Karyotype Evolution in Triatominae (Hemiptera, Reduviidae): The Role of Chromosomal Rearrangements in the Diversification of Chagas Disease Vectors.

Several cytogenetic studies have already been performed in Triatominae, such that different karyotypes could be characterized (ranging from 2n = 21 to 25 chromosomes), being the changes in the number of chromosomes related mainly to fusion and fission events. These changes have been associated with reproductive isolation and speciation events in other insect groups. Thus, we evaluated whether different karyotypes could act in the reproductive isolation of triatomines and we analyzed how the events of karyotypic evolution occurred along the diversification of these vectors. For this, experimental crosses were carried out between triatomine species with different karyotypes. Furthermore, based on a phylogeny with 88 triatomine taxa (developed with different molecular markers), a reconstruction of ancestral karyotypes and of anagenetic and cladogenetic events related to karyotypic alterations was performed through the ChromoSSE chromosomal evolution model. All crosses performed did not result in hybrids (prezygotic isolation in both directions). Our modeling results suggest that during Triatominae diversification, at least nine cladogenetic events may be associated with karyotype change. Thus, we emphasize that these alterations in the number of chromosomes can act as a prezygotic barrier in Triatominae (karyotypic isolation), being important evolutionary events during the diversification of the species of Chagas disease vectors.

A microscopy investigation of the complex problem of infertility of insect hybrids. Studies on the reproductive systems, eggshells, and karyotypes of the representatives of the genus Platymeris (Heteroptera, Reduviidae) and their hybrids.

Hybridisation is still a widely studied phenomenon that allows us to look at some processes differently. However, obtaining fertile hybrids, which we might consider in the long term as precursors of a new species, is still a field that requires research. Much of the research done so far indicates that hybrids are sterile - either sex or both. It is influenced by various mechanisms, both prezygotic and postzygotic reproductive isolation. Thanks to the use of light and transmission electron microscopy, our research has made it possible to understand, at least partially, the causes of infertility in male hybrids resulting from the crossing of two Afrotropical species of the genus Platymeris Laporte, 1833. The analysis of microscopic images showed that one of the possible causes of the infertility of hybrids might be spermatogenesis, during which sperm cells were not formed. In turn, the use of scanning electron microscopy revealed potential abnormalities in the structure of the hybrid eggshell. Moreover, karyotyping analyses suggest possible causes of infertility at the genetic level.

Biological, ecological, morphological and cytogenetic analyses, with taxonomic notes of emZelurus/em emochripennis/em (Stål, 1854) (Hemiptera: Heteroptera: Reduviidae: Reduviinae).

Based on specimens of Zelurus ochripennis (Stål, 1854) collected in the entrance zone of a small terrestrial cave, some biological, ecological, morphological and cytogenetic data, as well, as the general morphology of their nymphs are presented for the first time. A short taxonomic summary of the species is also provided.

Biogeographic origin and phylogenetic relationships of Mepraia (Hemiptera, Reduviidae) on islands of northern Chile.

Chagas disease is one of the main zoonoses mediated by vectors in America. The etiological agent is the protozoan Trypanosoma cruzi, transmitted mainly by hematophagous insects of the subfamily Triatominae. Mepraia species are triatomines endemic to Chile that play an important role in T. cruzi transmission in the wild cycle and are potential vectors for humans. In addition to the continental distribution, populations of Mepraia genus have been reported inhabiting islands of northern Chile. The presence of individuals of Mepraia in insular areas might be explained through passive dispersion by marine birds or by vicariance of an ancestral widespread population. To clarify the biogeographic origin and phylogenetic relationships of island individuals of Mepraia, mitochondrial COI and cyt b genes were sequenced in individuals from island and continental areas. Gene sequences were used to estimate phylogenetic relationships, divergence dates and migration rates between insular and continental populations. The dates of divergence estimates are congruent with sea level and tectonic changes that originated the islands during Pleistocene. Migration rates suggest symmetric historical island-continent gene flow. We suggest that the origin of island triatomines can be explained by both vicariance and dispersion. Phylogenetic relationships show that individuals from Santa María Island and the continent clustered in a clade different from those previously reported, indicating a new lineage of Mepraia genus. This study will contribute to understand the origin of the T. cruzi infection in coastal islands of northern Chile.

