The nuclear and mitochondrial genome assemblies of Tetragonisca angustula (Apidae: Meliponini), a tiny yet remarkable pollinator in the Neotropics.

BACKGROUND: The field of bee genomics has considerably advanced in recent years, however, the most diverse group of honey producers on the planet, the stingless bees, are still largely neglected. In fact, only eleven of the ~ 600 described stingless bee species have been sequenced, and only three using a long-read (LR) sequencing technology. Here, we sequenced the nuclear and mitochondrial genomes of the most common, widespread and broadly reared stingless bee in Brazil and other neotropical countries-Tetragonisca angustula (popularly known in Brazil as jataí). RESULTS: A total of 48.01 Gb of DNA data were generated, including 2.31 Gb of Pacific Bioscience HiFi reads and 45.70 Gb of Illumina short reads (SRs). Our preferred assembly comprised 683 contigs encompassing 284.49 Mb, 62.84 Mb of which (22.09%) corresponded to 445,793 repetitive elements. N50, L50 and complete BUSCOs reached 1.02 Mb, 91 contigs and 97.1%, respectively. We predicted that the genome of T. angustula comprises 17,459 protein-coding genes and 4,108 non-coding RNAs. The mitogenome consisted of 17,410 bp, and all 37 genes were found to be on the positive strand, an unusual feature among bees. A phylogenomic analysis of 26 hymenopteran species revealed that six odorant receptor orthogroups of T. angustula were found to be experiencing rapid evolution, four of them undergoing significant contractions. CONCLUSIONS: Here, we provided the first nuclear and mitochondrial genome assemblies for the ecologically and economically important T. angustula, the fourth stingless bee species to be sequenced with LR technology thus far. We demonstrated that even relatively small amounts of LR data in combination with sufficient SR data can yield high-quality genome assemblies for bees.

Decoding bee cleptoparasitism through comparative transcriptomics of Coelioxoides waltheriae and its host Tetrapedia diversipes.

Cleptoparasitism, also known as brood parasitism, is a widespread strategy among bee species in which the parasite lays eggs into the nests of the host species. Even though this behavior has significant ecological implications for the dynamics of several species, little is known about the molecular pathways associated with cleptoparasitism. To shed some light on this issue, we used gene expression data to perform a comparative analysis between two solitary neotropical bees: Coelioxoides waltheriae, an obligate parasite, and their specific host Tetrapedia diversipes. We found that ortholog genes involved in signal transduction, sensory perception, learning, and memory formation were differentially expressed between the cleptoparasite and the host. We hypothesize that these genes and their associated molecular pathways are engaged in cleptoparasitism-related processes and, hence, are appealing subjects for further investigation into functional and evolutionary aspects of cleptoparasitism in bees.

Cirugía de la ptosis palpebral: análisis de tres tipos de procedimientos quirúrgicos. Serie de Casos / Eyelid ptosis surgery: analysis of three types of surgical procedures. Number of cases

INTRODUCCIÓN. La ptosis palpebral se define como el descenso o disfunción del párpado con respecto a su posición considerada como normal, pudiendo causar alteraciones del campo visual. Puede dividirse en lesiones congénitas o adquiridas, por el grado de disfunción entre otros. Blefaroplastia, es el término para la corrección de la misma. Existen varias técnicas correctivas quirúrgicas y no quirúrgicas. CASOS. Se presenta una serie de 5 casos tratados mediante abordaje quirúrgico anterior; detallando la técnica quirúrgica utilizada, con corrección de las estructuras hipertrofiadas, sección, suspensión, sutura del elevador palpebral o su encortamiento; suspensión de índole estática con el uso de fascia del paciente y finalizando el procedimiento con la confección del surco palpebral superior. RESULTADOS. Los 5 pacientes fueron tratados con técnicas quirúrgicas individualizadas obteniéndose buenos resultados estéticos y funcionales, sin complicaciones durante o después del procedimiento. DISCUSIÓN. La técnica de blefaroplastia quirúrgica dependerá del grado de disfunción; se describen tres principales: Fasanella Servat, para ptosis leve y útil para ptosis congénita leve o síndrome de Horner; Reinserción de la aponeurosis del músculo elevador, técnica que respeta la anatomía y permite regular diferentes grados de ptosis; y Suspensión del párpado al músculo frontal pudiendo ser definitiva o reversible. La edad de intervención para ptosis congénita antes del año de edad es urgente, si tapa la pupila para así evitar ambliopía y tortícolis compensatoria, en el resto de casos se sugiere realizarla a partir de los 5 años de edad. CONCLUSIONES. El conocimiento adecuado de la anatomía palpebral, etiología de la dermatocalasia y blefaroptosis, permite realizar una correcta cirugía reparadora individualizada para cada paciente. Las técnicas presentadas en este trabajo son reproducibles.

