Expression and function of the vitellogenin receptor in the hypopharyngeal glands of the honey bee Apis mellifera (Hymenoptera: Apidae) workers.

The vitellogenin receptor (VgR) is essential for the uptake and transport of the yolk precursor, vitellogenin (Vg). Vg is synthesized in the fat body, released in the hemolymph, and absorbed in the ovaries, via receptor-mediated endocytosis. Besides its important role in the reproductive pathway, Vg occurs in nonreproductive worker honey bee, suggesting its participation in other pathways. The objective was to verify if the VgR occurs in the hypopharyngeal glands of Apis mellifera workers and how Vg is internalized by these cells. VgR occurrence in the hypopharyngeal glands was evaluated by qPCR analyses of VgR and immunohistochemistry in workers with different tasks. The VgR gene is expressed in the hypopharyngeal glands of workers with higher transcript levels in nurse honey bees. VgR is more expressed in 11-day-old workers from queenright colonies, compared to orphan ones. Nurse workers with developed hypopharyngeal glands present higher VgR transcripts than those with poorly developed glands. The immunohistochemistry results showed the co-localization of Vg, VgR and clathrin (protein that plays a major role in the formation of coated vesicles in endocytosis) in the hypopharyngeal glands, suggesting receptor-mediated endocytosis. The results demonstrate that VgR performs the transport of Vg to the hypopharyngeal glands, supporting the Ovary Ground Plan Hypothesis and contributing to the understanding of the role of this gland in the social context of honey bees.

Post-embryonic changes in the hindgut of honeybee Apis mellifera workers: Morphology, cuticle deposition, apoptosis, and cell proliferation.

In insects, the hindgut is a homeostatic region of the digestive tract, divided into pylorus, ileum, and rectum, that reabsorbs water, ions, and small molecules produced during hemolymph filtration. The hindgut anatomy in bee larvae is different from that of adult workers. This study reports the morphological changes and cellular events that occur in the hindgut during the metamorphosis of the honeybee Apis mellifera. We describe the occurrence of autophagosomes and the ultrastructure of the epithelial cells and cuticle, suggesting that cuticular degradation begins in prepupae, with the cuticle being reabsorbed and recycled by autophagosomes in white- and pink-eyed pupae, followed by the deposition of new cuticle in light-brown-eyed pupae. In L5S larvae and prepupae, the hindgut undergoes cell proliferation in the anterior and posterior ends. In the pupae, the pylorus, ileum, and rectum regions are differentiated, and cell proliferation ceases in dark-brown-eyed pupae. Apoptosis occurs in the hindgut from the L5S larval to the pink-eyed pupal stage. In light-brown- and dark-brown-eyed pupae, the ileum epithelium changes from pseudostratified to simple only after the production of the basal lamina, whereas the rectal epithelium is always flattened. In black-eyed pupae, ileum epithelial cells have large vacuoles and subcuticular spaces, while in adult forager workers these cells have long invaginations in the cell apex and many mitochondria, indicating a role in the transport of compounds. Our findings show that hindgut morphogenesis is a dynamic process, with tissue remodeling and cellular events taking place for the formation of different regions of the organ, the reconstruction of a new cuticle, and the remodeling of visceral muscles.

Genome assembly and variant analysis of two Saccharomyces cerevisiae strains isolated from stingless bee pollen.

