bordering the Amazonian biome. Conclusion: Malaria is widespread outside the Amazonian region of Brazil, including in more urbanized and industrialized states. This fact is concerning because these highly populated areas retain favourable conditions for spreading of the parasites and vectors. Control measures for both IMP and AU/IN malaria are essential in these high-risk areas.
- Artificial Neural Network applied as a methodology of mosquito species identification(2015) LORENZ, Camila; FERRAUDO, Antonio Sergio; SUESDEK, LincolnThere are about 200 species of mosquitoes (Culicidae) known to be vectors of pathogens that cause diseases in humans. Correct identification of mosquito species is an essential step in the development of effective control strategies for these diseases; recognizing the vectors of pathogens is integral to understanding transmission. Unfortunately, taxonomic identification of mosquitoes is a laborious task, which requires trained experts, and it is jeopardized by the high variability of morphological and molecular characters found within the Culicidae family. In this context, the development of an automatized species identification method would be a valuable and more accessible resource to non-taxonomist and health professionals. In this work, an artificial neural network (ANN) technique was proposed for the identification and classification of 17 species of the genera Anopheles, Aedes, and Culex, based on wing shape characters. We tested the hypothesis that classification using ANN is better than traditional classification by discriminant analysis (DA). Thirty-two wing shape principal components were used as input to a Multilayer Perceptron Classification ANN. The obtained ANN correctly identified species with accuracy rates ranging from 85.70% to 100%, and classified species more efficiently than did the traditional method of multivariate discriminant analysis. The results highlight the power of ANNs to diagnose mosquito species and to partly automatize taxonomic identification. These findings also support the hypothesis that wing venation patterns are species-specific, and thus should be included in taxonomic keys.
- Assessment of the correlation between wing size and body weight in captive Culex quinquefasciatus(2016) PETERSEN, Vivian; MARCHI, Marco Jacometto; NATAL, Delsio; MARRELLI, Mauro Toledo; BARBOSA, Admilson Clayton; SUESDEK, LincolnIntroduction: Mass production of mosquitoes under laboratory conditions allows implementing methods to control vector mosquitoes. Colony development depends on mosquito size and weight. Body size can be estimated from its correlation with wing size, whereas weight is more difficult to determine. Our goal was to test whether wing size can predict the weight. Methods: We compared dry weight and wing centroid size of Culex quinquefasciatus reared at different temperatures and four diets. Results: Weight and wing size were strongly correlated. The diets did not influence wing size. Conclusions: Wing centroid size is a good predictor of Cx. quinquefasciatus body weight.
- Effects of Wolbachia on ovarian apoptosis in Culex quinquefasciatus (Say, 1823) during the previtellogenic and vitellogenic periods(2017) ALMEIDA, Fabio; SUESDEK, LincolnBackground: Apoptosis is programmed cell death that ordinarily occurs in ovarian follicular cells in various organisms. In the best-studied holometabolous insect, Drosophila, this kind of cell death occurs in all three cell types found in the follicles, sometimes leading to follicular atresia and egg degeneration. On the other hand, egg development, quantity and viability in the mosquito Culex quinquefasciatus are disturbed by the infection with the endosymbiont Wolbachia. Considering that Wolbachia alters reproductive traits, we hypothesised that such infection would also alter the apoptosis in the ovarian cells of this mosquito. The goal of this study was to comparatively describe the occurrence of apoptosis in Wolbachia-infected and uninfected ovaries of Cx. quinquefasciatus during oogenesis and vitellogenesis. For this, we recorded under confocal microscopy the occurrence of apoptosis in all three cell types of the ovarian follicle. In the first five days of adult life we observed oogenesis and, after a blood meal, the initiation step of vitellogenesis. Results: Apoptoses in follicular cells were found at all observation times during both oogenesis and vitellogenesis, and less commonly in nurse cells and the oocyte, as well as in atretic follicles. Our results suggested that apoptosis in follicular cells occurred in greater numbers in infected mosquitoes than in uninfected ones during the second and third days of adult life and at the initiation step of vitellogenesis. Conclusions: The presence of Wolbachia leads to an increase of apoptosis occurrence in the ovaries of Cx. quinquefasciatus. Future studies should investigate if this augmented apoptosis frequency is the cause of the reduction in the number of eggs laid by Wolbachia-infected females. Follicular atresia is first reported in the previtellogenic period of oogenesis. Our findings may have implications for the use of Wolbachia as a mosquito and pathogens control strategy.