INTRODUCTION. Palpebral ptosis is defined as the descent or dysfunction of the eyelid with respect to its position considered as normal, which can cause alterations in the visual field. It can be divided into congenital or acquired lesions, according to the degree of dysfunction, among others. Blepharoplasty is the term for its correction. There are several surgical and non-surgical corrective techniques. CASES. We present a series of 5 cases treated by anterior surgical approach; detailing the surgical technique used, with correction of the hypertrophied structures, section, suspension, suture of the palpebral elevator or its shortening; static suspension with the use of the patient's fascia and ending the procedure with the confection of the superior palpebral sulcus. RESULTS. All 5 patients were treated with individualized surgical techniques obtaining good aesthetic and functional results, without complications during or after the procedure. DISCUSSION. The surgical blepharoplasty technique will depend on the degree of dysfunction; three main techniques are described: Fasanella Servat, for mild ptosis and useful for mild congenital ptosis or Horner syndrome; Reinsertion of the levator muscle aponeurosis, a technique that respects the anatomy and allows regulating different degrees of ptosis; and Eyelid suspension to the frontalis muscle, which can be definitive or reversible. The age of intervention for congenital ptosis before one year of age is urgent, if it covers the pupil in order to avoid amblyopia and compensatory torticollis, in the rest of cases it is suggested to perform it from 5 years of age. CONCLUSIONS. The adequate knowledge of the palpebral anatomy, etiology of dermatochalasis and blepharoptosis, allows a correct individualized reparative surgery for each patient. The techniques presented in this work are reproducible.

Reconstrucción de la fosa poplítea con colgajo fasciocutáneo de la arteria safena / Reconstruction of the popliteal fossa with fasciocutaneous flap of the saphenous artery

INTRODUCCIÓN. Los defectos de la fosa poplítea suponen un desafío reconstructivo para el cirujano plástico, dada la relación íntima de esta área con la articulación de la rodilla y la neurovasculatura vital subyacente. El propósito de éste informe fie compartir la experiencia en la utilización de un colgajo fasciocutaneode la arteria safena. CASO CLÍNICO. Paciente de 5 años de edad que sufrió quemadura térmica en miembros inferiores con dos años y medio de evolución, ameritó injertos, presentó contractura por tejido cicatricial en fosa poplítea, dificultó la deambulación y desarrollo neuro osteomuscular. Se realizó reconstrucción de la fosa poplítea con colgajo fasciocutáneo de la arteria safena. RESULTADOS. El colgajo fasciocutáneo de la arteria safena dio cobertura al defecto en fosa poplítea izquierda tras retiro del tejido cicatricial que producía contractura, limitaba la marcha y el desarrollo neuro osteo-muscular. Seis meses postquirúrgicos brindó cobertura cutánea definitiva y estable en el área crítica, que permitió la deambulación con movimientos de extensión y flexión de rodilla conservados. DISCUSIÓN. Este colgajo al igual que en otros estudios que respaldan su ejecución brindó excelentes resultados en defectos de partes blandas a nivel de la articulación de la rodilla. CONCLUSIÓN. El colgajo fasciocutáneo de la arteria safena demostró utilidad para la reconstrucción del defecto de la fosa poplítea, posibilitó una cobertura definitiva, funcional y estética, restableció los ángulos de movilidad y favoreció el desarrollo pondoestatural del paciente.