Products from stingless bees are rich reservoirs of microbial diversity, including yeasts with fermentative potential. Previously, two Saccharomyces cerevisiae strains, JP14 and IP9, were isolated from Jataí (Tetragonisca angustula) and Iraí (Nannotrigona testaceicornis) bees, respectively, aiming at mead production. Both strains presented great osmotic and sulfite tolerance, and ethanol production, although they have a high free amino nitrogen demand. Herein, their genomes were sequenced, assembled, and annotated, and the variants were compared to the S. cerevisiae S288c reference strain. The final assembly of IP9 and JP14 presented high N50 and BUSCO scores, and more than 6430 protein-coding genes. Additionally, nQuire predicted the ploidy of IP9 as diploid, but the results were not enough to determine the ploidy of JP14. The mitochondrial genomes of IP9 and JP14 presented the same gene content as S288c but the genes were rearranged and fragmented in different patterns. Meanwhile, the genes with mutations of high impact (e.g., indels, gain of stop codon) for both yeasts were enriched for transmembrane transport, electron transfer, oxidoreductase, heme binding, fructose, mannose, and glucose transport, activities related to the respiratory chain and sugar metabolism. The IP9 strain presented copy number gains in genes related to sugar transport and cell morphogenesis; in JP14, genes were enriched for disaccharide metabolism and transport, response to reactive oxygen species, and polyamine transport. On the other hand, IP9 presented copy number losses related to disaccharide, thiamine, and aldehyde metabolism, while JP14 presented depletions related to disaccharide, oligosaccharide, asparagine, and aspartate metabolism. Notably, both strains presented a killer toxin gene, annotated from the assembling of unmapped reads, representing a potential mechanism for the control of other microorganisms population in the environment. Therefore, the annotated genomes of JP14 and IP9 presented a high selective pressure for sugar and nitrogen metabolism and stress response, consistent with their isolation source and fermentative properties.

Melipona stingless bees and honey microbiota reveal the diversity, composition, and modes of symbionts transmission.

The Melipona gut microbiota differs from other social bees, being characterized by the absence of crucial corbiculate core gut symbionts and a high occurrence of environmental strains. We studied the microbial diversity and composition of three Melipona species and their honey to understand which strains are obtained by horizontal transmission (HT) from the pollination environment, represent symbionts with HT from the hive/food stores or social transmission (ST) between nestmates. Bees harbored higher microbial alpha diversity and a different and more species-specific bacterial composition than honey. The fungal communities of bee and honey samples are also different but less dissimilar. As expected, the eusocial corbiculate core symbionts Snodgrassella and Gilliamella were absent in bees that had a prevalence of Lactobacillaceae - including Lactobacillus (formerly known as Firm-5), Bifidobacteriaceae, Acetobacteraceae, and Streptococcaceae - mainly strains close to Floricoccus, a putative novel symbiont acquired from flowers. They might have co-evolved with these bees via ST, and along with environmental Lactobacillaceae and Pectinatus (Veillonellaceae) strains obtained by HT, and Metschnikowia and Saccharomycetales yeasts acquired by HT from honey or the pollination environment, including plants/flowers, possibly compose the Melipona core microbiota. This work contributes to the understanding of Melipona symbionts and their modes of transmission.

Cuticle melanization and the expression of immune-related genes in the honeybee Apis mellifera (Hymenoptera: Apidae) adult workers.

The global decline of bee populations has several factors, including pathogens, which need overcome the insect defenses such as the physical barriers, the body cuticle and peritrophic matrix (primary defenses), as well as the secondary defenses with antimicrobial peptides (AMPs) and the enzyme lysozyme. The regulation of immune defenses according to the infection risks raises questions about the immunity of social bees due to their exposition to different pathogens pressures during the adult lifespan and tasks performed. This study evaluated the primary (body cuticle melanization, peritrophic matrix and cpr14 expression) and secondary (AMPs and lysozyme expression) defenses of the honeybee Apis mellifera workers according to the age and tasks. The expression of malvolio was used to detect precocious forage tasks outside the colony. Forager workers have higher amount of cuticular melanization in the body cuticle than nurse, but not when the age effect is retired, indicating the gradual acquisition of this compound in the integument of adult bees. The relative value of chitin in the peritrophic matrix and cpr14 mRNA are similar in all bees evaluated, suggesting that these components of primary defenses do not change according to the task and age. Differential expression of genes for AMPs in workers performing different tasks, within the same age group, indicates that the behavior stimulates expression of genes related to secondary immune defense. The expression of malvolio gene, accelerating the change in workers behavior, and those related to immune defense suggest the investment in secondary defense mechanisms when the primary defense of the body cuticle is not yet completed.

Extinction of anciently associated gut bacterial symbionts in a clade of stingless bees.