Invasion of the assassin bug Agriosphodrus dohrni (Hemiptera: Reduviidae) to Japan: Source estimation inferred from mitochondrial and nuclear gene sequences.

A large-sized assassin bug Agriosphodrus dohrni (Signoret), has been recorded from India, Vietnam, China and Japan. It is one of the potential biological control agents against some important agricultural and forest pests. This species is speculated to have invaded Japan from its native range in China about 60 years ago. We used three mitochondrial gene fragments (COI, Cytb, and ND5) and one nuclear gene fragment (EF-1α) to clarify the invasion history of A. dohrni and assess the effects of geographic events and associated ecological adaptation on the distribution pattern. The native populations of A. dohrni in China are divided into three distinct groups, which might be molded by the Early Pleistocene glaciation event and diverged during the Calabrian Stage. However, consistent with the hypothesis of a recent invasion, extremely low level of genetic variation was detected in the Japanese populations, with only two haplotypes for the combined mitochondrial genes. Both the splits network and the ML/BI phylogenetic trees revealed that haplotypes of Japan were more closely-related to those from eastern China. Therefore, we postulate that there has been only one introduction event, probably from somewhere around the Nanjing (NJ) and Lin'an (LA) populations of eastern China.

Pathogen-insect interaction candidate molecules for transmission-blocking control strategies of vector borne diseases.

OBJECTIVE: To analyze the current knowledge of pathogen-insect interactions amenable for the design of molecular-based control strategies of vector-borne diseases. MATERIALS AND METHODS: We examined malaria, dengue, and Chagas disease pathogens and insect molecules that participate in interactions during their vectors infection. RESULTS: Pathogen molecules that participate in the insect intestine invasion and induced vector immune molecules are presented, and their inclusion in transmission blocking vaccines (TBV) and in genetically modify insect (GMI) vectors or symbiotic bacteria are discussed. CONCLUSIONS: Disruption of processes by blocking vector-pathogen interactions provides several candidates for molecular control strategies, but TBV and GMI efficacies are still limited and other secondary effects of GMI (improving transmission of other pathogens, affectation of other organisms) should be discarded.

The assassin bug Pristhesancus plagipennis produces two distinct venoms in separate gland lumens.

The assassin bug venom system plays diverse roles in prey capture, defence and extra-oral digestion, but it is poorly characterised, partly due to its anatomical complexity. Here we demonstrate that this complexity results from numerous adaptations that enable assassin bugs to modulate the composition of their venom in a context-dependent manner. Gland reconstructions from multimodal imaging reveal three distinct venom gland lumens: the anterior main gland (AMG); posterior main gland (PMG); and accessory gland (AG). Transcriptomic and proteomic experiments demonstrate that the AMG and PMG produce and accumulate distinct sets of venom proteins and peptides. PMG venom, which can be elicited by electrostimulation, potently paralyses and kills prey insects. In contrast, AMG venom elicited by harassment does not paralyse prey insects, suggesting a defensive role. Our data suggest that assassin bugs produce offensive and defensive venoms in anatomically distinct glands, an evolutionary adaptation that, to our knowledge, has not been described for any other venomous animal.

Pathogen-insect interaction candidate molecules for transmission-blocking control strategies of vector borne diseases / Moléculas candidatas para el control de enfermedades transmitidas por vector mediante el bloqueo de interacciones patógeno-insecto

Abstract: Objective: To analyze the current knowledge of pathogen-insect interactions amenable for the design of molecular-based control strategies of vector-borne diseases. Materials and methods: We examined malaria, dengue, and Chagas disease pathogens and insect molecules that participate in interactions during their vectors infection. Results: Pathogen molecules that participate in the insect intestine invasion and induced vector immune molecules are presented, and their inclusion in transmission blocking vaccines (TBV) and in genetically modify insect (GMI) vectors or symbiotic bacteria are discussed. Conclusion: Disruption of processes by blocking vector-pathogen interactions provides several candidates for molecular control strategies, but TBV and GMI efficacies are still limited and other secondary effects of GMI (improving transmission of other pathogens, affectation of other organisms) should be discarded.