INTRODUCTION. Defects of the popliteal fossa pose a reconstructive challenge for the plastic surgeon, because of the intimate relation of this area with the knee joint and the near vital neurovasculature; the purpose of this report was to share the experience of using a fasciocutaneous flap of the saphenous artery. CLINICAL CASE. A 5 year old patient who suffered thermal burn in lower limbs with two and a half years of evolution, he nedeed grafts and presented tissue contracture because the scar in the popliteal fossa hindered ambulation and neuro-osteomuscular growth. Reconstruction of the popliteal fossa was made it with a fasciocutaneous flap of the saphenous artery. RESULTS. The fasciocutaneous flap of the saphenous artery covered the defect in the left popliteal fossa after removal of the scar tissue that caused contracture, limited to walk and growth. Six months after surgery the flap provided definitive and secure skin coverage in the critical area, which allowed to walk with preserved knee extension and flexion movements. DISCUSSION. This flap was useful for the recons-truction of the defect of the popliteal fossa and provided excellent results in soft tissue defects in this area of the knee joint. CONCLUSION. The fasciocutaneous flap of the saphenous artery proved useful for the reconstruction of the popliteal fossa defect, it permited a definitive, functional and esthetic coverage, reestablished the angles of mobility and helped with the patient growth

Gene expression and epigenetics reveal species-specific mechanisms acting upon common molecular pathways in the evolution of task division in bees.

A striking feature of advanced insect societies is the existence of workers that forgo reproduction. Two broad types of workers exist in eusocial bees: nurses who care for their young siblings and the queen, and foragers who guard the nest and forage for food. Comparisons between these two worker subcastes have been performed in honeybees, but data from other bees are scarce. To understand whether similar molecular mechanisms are involved in nurse-forager differences across distinct species, we compared gene expression and DNA methylation profiles between nurses and foragers of the buff-tailed bumblebee Bombus terrestris and the stingless bee Tetragonisca angustula. These datasets were then compared to previous findings from honeybees. Our analyses revealed that although the expression pattern of genes is often species-specific, many of the biological processes and molecular pathways involved are common. Moreover, the correlation between gene expression and DNA methylation was dependent on the nucleotide context, and non-CG methylation appeared to be a relevant factor in the behavioral changes of the workers. In summary, task specialization in worker bees is characterized by a plastic and mosaic molecular pattern, with species-specific mechanisms acting upon broad common pathways across species.

Conserved numts mask a highly divergent mitochondrial-COI gene in a species complex of Australian stingless bees Tetragonula (Hymenoptera: Apidae).

Tetragonula carbonaria, Tetragonula davenporti, Tetragonula hockingsi and Tetragonula mellipes comprise a species complex of Australian stingless bee species known as the 'Carbonaria' group. The species are difficult to distinguish morphologically and the major species-defining characters relate to comb architecture and nest entrance ornamentation. The taxonomy of the group is further complicated by likely nuclear mitochondrial pseudogenes (numts) and inter-specific hybrids. Here we demonstrate the existence of COI numts and isolate and characterize the 'true' mt-COI gene in T. carbonaria and T. hockingsi. Numts were isolated from enriched-nuclear DNA extraction followed by PCR amplification and Sanger sequencing, and were recognized by the presence of deletions and/or premature stop codons in the translated sequences. The mt-COI sequences were obtained from NGS sequencing using purified mtDNA. In T. carbonaria, two numts (numt1 and numt2) were identified and a third (numt3) was identified in T. hockingsi. Numt2 and numt3 are similar (1.2% sequence divergence), indicating a recent common origin. The genetic distance between the mt-COI of the two Tetragonula species was higher than might be expected for closely related species, 16.5%, corroborating previous studies in which T. carbonaria and T. hockingsi were regarded as separate species. The three numts are more similar to the COI of other stingless bee species, including Australian Austroplebia australis and South American Melipona bicolor (81.7-83.9%) than to the mt-COI of their own species (70-71.4%). This is because the mt-COI of T. carbonaria and T. hockingsi differ greatly from other Meliponinae. Our findings explain some formerly puzzling aspects of Carbonaria biogeography, and misinterpreted amplifications.