Animal-microbe symbioses are often stable for millions of years. An example is the clade consisting of social corbiculate bees-honeybees, bumblebees, and stingless bees-in which a shared ancestor acquired specialized gut bacteria that subsequently diversified with hosts. This model may be incomplete, however, as few microbiomes have been characterized for stingless bees, which are diverse and ecologically dominant pollinators in the tropics. We surveyed gut microbiomes of Brazilian stingless bees, focusing on the genus Melipona, for which we sampled multiple species and biomes. Strikingly, Melipona lacks Snodgrassella and Gilliamella, bacterial symbionts ubiquitous in other social corbiculate bees. Instead, Melipona species harbor more environmental bacteria and bee-specific Starmerella yeasts. Loss of Snodgrassella and Gilliamella may stem from ecological shifts in Melipona or the acquisition of new symbionts as functional replacements. Our findings demonstrate the value of broadly sampling microbiome biodiversity and show that even ancient symbioses can be lost.

Selection of yeasts from bee products for alcoholic beverage production.

The use of appropriate yeast strains allows to better control the fermentation during beverage production. Bee products, especially of stingless bees, are poorly explored as sources of fermenting microorganisms. In this work, yeasts were isolated from honey and pollen from Tetragonisca angustula (Jataí), Nannotrigona testaceicornis (Iraí), Frieseomelitta varia (Marmelada), and honey of Apis mellifera bees and screened according to morphology, growth, and alcohol production. Bee products showed to be potential sources of fermenting microorganisms. From 55 isolates, one was identified as Papiliotrema flavescens, two Rhodotorula mucilaginosa, five Saccharomyces cerevisiae, and nine Starmerella meliponinorum. The S. cerevisiae strains were able to produce ethanol and glycerol at pH 4.0-8.0 and temperature of 10-30 °C, with low or none production of undesirable compounds, such as acetic acid and methanol. These strains are suitable for the production of bioethanol and alcoholic beverages due to their high ethanol production, similar or superior to the commercial strain, and in a broad range of conditions like as 50% (m/v) glucose, 10% (v/v) ethanol, or 500 mg L-1 of sodium metabisulfite.

Post-embryonic development of the Malpighian tubules in Apis mellifera (Hymenoptera) workers: morphology, remodeling, apoptosis, and cell proliferation.

The honeybee Apis mellifera has ecological and economic importance; however, it experiences a population decline, perhaps due to exposure to toxic compounds, which are excreted by Malpighian tubules. During metamorphosis of A. mellifera, the Malpighian tubules degenerate and are formed de novo. The objective of this work was to verify the cellular events of the Malpighian tubule renewal in the metamorphosis, which are the gradual steps of cell remodeling, determining different cell types and their roles in the excretory activity in A. mellifera. Immunofluorescence and ultrastructural analyses showed that the cells of the larval Malpighian tubules degenerate by apoptosis and autophagy, and the new Malpighian tubules are formed by cell proliferation. The ultrastructure of the cells in the Malpighian tubules suggest that cellular remodeling only occurs from dark-brown-eyed pupae, indicating the onset of excretion activity in pupal Malpighian tubules. In adult forager workers, two cell types occur in the Malpighian tubules, one with ultrastructural features (abundance of mitochondria, vacuoles, microvilli, and narrow basal labyrinth) for primary urine production and another cell type with dilated basal labyrinth, long microvilli, and absence of spherocrystals, which suggest a role in primary urine re-absorpotion. This study suggests that during the metamorphosis, Malpighian tubules are non-functional until the light-brown-eyed pupae, indicating that A. mellifera may be more vulnerable to toxic compounds at early pupal stages. In addition, cell ultrastructure suggests that the Malpighian tubules may be functional from dark-brown-eyed pupae and acquire greater complexity in the forager worker bee.