Resumen: Objetivo: Analizar el conocimiento actual de las interacciones patógeno-insecto susceptibles a incluirse en el diseño de estrategias moleculares para el control de enfermedades transmitidas por vectores. Material y métodos: Se examinaron los agentes causales de la malaria, el dengue y la enfermedad de Chagas, y las moléculas de insectos que participan en interacciones durante la infección de sus vectores. Resultados: Se presentan moléculas de patógenos que participan en la invasión del intestino del insecto y moléculas inmunes inducidas en los vectores. Se discute su inclusión en vacunas bloqueadoras de transmisión (VBT) y en la modificación genética de vectores (MGI) o de sus bacterias simbióticas. Conclusión: La interrupción de procesos mediante el bloqueo de las interacciones patógeno-vector proporciona varios candidatos para las estrategias de control molecular, pero la eficacia de VBT y MGI es aún limitada y los efectos secundarios de MGI (aumento de la transmisión de otros patógenos y afectación de otros organismos) deben descartase.

Temporal transcriptomic profiling of the ant-feeding assassin bug Acanthaspis cincticrus reveals a biased expression of genes associated with predation in nymphs.

Acanthaspis cincticrus (Stål) is an assassin bug with a specialized camouflaging behavior to ambush ants in the nymphal stages. In this study, we comprehensively sequenced all the life stages of A. cincticrus, including the eggs, five nymph instars, female and male adults using Illumina HiSeq technology. We obtained 176 million clean sequence reads. The assembled 84,055 unigenes were annotated and classified functionally based on protein databases. Among the unigenes, 29.03% were annotated by one or more databases, suggesting their well-conserved functions. Comparison of the gene expression profiles in the egg, nymph and adult stages revealed certain bias. Functional enrichment analysis of significantly differentially expressed genes (SDEGs) showed positive correlation with specific physiological processes within each stage, including venom, aggression, olfactory recognition as well as growth and development. Relative expression of ten SDEGs involved in predation process was validated using quantitative real-time PCR (qRT-PCR).

Molecular phylogenetics and biogeography of the ambush bugs (Hemiptera: Reduviidae: Phymatinae).

The ambush bugs (Heteroptera: Reduviidae: Phymatinae) are a diverse clade of predators known for their cryptic hunting behavior and morphologically diverse raptorial forelegs. Despite their striking appearance, role as pollinator predators, and intriguing biogeographic distribution, phylogenetic relationships within Phymatinae are largely unknown and the evolutionary history of the subfamily has remained in the dark. We here utilize the most extensive molecular phylogeny of ambush bugs to date, generated from a 3328 base pair molecular dataset, to refine our understanding of phymatine relationships, estimate dates of divergence (BEAST 2), and uncover historical biogeographic patterns (S-DIVA and DEC). This taxon set (39 species of Phymatinae and six outgroups) allowed reevaluation of the proposed sister group of Phymatinae and tribal-level relationships within the group, and for the first time proposes species-level relationships within Phymata Latreille, the largest genus of ambush bugs (∼109spp.). Available evidence suggests that Phymata originated in the Neotropical region, with subsequent dispersals to the Nearctic and Palearctic regions. This study provides a framework for future research investigating the evolutionary history of ambush bugs, as well as ecological and microevolutionary investigations.

Melt With This Kiss: Paralyzing and Liquefying Venom of The Assassin Bug Pristhesancus plagipennis (Hemiptera: Reduviidae).