Loss of developmental diapause as prerequisite for social evolution in bees.

Diapause is a physiological arrest of development ahead of adverse environmental conditions and is a critical phase of the life cycle of many insects. In bees, diapause has been reported in species from all seven taxonomic families. However, they exhibit a variety of diapause strategies. These different strategies are of particular interest since shifts in the phase of the insect life cycle in which diapause occurs have been hypothesized to promote the evolution of sociality. Here we provide a comprehensive evaluation of this hypothesis with phylogenetic analysis and ancestral state reconstruction (ASR) of the ecological and evolutionary factors associated with diapause phase. We find that social lifestyle, latitude and voltinism are significant predictors of the life stage in which diapause occurs. ASR revealed that the most recent common ancestor of all bees likely exhibited developmental diapause and shifts to adult, reproductive, or no diapause have occurred in the ancestors of lineages in which social behaviour has evolved. These results provide fresh insight regarding the role of diapause as a prerequisite for the evolution of sociality in bees.

Fidelity of DNA polymerases in the detection of intraindividual variation of mitochondrial DNA.

Here we investigated the consequences of PCR amplification errors in the identification of intraindividual mtDNA variation. The bumblebee Bombus morio was chosen as model for the COI gene amplification tests with two DNA polymerases (Taq and Q5) presenting different error rates. The amplifications using Taq resulted in a significant increase of singleton haplotypes per individual in comparison to Q5. The sequence characteristics indicated that Taq resulted haplotypes are mostly due to amplification errors. Studies focusing on intraindividual variability should address special attention to the DNA polymerase fidelity to avoid overestimation of heteroplasmic haplotypes.

Diapause in a tropical oil-collecting bee: molecular basis unveiled by RNA-Seq.

BACKGROUND: Diapause is a natural phenomenon characterized by an arrest in development that ensures the survival of organisms under extreme environmental conditions. The process has been well documented in arthropods. However, its molecular basis has been mainly studied in species from temperate zones, leaving a knowledge gap of this phenomenon in tropical species. In the present study, the Neotropical and solitary bee Tetrapedia diversipes was employed as a model for investigating diapause in species from tropical zones. Being a bivoltine insect, Tetrapedia diversipes produce two generations of offspring per year. The first generation, normally born during the wet season, develops faster than individuals from the second generation, born after the dry season. Furthermore, it has been shown that the development of the progeny, of the second generation, is halted at the 5th larval instar, and remains in larval diapause during the dry season. Towards the goal of gaining a better understanding of the diapause phenomenon we compared the global gene expression pattern, in larvae, from both reproductive generations and during diapause. The results demonstrate that there are similarities in the observed gene expression patterns to those already described for temperate climate models, and also identify diapause-related genes that have not been previously reported in the literature. RESULTS: The RNA-Seq analysis identified 2275 differentially expressed transcripts, of which 1167 were annotated. Of these genes, during diapause, 352 were upregulated and 815 were downregulated. According to their biological functions, these genes were categorized into the following groups: cellular detoxification, cytoskeleton, cuticle, sterol and lipid metabolism, cell cycle, heat shock proteins, immune response, circadian clock, and epigenetic control. CONCLUSION: Many of the identified genes have already been described as being related to diapause; however, new genes were discovered, for the first time, in this study. Among those, we highlight: Niemann-Pick type C1, NPC2 and Acyl-CoA binding protein homolog (all involved in ecdysteroid synthesis); RhoBTB2 and SASH1 (associated with cell cycle regulation) and Histone acetyltransferase KAT7 (related to epigenetic transcriptional regulation). The results presented here add important findings to the understanding of diapause in tropical species, thus increasing the comprehension of diapause-related molecular mechanisms.

Comparative phylogeography in the Atlantic forest and Brazilian savannas: pleistocene fluctuations and dispersal shape spatial patterns in two bumblebees.

