Immunity and physiological changes in adult honey bees (Apis mellifera) infected with Nosema ceranae: The natural colony environment.

Nosema ceranae is a microsporidium that infects Apis mellifera, causing diverse physiological and behavioral alterations. Given the existence of individual and social mechanisms to reduce infection and fungal spread in the colony, bees may respond differently to infection depending on their rearing conditions. In this study, we investigated the effect of N. ceranae in honey bee foragers naturally infected with different fungal loads in a tropical region. In addition, we explored the effects of N. ceranae artificially infected young bees placed in a healthy colony under field conditions. Honey bees naturally infected with higher loads of N. ceranae showed downregulation of genes from Toll and IMD immune pathways and antimicrobial peptide (AMP) genes, but hemolymph total protein amount and Vitellogenin (Vg) titers were not affected. Artificially infected bees spread N. ceranae to the controls in the colony, but fungal loads were generally lower than those observed in cages, probably because of social immunity. Although no significant changes in mRNA levels of AMP-encoding were observed, N. ceranae artificially infected bees showed downregulation of miR-989 (an immune-related microRNA), lower vitellogenin gene expression, and decreased hemolymph Vg titers. Our results demonstrate for the first time that natural infection by N. ceranae suppresses the immune system of honey bee foragers in the field. This parasite is detrimental to the immune system of young and old bees, and disease spread, mitigation and containment will depend on the colony environment.

The nuclear and mitochondrial genomes of Frieseomelitta varia - a highly eusocial stingless bee (Meliponini) with a permanently sterile worker caste.

BACKGROUND: Most of our understanding on the social behavior and genomics of bees and other social insects is centered on the Western honey bee, Apis mellifera. The genus Apis, however, is a highly derived branch comprising less than a dozen species, four of which genomically characterized. In contrast, for the equally highly eusocial, yet taxonomically and biologically more diverse Meliponini, a full genome sequence was so far available for a single Melipona species only. We present here the genome sequence of Frieseomelitta varia, a stingless bee that has, as a peculiarity, a completely sterile worker caste. RESULTS: The assembly of 243,974,526 high quality Illumina reads resulted in a predicted assembled genome size of 275 Mb composed of 2173 scaffolds. A BUSCO analysis for the 10,526 predicted genes showed that these represent 96.6% of the expected hymenopteran orthologs. We also predicted 169,371 repetitive genomic components, 2083 putative transposable elements, and 1946 genes for non-coding RNAs, largely long non-coding RNAs. The mitochondrial genome comprises 15,144 bp, encoding 13 proteins, 22 tRNAs and 2 rRNAs. We observed considerable rearrangement in the mitochondrial gene order compared to other bees. For an in-depth analysis of genes related to social biology, we manually checked the annotations for 533 automatically predicted gene models, including 127 genes related to reproductive processes, 104 to development, and 174 immunity-related genes. We also performed specific searches for genes containing transcription factor domains and genes related to neurogenesis and chemosensory communication. CONCLUSIONS: The total genome size for F. varia is similar to the sequenced genomes of other bees. Using specific prediction methods, we identified a large number of repetitive genome components and long non-coding RNAs, which could provide the molecular basis for gene regulatory plasticity, including worker reproduction. The remarkable reshuffling in gene order in the mitochondrial genome suggests that stingless bees may be a hotspot for mtDNA evolution. Hence, while being just the second stingless bee genome sequenced, we expect that subsequent targeting of a selected set of species from this diverse clade of highly eusocial bees will reveal relevant evolutionary signals and trends related to eusociality in these important pollinators.

Evaluation of reference genes for gene expression analysis by real-time quantitative PCR (qPCR) in three stingless bee species (Hymenoptera: Apidae: Meliponini).

Stingless bees are generalist pollinators distributed through the pantropical region. There is growing evidence that their wild populations are experiencing substantial decline in response to habitat degradation and pesticides. Policies for conservation of endangered species will benefit from studies focusing on genetic and molecular aspects of their development and behavior. The most common method for looking at gene expression is real-time quantitative polymerase chain reaction preceded by reverse transcription (RT-qPCR) of the mRNA of interest. This method requires the identification of reliable reference genes to correctly estimate fluctuations in transcript levels. To contribute to molecular studies on stingless bees, we used Frieseomelitta varia, Melipona quadrifasciata, and Scaptotrigona bipunctata species to test the expression stability of eight reference genes (act, ef1-α, gapdh, rpl32, rps5, rps18, tbp, and tbp-af) in RT-qPCR procedures in five physiological and experimental conditions (development, sex, tissues, bacteria injection, and pesticide exposure). In general, the rpl32, rps5 and rps18 ribosomal protein genes and tpb-af gene showed the highest stability, thus being identified as suitable reference genes for the three stingless bee species and defined conditions. Our results also emphasized the need to evaluate the stability of candidate genes for any designed experimental condition and stingless bee species.

Integrative taxonomy of a new Redudasys species (Gastrotricha: Macrodasyida) sheds light on the invasion of fresh water habitats by macrodasyids.