Assassin bugs (Hemiptera: Heteroptera: Reduviidae) are venomous insects, most of which prey on invertebrates. Assassin bug venom has features in common with venoms from other animals, such as paralyzing and lethal activity when injected, and a molecular composition that includes disulfide-rich peptide neurotoxins. Uniquely, this venom also has strong liquefying activity that has been hypothesized to facilitate feeding through the narrow channel of the proboscis-a structure inherited from sap- and phloem-feeding phytophagous hemipterans and adapted during the evolution of Heteroptera into a fang and feeding structure. However, further understanding of the function of assassin bug venom is impeded by the lack of proteomic studies detailing its molecular composition.By using a combined transcriptomic/proteomic approach, we show that the venom proteome of the harpactorine assassin bug Pristhesancus plagipennis includes a complex suite of >100 proteins comprising disulfide-rich peptides, CUB domain proteins, cystatins, putative cytolytic toxins, triabin-like protein, odorant-binding protein, S1 proteases, catabolic enzymes, putative nutrient-binding proteins, plus eight families of proteins without homology to characterized proteins. S1 proteases, CUB domain proteins, putative cytolytic toxins, and other novel proteins in the 10-16-kDa mass range, were the most abundant venom components. Thus, in addition to putative neurotoxins, assassin bug venom includes a high proportion of enzymatic and cytolytic venom components likely to be well suited to tissue liquefaction. Our results also provide insight into the trophic switch to blood-feeding by the kissing bugs (Reduviidae: Triatominae). Although some protein families such as triabins occur in the venoms of both predaceous and blood-feeding reduviids, the composition of venoms produced by these two groups is revealed to differ markedly. These results provide insights into the venom evolution in the insect suborder Heteroptera.

Would Nesotriatoma bruneri Usinger, 1944 be a valid species?

Variation in the size and morphological differences intraspecific of Nesotriatoma flavida led to the description of N. bruneri. However, two years later the same author proposed the synonymization N. bruneri with N. flavida. N. bruneri was revalidated through morphological analysis after 35 years. Thus, given the existing taxonomic questioning between these Cuban triatomines, we analyzed new parameters such as genetic distance from the mitochondrial 16S rDNA deposited in Genbank and cytogenetic characterization, through the constitutive heterochromatin pattern, in order to reassess the specific status of N. bruneri. The analysis of the disposition of constitutive heterochromatin in the genome of these triatomines allowed observing that only the sex chromosome Y is heterochromatic, and the autosomes and the sex chromosomes X are euchromatic. These characteristics are identical to those described for N. flavida. By means of analysis of genetic distance matrix, we found that the genetic distance between N. bruneri and N. flavida was only 0.04%. Thus, by means of extremely low genetic distance and identical cytogenetic characteristics, we suggest that N. bruneri should be back again synonymized with N. flavida. However, we recommend that experimental hybrid crosses and new molecular analysis should be conducted, focusing mainly in the genetic distance based on other genes, on the rate of fertility of eggs and viability of hybrids to confirm the proposed of synonymization.

Duplication and Remolding of tRNA Genes in the Mitochondrial Genome of Reduvius tenebrosus (Hemiptera: Reduviidae).

Most assassin bugs are predators that act as important natural enemies of insect pests. Mitochondrial (mt) genomes of these insects are double-strand circular DNAs that encode 37 genes. In the present study, we explore the duplication and rearrangement of tRNA genes in the mt genome of Reduvius tenebrosus, the first mt genome from the subfamily Reduviinae. The gene order rearranges from CR (control region)-trnI-trnQ-trnM-ND2 to CR-trnQ-trnI2-trnI1-trnM-ND2. We identified 23 tRNA genes, including 22 tRNAs commonly found in insects and an additional trnI (trnI2), which has high sequence similarity to trnM. We found several pseudo genes, such as pseudo-trnI, pseudo-CR, and pseudo-ND2, in the hotspot region of gene rearrangement (between the control region and ND2). These features provided evidence that this novel gene order could be explained by the tandem duplication/random loss (TDRL) model. The tRNA duplication/anticodon mutation mechanism further explains the presence of trnI2, which is remolded from a duplicated trnM in the TDRL process (through an anticodon mutation of CAT to GAT). Our study also raises new questions as to whether the two events proceed simultaneously and if the remolded tRNA gene is fully functional. Significantly, the duplicated tRNA gene in the mitochondrial genome has evolved independently at least two times within assassin bugs.

Transmission Capacity of Trypanosoma cruzi (Trypanosomatida: Trypanosomatidae) by Three Subspecies of Meccus phyllosomus (Heteroptera: Reduviidae) and Their Hybrids.