BACKGROUND: Bombus morio and B. pauloensis are sympatric widespread bumblebee species that occupy two major Brazilian biomes, the Atlantic forest and the savannas of the Cerrado. Differences in dispersion capacity, which is greater in B. morio, likely influence their phylogeographic patterns. This study asks which processes best explain the patterns of genetic variation observed in B. morio and B. pauloensis, shedding light on the phenomena that shaped the range of local populations and the spatial distribution of intra-specific lineages. RESULTS: Results suggest that Pleistocene climatic oscillations directly influenced the population structure of both species. Correlative species distribution models predict that the warmer conditions of the Last Interglacial contributed to population contraction, while demographic expansion happened during the Last Glacial Maximum. These results are consistent with physiological data suggesting that bumblebees are well adapted to colder conditions. Intra-specific mitochondrial genealogies are not congruent between the two species, which may be explained by their documented differences in dispersal ability. CONCLUSIONS: While populations of the high-dispersal B. morio are morphologically and genetically homogeneous across the species range, B. pauloensis encompasses multiple (three) mitochondrial lineages, and show clear genetic, geographic, and morphological differences. Because the lineages of B. pauloensis are currently exposed to distinct climatic conditions (and elevations), parapatric diversification may occur within this taxon. The eastern portion of the state of São Paulo, the most urbanized area in Brazil, represents the center of genetic diversity for B. pauloensis.

A protocol for isolating insect mitochondrial genomes: a case study of NUMT in Melipona flavolineata (Hymenoptera: Apidae).

Nuclear mitochondrial DNA insertions (NUMTs) are mitochondrial DNA sequences that have been transferred into the nucleus and are recognized by the presence of indels and stop codons. Although NUMTs have been identified in a diverse range of species, their discovery was frequently accidental. Here, our initial goal was to develop and standardize a simple method for isolating NUMTs from the nuclear genome of a single bee. Subsequently, we tested our new protocol by determining whether the indels and stop codons of the cytochrome c oxidase subunit I (COI) sequence of Melipona flavolineata are of nuclear origin. The new protocol successfully demonstrated the presence of a COI NUMT. In addition to NUMT investigations, the protocol described here will also be very useful for studying mitochondrial mutations related to diseases and for sequencing complete mitochondrial genomes with high read coverage by Next-Generation technology.

Molecular genetic diversity in populations of the stingless bee Plebeia remota: A case study.

Genetic diversity is a major component of the biological diversity of an ecosystem. The survival of a population may be seriously threatened if its genetic diversity values are low. In this work, we measured the genetic diversity of the stingless bee Plebeia remota based on molecular data obtained by analyzing 15 microsatellite loci and sequencing two mitochondrial genes. Population structure and genetic diversity differed depending on the molecular marker analyzed: microsatellites showed low population structure and moderate to high genetic diversity, while mitochondrial DNA (mtDNA) showed high population structure and low diversity in three populations. Queen philopatry and male dispersal behavior are discussed as the main reasons for these findings.

Very low mitochondrial variability in a stingless bee endemic to cerrado.

Partamona mulata is a stingless bee species endemic to cerrado, a severely threatened phytogeographical domain. Clearing for pasture without proper soil treatment in the cerrado facilitates the proliferation of termite ground nests, which are the nesting sites for P. mulata. The genetic consequences of these changes in the cerrado environment for bee populations are still understudied. In this work, we analyzed the genetic diversity of 48 colonies of P. mulata collected throughout the species' distribution range by sequencing two mitochondrial genes, cytochrome oxidase I and cytochrome B. A very low polymorphism rate was observed when compared to another Partamona species from the Atlantic forest. Exclusive haplotypes were observed in two of the five areas sampled. The sharing of two haplotypes between collection sites separated by a distance greater than the flight range of queens indicates an ancient distribution for these haplotypes. The low haplotype and nucleotide diversity observed here suggests that P. mulata is either a young species or one that has been through population bottlenecks. Locally predominant and exclusive haplotypes (H2 and H4) may have been derived from local remnants through cerrado deforestation and the expansion of a few colonies with abundant nesting sites.