Flight a activity of Tetragona clavipes (Fabricius, 1804) (Hymenoptera, Apidae, Meliponini) at the São Paulo University campus in Ribeirão Preto / Atividade de vôo de Tetragona clavipes (Fabricius, 1804) (Hymenopetra, Apidae, Meliponini) no campus da Universidade de São Paulo em Ribeirão Preto

The Tetragona clavipes (Fabricius, 1804) bees, knowing as “borá”, is found in all Brazil country until south of Parana state. However in the São Paulo state, this species is not very common. Our main aim was, in São Paulo University Campus at Ribeirão Preto, to realize the characterization of the flight activity of this bee, showing possible relations some environmental factors, such as temperature, relative humidity and “A” type ultraviolet radiation. Data were obtained during September and October months (Spring) and showed that there is a high correlation between the start and finish of the flight activity and environmental temperature. However the UVA radiation and specially air relative humidity did not show relevant role on this behavior and they were considered secondary factors.

A abelha Tetragona clavipes (Fabricius, 1804), também conhecida como Borá, é encontrada em todo Brasil, até os limites sul do estado do Paraná. Embora ocorra no estado de São Paulo, ela não é uma espécie muito comum. Nosso principal objetivo foi realizar, no Campus da USP em Ribeirão Preto, a caracterização das atividades de vôo dessa abelha, mostrando suas possíveis relações com alguns fatores climáticos, tais como temperatura, umidade relativa do ar e radiação ultravioleta do tipo A. Após coleta e análise dos dados durante os meses de Setembro e Outubro (primavera), pôde-se afirmar que existe uma alta correlação entre o início e o término das atividades de vôo e a temperatura ambiente. Porém, os índices de radiação UVA e principalmente a umidade relativa do ar não apresentaram papel relevante neste tipo de comportamento e foram considerados como parâmetros secundários.

Abelhas para a melhor idade: curso de meliponíneos, alfabetização técnica para a conservação / Bees for the best age: stingless bees course, technical teaching for conservation

O presente trabalho é resultado de uma experiência inédita desenvolvida por alunos do Programa de Pós-graduação em Entomologia da FFCLRP-USP, apresentando as abelhas sociais a grupos de pessoas da terceira idade, ministrando dois cursos de difusão cultural. Os objetivos, nestes cursos, foram despertar e desenvolver a consciência para a preservação dos recursos naturais, da diversidade da flora e fauna, redimensionar a prática pedagógica, com a universidade indo diretamente à comunidade unindo teoria e prática, utilizando como pano de fundo o estudo e o manejo de meliponíneos - abelhas sociais sem ferrão. Os resultados obtidos foram analisados pelo método qualitativo, considerando que as pessoas na terceira idade estão em uma fase de transição na qual deixam a obrigatoriedade das atividades escolhidas na juventude e têm a possibilidade de escolher outra atividade à qual queiram se dedicar só por prazer ou como atividade produtiva. A experiência com os grupos mostrou que a maneira como os conceitos foram trabalhados durante as aulas tornou muito fácil a compreensão e a prática, independentemente do nível de escolaridade dos alunos, visto que nos grupos havia desde pessoas com pós-graduação até pessoas que eram somente alfabetizadas. Outro aspecto educacional relevante é que se iniciou, a partir destes cursos, um grupo de discussões permanente com reuniões periódicas para aperfeiçoar e discutir propostas de preservação ambiental, caracterizando uma formação continuada.

The present work is the result of an unpublished experience developed by students of the Pos-Graduation Program in Entomology from FFCLRP-USP, presenting the social bees to senior citizen groups, carrying out two courses of cultural diffusion. The objectives, in these courses, were to arouse and develop the conscientious to preservation of natural resources, diversity of flora and fauna, resize the pedagogical practice, with the University acting directly in the community life joining theory and practice, having as a base the study and handling of stingless bees. The results were analyzed by the qualitative method, considering that senior citizen are in a transitory stage, on which they have already left the duties chosen at youth and have the possibility of choose another activity where they want to dedicate as a hobby or as a lucrative activity. The experience with the groups shown that the manner how the professors deal with some concepts during the classes made much easier the comprehension and practice, independently of the students scholar level, whereas in the groups there were pos graduated people until those that were just literate. Another important educational aspect is the initiation of a permanent discussion group with periodic meetings to improve and discuss proposal of environmental conservation characterizing a progressive learning.