Three behaviors of epidemiological importance: the time lapse for the onset of feeding, actual feeding, and defecation time for Meccus phyllosomus pallidipennis (Stål), Meccus phyllosomus longipennis (Usinger), Meccus phyllosomus picturatus (Usinger), and their laboratory hybrids were evaluated in this study. The mean time lapse for the beginning of feeding was between 0.5 and 8.3 min considering all instars in each cohort, with highly significant differences only among fifth-instar nymphs, females, and males of M. p. pallidipennis and M. p. longipennis relative to the hybrid cohorts. Four hybrid (LoPa [M. p. longipennis and M. p. pallidipennis] and LoPi [M. p. longipennis and M. p. picturatus] and their reciprocal experimental crosses) cohorts had similar mean feeding times to one of the parental subspecies, but longer than the other one. The remaining hybrid cohort (PaPi [M. p. pallidipennis and M. p. picturatus]) had longer feeding times than both of its parental subspecies. The specimens of the LoPa and LoPi hybrid cohorts defecated faster than the respective instars of the three parental cohorts. With exception of first- and fifth-instar nymphs, PaPi cohorts defecated faster than the remaining seven cohorts. More than 60% of defecation events occurred during feeding in the six hybrid cohorts. Our results indicate that hybrid cohorts have more potential to acquire infection and transmit Trypanosoma cruzi Chagas than their parental cohorts.

Phylogenetics and biogeography of the endemic Madagascan millipede assassin bugs (Hemiptera: Reduviidae: Ectrichodiinae).

For at least the past 80my, Madagascar, a major biodiversity hotspot, has been isolated from all other landmasses. This long-term isolation, along with geologic and climatic factors within Madagascar and throughout the Indian Ocean, has undoubtedly influenced the evolution of the island's biota. However, few systematic analyses incorporating modern divergence dating and biogeographic analyses have focused on Madagascan insects. The diverse Madagascan millipede assassin bugs (Heteroptera: Reduviidae: Ectrichodiinae) offer an opportunity to contribute to a limited body of insect-related research that explores Madagascar's historical biogeography. A molecular dataset (COI mtDNA and 18S, 28S D2 and D3-D5 rDNAs) for 56 taxa (39 ingroup) and a combined morphological (145 characters) and molecular dataset for 110 taxa (93 ingroup) are analyzed with maximum likelihood (ML) and parsimony approaches. Based on the molecular ML phylogeny, divergence times were estimated using fossil and secondary calibrations and biogeographic analyses performed using DIVA, DEC, and DEC+j models to determine the role and patterns of vicariance and dispersal in the origin of Madagascan Ectrichodiinae. Results indicate that Ectrichodiinae in Madagascar do not form a monophyletic group, different clades are closely related to Afrotropical and Oriental lineages, and have colonized the island via transoceanic dispersal at least twice from the Oriental region and once from the Afrotropical region in the last ∼68my. Additionally, the DEC+j and DIVA models infer a single out-of-Madagascar dispersal event to the Afrotropical region. Oceanic and geologic factors that may have facilitated dispersal between these three regions are discussed. Results of the combined analyses are used to explore character support for Madagascan taxa and inform taxonomic diagnoses. Our results are congruent with the small but growing body of biogeographic research supporting Cenozoic transoceanic dispersal for Madagascan invertebrates to and from Oriental and Afrotropical regions.

Efficient capture of natural history data reveals prey conservatism of cryptic termite predators.

Stenophagy, specialization of a clade on a narrow range of taxa, has not been well studied in speciose clades of predators, principally due to the difficulty of obtaining adequate natural history data. The pantropical Salyavatinae (Hemiptera: Reduviidae; 17 genera, 107 species) contains members with enigmatic morphology and specialized behavior for feeding on termites. All Salyavatinae are suspected specialist termite predators; however, existing observations are limited to seven species. Prior analyses indicate that Salyavatinae may be paraphyletic with respect to another subfamily, Sphaeridopinae, also hypothesized to feed on termites. A molecular phylogeny of these putative termite assassins is here constructed using seven loci from 28 species in nine genera and is used in a dating analysis to shed light on the timing of Neotropical colonization by this primarily Old World clade. DNA extracted from gut contents of 50 individuals was assayed using PCR with prey-specific primers.Molecular assays, along with recent photographs and observations, provide substantial evidence that this clade feeds specifically upon termites, documenting 28 new individual associations. Our phylogeny supports a sister group relationship of the Neotropical genus Salyavata with Sphaeridopinae. Termite association data combined with our phylogeny provide evidence of previously unknown prey conservatism among clades of one of the most diverse groups of specialist termite predators.

