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.

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 genusApis,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 singleMeliponaspecies only. We present here the genome sequence ofFrieseomelitta 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 forF. variais 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.

Insect development is primarily controlled by juvenile hormone (JH) and 20-hydroxyecdysone (20E), which regulate gene cascades leading to changes in phenotype, physiology, and behavior. Besides these hormones, microRNAs play a crucial role in insect development by regulating gene expression at the post-transcriptional level. To advance the molecular understanding of holometabolous developmental events, we investigate the pupal phase in the honeybee, Apis mellifera. In this study, we assessed the expression profiles of genes components of JH and 20E cascades – Usp, ftz-f1, EcR, Met, Chd64, InR-2, Kr-h1 and Tai – as well as the microRNAs miRNA-34 and miRNA-281 during pupal development of A. mellifera. We then analyzed the impact of JH and 20E treatments on the expression of these developmental genes and their putative regulators, the microRNAs. Overall, the selected genes and miRNAs remained stable or were downregulated following 20E treatment, while treatments with JH, upregulated most of our candidate developmental genes and microRNAs. Notably, the expression profile of Met, an intracellular receptor of JH, showed a strong correlation with fluctuations in 20E titers during pupal development. Furthermore, a computational analysis, followed by experimental assays, points to both miR-34 and miR-281 as potential regulators of pupal development in A. mellifera. This study paves the way for a better understanding of how JH and 20E hormones interact with developmental genes and microRNAs (miR-34 and miR-281) to regulate pupal development in honeybees, elucidating a piece of this complex network of interactions.

The effector molecule is a short RNA duplex, composed of two strands of approximately 21 nucleotides, with two overhanging bases at the 3’ end (^{Elbashir et al., 2001}). However, several types of precursors can trigger gene silencing, such as long double-stranded RNAs (dsRNAs) from 200 to 800 base pairs (bp) (^{Guo et al., 2018}), hairpin RNAs (hpRNAs) (^{Hussain et al., 2019}), small interfering RNAs (siRNAs) (^{Guo et al., 2018}), short hairpin RNAs (shRNAs) (^{Wang et al., 2018}), among others. Notoriously, an ingenious and inexpensive way to produce shRNAs in large quantities was developed, via in vitro transcription of a circularized DNA template, generating multimeric shRNAs (mshRNAs) (^{Seyhan et al., 2006}; ^{Abe et al., 2012}). In the last decade, such multimeric shRNAs generated by rolling circle transcription have been used by other groups and have been shown to be an interesting option for RNAi (^{Wang et al., 2015}; ^{Shopsowitz et al., 2016}; ^{Wu et al., 2016}; ^{Kim et al., 2018}).^{7 8}

Hydropower plants have significant impacts on aquatic biodiversity, particularly on migratory fish species. Effectively managing these impacts requires a comprehensive understanding of fish reproduction and recruitment within altered river systems, which can be assessed through ichthyoplankton studies. However, traditional morphological methods for identifying fish eggs and larvae present considerable challenges due to morphological ambiguity and developmental constraints. In this study, we applied DNA barcoding to characterize the ichthyoplankton community within a relictual lotic stretch downstream of the Capivara Dam, located on the Paranapanema River in Southern Brazil. Cytochrome oxidase I (COI) gene sequences from 79 samples were compared against the Barcode of Life Data System (BOLD) and GenBank databases, resulting in successful species-level identification for all samples, each exhibiting around 99.8% similarity. The identified specimens comprised eight species, six genera, four families, and two orders. Species from the order Siluriformes accounted for 60.5% of the total abundance, predominantly including migratory species such as Pimelodus ornatus, Pimelodus maculatus, Leporinus friderici, and Pinirampus pirinampu, the latter a species rarely observed in the basin. These findings highlight the importance of lotic stretches as spawning grounds and emphasize the need for their conservation. DNA barcoding proved to be an efficient method for species identification, providing essential data for environmental assessments and conservation strategies targeting local fish populations.