- Morphogenetic characterisation, date of divergence, and evolutionary relationships of malaria vectors Anopheles cruzii and Anopheles homunculus(2015) LORENZ, Camila; PATANE, Jose S. L.; SUESDEK, LincolnThe mosquito species Anopheles cruzii and Anopheles homunculus are co-occurring vectors for etiological agents of malaria in southeastern Brazil, a region known to be a major epidemic spot for malaria outside Amazon region. We sought to better understand the biology of these species in order to contribute to future control efforts by (1) improving species identification, which is complicated by the fact that the females are very similar, (2) investigating genetic composition and morphological differences between the species, (3) inferring their phylogenetic histories in comparison with those of other Anophelinae, and (4) dating the evolutionary divergence of the two species. To characterise the species we used wing geometry and mitochondrial cytochrome oxidase subunit I (COI) gene as morphological and genetic markers, respectively. We also used the genes white, 28S, ITS2, Cytb, and COI in our phylogenetic and dating analyses. A comparative analysis of wing thin-plate splines revealed species-specific wing venation patterns, and the species An. cruzii showed greater morphological diversity (8.74) than An. homunculus (5.58). Concerning the COI gene, An. cruzii was more polymorphic and also showed higher haplotype diversity than An. homunculus, with many rare haplotypes that were displayed by only a few specimens. Phylogenetic analyses revealed that all tree topologies converged and showed [Anopheles bellator + An. homunculus] and [Anopheles laneanus + An. cruzii] as sister clades. Diversification within the subgenus Kerteszia occurred 2-14.2 million years ago. The landmark data associated with wing shape were consistent with the molecular phylogeny, indicating that this character can distinguish higher level phylogenetic relationships within the Anopheles group. Despite their morphological similarities and cooccurrence, An. cruzii and An. homunculus show consistent differences. Phylogenetic analysis revealed that the species are not sister-groups but species that recently diverged within the Kerteszia group, perhaps concomitantly with the radiation of bromeliads in South America or during the Pleistocene climate oscillations.
- Transcriptome profiling and Calreticulin expression in Zika virus -infected Aedes aegypti(2023) ALMEIDA, Laisa Silva de; NISHIYAMA-JR, Milton Yutaka; PEDROSO, Aurelio; COSTA-DA-SILVA, Andre Luis; IOSHINO, Rafaella Sayuri; CAPURRO, Margareth Lara; SUESDEK, LincolnZika virus (ZIKV) may cause febrile illness and neurological damage, such as microcephaly in fetuses. ZIKV is transmitted to humans by Aedes aegypti, a nearly cosmopolitan mosquito. Understanding the virus-vector molecular interactions has been promising to enhance the knowledge towards disease mitigation. Since ZIKV infection alters gene physiology of mosquitoes, we examined the expression profile of ZIKV-infected Ae. aegypti by several approaches to identify genes altered by viral infection. Transcriptomics were performed by comparing between ZIKV-infected and uninfected Ae. aegypti females, which revealed some differentially expressed genes. Most of these genes appear to be involved with immune response as evidenced by an interactome analysis, and a prominent finding was a calreticulin-like (CRT) gene, which was upregulated during the infection. Expression of CRT was also experimentally quantified by qPCR, however, it revealed no significant differences between infected and uninfected females. Instead, expression levels were highly variable among individuals and negatively correlated to viral load. We also tested the possibility of this gene to be silenced, but the doublestranded RNA did not reduce CRT expression, and actually increased the inter-individuals' expressional variability. Present results differed from our original hypothesis of upregulation by infection. They also diverged between them (comparing qPCR to Transcriptomics) and from the literature which reported augmented CRT levels in Aedes species during viral infection. Present case probably underlies a more complex virus-host interaction system than we expected. Regulation of this gene seems not to be a linear correlation between expression and viremy. As infection takes place, a complex homeostatic mechanism may act to prevent expression and other cellular tasks from drifting. It is also possible that CRT expression is simply randomly disturbed by viral infection. Taken together, results show that CRT expression profile during ZIKV infection is complex and requires different investigative approaches to be understood. Studies focused on the biochemical function of CRT protein and on its role in the native mosquito metabolic network could unravel how it is actually influenced by ZIKV. Current work contributes more by getting incidental findings and by posing new hypotheses than by answering the original questions.
- The use of wing shape for characterising macroevolution in mosquitoes (Diptera: Culicidae)(2020) LORENZ, Camila; SUESDEK, LincolnThe wing form of culicid mosquitoes shows considerable variation among groups: this phenomenon has been addressed by several studies through space-time analyses in mosquito populations, species, and genera. The observed variation results from a combination of two distinct factors: heredity and phenotypic plasticity. The first is usually related to wing shape, a complex character that may serve as a taxonomic marker in specific cases. We hypothesized that wing shape might be phylogenetically meaningful in Culicidae. In this study, we applied a geometric morphometrical approach based on 18 landmarks in 81 species of mosquitoes, representing 19 different genera, to investigate whether wing shape can help retrieve macroevolutionary patterns or identify any phylogenetic signals. We observed that wing shape differed considerably among groups, especially between Anophelinae and Culicinae subfamilies; thus, some wing shape elements may be synapomorphic. Comparisons among wing consensus after Procrustes superimposition revealed that landmark #1, located between the veins RS and R1, was the most variable. Sabethini tribe was distinguished from other taxa owing to a strong phylogenetic signal of its wings, whereas other culicids presented weaker signals and were not that distinguishable. Evolutionary forces such as natural selection, evolutionary limitation/constraint, or canalization mechanisms might drive the evolution of wing phenotype. These findings suggest that the wing undergoes evolution over long periods, but is not neutral enough to reconstruct the phylogenetic history of these insects. Gene-based studies should be performed to understand the driving forces in wing evolution.