The mitochondrial genome of the stingless bee Melipona bicolor (Hymenoptera, Apidae, Meliponini): sequence, gene organization and a unique tRNA translocation event conserved across the tribe Meliponini

At present a complete mtDNA sequence has been reported for only two hymenopterans, the Old World honey bee, Apis mellifera and the sawfly Perga condei. Among the bee group, the tribe Meliponini (stingless bees) has some distinction due to its Pantropical distribution, great number of species and large importance as main pollinators in several ecosystems, including the Brazilian rain forest. However few molecular studies have been conducted on this group of bees and few sequence data from mitochondrial genomes have been described. In this project, we PCR amplified and sequenced 78 percent of the mitochondrial genome of the stingless bee Melipona bicolor (Apidae, Meliponini). The sequenced region contains all of the 13 mitochondrial protein-coding genes, 18 of 22 tRNA genes, and both rRNA genes (one of them was partially sequenced). We also report the genome organization (gene content and order), gene translation, genetic code, and other molecular features, such as base frequencies, codon usage, gene initiation and termination. We compare these characteristics of M. bicolor to those of the mitochondrial genome of A. mellifera and other insects. A highly biased A+T content is a typical characteristic of the A. mellifera mitochondrial genome and it was even more extreme in that of M. bicolor. Length and compositional differences between M. bicolor and A. mellifera genes were detected and the gene order was compared. Eleven tRNA gene translocations were observed between these two species. This latter finding was surprising, considering the taxonomic proximity of these two bee tribes. The tRNA Lys gene translocation was investigated within Meliponini and showed high conservation across the Pantropical range of the tribe.

Mitochondrial tRNA gene translocations in highly eusocial bees

Mitochondrial gene rearrangement events, especially involving tRNA genes, have been described more frequently as more complete mitochondrial genome sequences are becoming available. In the present work, we analyzed mitochondrial tRNA gene rearrangements between two bee species belonging to the tribes Apini and Meliponini within the "corbiculate Apidae". Eleven tRNA genes are in different genome positions or strands. The molecular events responsible for each translocation are explained. Considering the high number of rearrangements observed, the data presented here contradict the general rule of high gene order conservation among closely related organisms, and also represent a powerful molecular tool to help solve questions about phylogeny and evolution in bees.

Validación de la escala de envolvimiento emocional y criticismo familiar (FEICS) en la población clínica psiquiátrica colombiana / Validation of the Family Emotional Involvement and Criticism Scale (FEICS) in Colombian Psychiatric Clinical Population

Objetivo: validar la escala de envolvimiento emocional y crítica familiar (FEICS) en la población clínica psiquiátrica colombiana. Métodos: se tradujo la escala y luego se hizo el mismo proceso en sentido inverso. Se evaluaron: validez de apariencia por un grupo de expertos, validez de contenido y conceptos mediante análisis factorial de componentes principales, validez concurrente mediante comparación con "monólogo de cinco minutos" y subescala de cohesión de la Escala de Evaluación de Adaptabilidad y Cohesión Familiar (FACES-III), consistencia interna y reproducibilidad test-retest. Resultados: en el análisis factorial se encontraron cuatro dominios: censura, envolvimiento, intrusión y apoyo, que explicaron el 57,4 de la varianza. La consistencia interna de la subescala de crítica fue buena con una alfa de Crombach de 0,76 y la subescala de envolvimiento fue baja con un alfa de Crombach de 0,54. La reproducibilidad test-retest de la subescala de criticismo fue excelente (CCI = 0,92) y de la subescala de envolvimiento, buena (CCI = 0,62). Hubo una correlación significativa entre la subescala de crítica y el número de comentarios críticos del "monólogo de cinco minutos" (r = 0,31, p = 0,0001). Conclusiones: la FEICS fue de fácil aplicación y mostró cuatro dominios, a diferencia de los dos de la escala original. La subescala de crítica tiene gran consistencia interna, reproducibilidad test-retest y validez de concepto, lo cual la hace útil para la práctica clínica y de investigación. A pesar de que la subescala de envolvimiento se correlacionó con la FACESIII y su reproducibilidad test-retest fue buena, su escasa consistencia interna hace pensar en la necesidad de estudiar con mayor profundidad factores culturales asociados con el envolvimiento para posiblemente modificar los ítems de esta subescala...