Evolution of sexual dimorphism in phenotypic covariance structure in Phymata.

Sexual dimorphism is a consequence of both sex-specific selection and potential constraints imposed by a shared genetic architecture underlying sexually homologous traits. However, genetic architecture is expected to evolve to mitigate these constraints, allowing the sexes to approach their respective optimal mean phenotype. In addition, sex-specific selection is expected to generate sexual dimorphism of trait covariance structure (e.g., the phenotypic covariance matrix, P), but previous empirical work has not fully addressed this prediction. We compared patterns of phenotypic divergence, for three traits in seven taxa in the insect genus Phymata (Reduviidae), to ask whether sexual dimorphism in P is common and whether its magnitude relates to the extent of sexual dimorphism in trait means. We found that sexual dimorphism in both mean and covariance structure was pervasive but also that the multivariate distance between sex-specific means was correlated with sex differences in the leading eigenvector of P, while accounting for uncertainty in phylogenetic relationships. Collectively, our findings suggest that sexual dimorphism in covariance structure may be a common but underappreciated feature of dioecious populations.

Comparative mitogenomics of the assassin bug genus Peirates (Hemiptera: Reduviidae: Peiratinae) reveal conserved mitochondrial genome organization of P. atromaculatus, P. fulvescens and P. turpis.

In this study, we sequenced four new mitochondrial genomes and presented comparative mitogenomic analyses of five species in the genus Peirates (Hemiptera: Reduviidae). Mitochondrial genomes of these five assassin bugs had a typical set of 37 genes and retained the ancestral gene arrangement of insects. The A+T content, AT- and GC-skews were similar to the common base composition biases of insect mtDNA. Genomic size ranges from 15,702 bp to 16,314 bp and most of the size variation was due to length and copy number of the repeat unit in the putative control region. All of the control region sequences included large tandem repeats present in two or more copies. Our result revealed similarity in mitochondrial genomes of P. atromaculatus, P. fulvescens and P. turpis, as well as the highly conserved genomic-level characteristics of these three species, e.g., the same start and stop codons of protein-coding genes, conserved secondary structure of tRNAs, identical location and length of non-coding and overlapping regions, and conservation of structural elements and tandem repeat unit in control region. Phylogenetic analyses also supported a close relationship between P. atromaculatus, P. fulvescens and P. turpis, which might be recently diverged species. The present study indicates that mitochondrial genome has important implications on phylogenetics, population genetics and speciation in the genus Peirates.

Complete mitochondrial genome of the assassin bug Oncocephalus breviscutum (Hemiptera: Reduviidae).

The mitochondrial genome of an assassin bug, Oncocephalus breviscutum Reuter, is a typical circular DNA molecule of 15,984 bp with 37 genes and a large control region. The gene order is identical to that of the putative ancestral arrangement of insects. Twelve protein-coding genes start with ATN codon and ND4L uses GTG. All of the 22 tRNAs, ranging from 61 to 70 bp, have the clover-leaf structure except for the dihydrouridine (DHU) arm of trnS2 forms a simple loop. The control region is 1345 bp in length and includes six tandem repeats of three 31-nt and three 145-nt units.

DNA barcoding does not separate South American Triatoma (Hemiptera: Reduviidae), Chagas Disease vectors.

BACKGROUND: DNA barcoding assumes that a biological entity is completely separated from its closest relatives by a barcoding gap, which means that intraspecific genetic distance (from COI sequences) should never be greater than interspecific distances. We investigated the applicability of this strategy in identifying species of the genus Triatoma from South America. FINDINGS: We calculated intra and interspecific Kimura-2-parameter distances between species from the infestans, matogrossensis, sordida and rubrovaria subcomplexes. In every subcomplex examined we observed at least one intraspecific distance greater than interspecific distances. CONCLUSIONS: Although DNA barcoding is a straightforward approach, it was not applicable for identifying Southern American Triatoma species, which may have diverged recently. Thus, caution should be taken in identifying vector species using this approach, especially in groups where accurate identification of taxa is fundamentally linked to public health issues.