The order Macrodasyida (Gastrotricha) includes over 350 marine species, and only 3 freshwater species (Marinellina flagellata, Redudasys fornerise, R. neotemperatus). Herein we describe a new freshwater species of Macrodasyida, Redudasys brasiliensis sp. nov., from Brazil through an integrative taxonomic approach. The external morphology and internal anatomy were investigated using differential interference contrast microscopy, confocal microscopy, scanning and transmission electron microscopy. The systematization of the new taxon was inferred by nuclear (18S and 28S) and mitochondrial (COI) genes, and its intra-order relationships were assessed using data from most of available macrodasyids. Phylogenetic analyses yielded congruent trees, in which the new taxon is nested within the family Redudasyidae, but it was genetically distinct from the other species of the genus Redudasys. The new species shares the gross morphology and reproductive traits with other Redudasyidae and the presence of only 1 anterior adhesive tube per side with Redudasys neotemperatus, but it has a specific pattern of ventral ciliation and muscle organization. Results support the hypothesis that dispersion into fresh water habitats by Macrodasyida and Chaetonotida taxa occurred independently and that within Macrodasyida a single lineage invaded the freshwater environment only once. Furthermore, the Neotropical region seems to be peculiar for the evolution of the freshwater macrodasyid clade.

Bacterial infection activates the immune system response and dysregulates microRNA expression in honey bees.

In insects, a rapid and massive synthesis of antimicrobial peptides (AMPs) is activated through signaling pathways (Toll and Imd) to combat invading microbial pathogens. However, it is still unclear whether different types of bacteria provoke specific responses. Immune response mechanisms and the activation of specific genes were investigated by challenging Apis mellifera workers with the Gram-negative bacterium Serratia marcescens or the Gram-positive bacterium Micrococcus luteus. The immune system responded by activating most genes of the Toll and Imd pathways, particularly AMP genes. However, genes specifically regulated by M. luteus or S. marcescens were not detected, suggesting an interaction between the signaling pathways that lead to immune effectors synthesis. Despite this finding, kappaB motifs in the 5'-UTRs of selected genes suggest a pathway-specific control of AMP and transferrin-1 gene expression. Regulation by miRNAs was also investigated and revealed a number of candidates for the post-transcriptional regulation of immune genes in bees.

New data on freshwater psammic Gastrotricha from Brazil.

Current knowledge of freshwater gastrotrich fauna from Brazil is underestimated as only two studies are available. The present communication is a taxonomic account of the first-ever survey of freshwater Gastrotricha in Minas Gerais State. Samplings were carried out yielding six species of three Chaetonotidae genera: Aspidiophorus cf. pleustonicus, Ichthydium cf. chaetiferum, Chaetonotus acanthocephalus, Chaetonotus heideri, Chaetonotus cf. succinctus, Chaetonotus sp., and also an undescribed species belonging to the genus Redudasys (incertae sedis): this is the first finding of specimens of Redudasys outside of original type locality. These preliminary observations suggest that the knowledge of the biodiversity of Gastrotricha in the Minas Gerais State, as well as in the whole Brazil, will certainly increase as further investigations are undertaken, and that freshwater Macrodasyida may be more common than previously thought.

Trade-off between immune stimulation and expression of storage protein genes.

Proteins stored in insect hemolymph may serve as a source of amino acids and energy for metabolism and development. The expression of the main storage proteins was assessed in bacterial-challenged honey bees using real-time (RT)-PCR and Western blot. After ensuring that the immune system had been activated by measuring the ensuing expression of the innate immune response genes, defensin-1 (def-1) and prophenoloxidase (proPO), we verified the expression of four genes encoding storage proteins. The levels of vitellogenin (vg) mRNA and of the respective protein were significantly lowered in bees injected with bacteria or water only (injury). An equivalent response was observed in orally-infected bees. The levels of apolipophorin II/I (apoLp-II/I) and hexamerin (hex 70a) mRNAs did not significantly change, but levels of Hex 70a protein subunit showed a substantial decay after bacterial challenge or injury. Infection also caused a strong reduction in the levels of apoLp-III transcripts. Our findings are consistent with a down-regulation of the expression and accumulation of storage proteins as a consequence of activation of the immune system, suggesting that this phenomenon represents a strategy to redirect resources to combat injury or infection.

Vitellogenin expression in queen ovaries and in larvae of both sexes of Apis mellifera.

In the honeybee, Apis mellifera, vitellogenin (Vg) expression has been detected in the ovary of queens, but not in that of workers. In addition, larvae of both sexes produce Vg in significant amounts, which suggest that Vg serves for functions additional to oocyte growth and energy supply to the embryo. In vivo hormone treatment experiments suggest that the decrease of 20-hydroxyecdysone concentration occurring in previtellogenic phases allows Vg production. Southern analysis indicates that the Vg gene is present as a single copy in the honeybee genome.

Phenoloxidase activity in Apis mellifera honey bee pupae, and ecdysteroid-dependent expression of the prophenoloxidase mRNA.

Phenoloxidase (monophenol, l-dopa: oxygen oxidoreductase, EC 1.14.18.1) is a multicopper oxidase, which plays an important role in melanin synthesis, necessary for defense against intruding microorganisms and parasites, wound healing and cuticle pigmentation. A phenoloxidase from the hemolymph of honey bee pupae exhibited an apparent molecular mass of 70 kDa, as estimated by gel filtration and SDS-PAGE. Optimal pH and temperature were 6.5 and 20 degrees C, respectively. Activity was fully stable for 30 min at 50 degrees C. Like phenoloxidases from the hemolymph of other insects, the honey bee enzyme was activated by trypsin and inhibited by protease inhibitors and phenylthiourea. Only high concentrations of sodium azide effectively inhibited the detected activity. A low concentration (5 microM) of Ca2+, Mg2+, and Mn2+ had a stimulatory effect on the activity. Single Michaelis-Menten curves were observed for l-dopa and dopamine oxidation, but the affinity of the enzyme for dopamine was greater than for L-dopa. Semiquantitative RT-PCR and Southern blot analysis using a 359 bp labeled probe, and quantification of the prophenoloxidase mRNA levels by real-time PCR showed increased amounts of transcripts in hemocytes and integument from young pupae injected with 20-hydroxyecdysone.