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. 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. 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.

The extreme conservation of mitochondrial genomes in metazoans poses a significant challenge to understanding mitogenome evolution. However, the presence of variation in gene order or genome structure, found in a small number of taxa, can provide unique insights into this evolution. Previous work on two stingless bees in the genus Tetragonula (T. carbonaria and T. hockingsi) revealed highly divergent CO1 regions between them and when compared to the bees from the same tribe (Meliponini), indicating rapid evolution. Using mtDNA isolation and Illumina sequencing, we elucidated the mitogenomes of both species. In both species, there has been a duplication of the whole mitogenome to give a total genome size of 30,666 bp in T. carbonaria; and 30,662 bp in T. hockingsi. These duplicated genomes present a circular structure with two identical and mirrored copies of all 13 protein coding genes and 22 tRNAs, with the exception of a few tRNAs that are present as single copies. In addition, the mitogenomes are characterized by rearrangements of two block of genes. We believe that rapid evolution is present in the whole Indo-Malay/Australasian group of Meliponini but is extraordinarily elevated in T. carbonaria and T. hockingsi, probably due to founder effect, low effective population size and the mitogenome duplication. All these features - rapid evolution, rearrangements, and duplication - deviate significantly from the vast majority of the mitogenomes described so far, making the mitogenomes of Tetragonula unique opportunities to address fundamental questions of mitogenome function and evolution.

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.

The evolution of mitochondrial genomes in the stingless bees is surprisingly dynamic, making them a model system to understand mitogenome structure, function, and evolution. Out of the seven mitogenomes available in this group, five exhibit atypical characteristics, including extreme rearrangements, rapid evolution and complete mitogenome duplication. To further explore the mitogenome diversity in these bees, we utilized isolated mtDNA and Illumina sequencing to assemble the complete mitogenome of Trigonisca nataliae, a species found in Northern Brazil. The mitogenome of T. nataliae was highly conserved in gene content and structure when compared to Melipona species but diverged in the control region (CR). Using PCR amplification, cloning and Sanger sequencing, six different CR haplotypes, varying in size and content, were recovery. These findings indicate that heteroplasmy, where different mitochondrial haplotypes coexist within individuals, occurs in T. nataliae. Consequently, we argue that heteroplasmy might indeed be a common phenomenon in bees that could be associated with variations in mitogenome size and challenges encountered during the assembly process.

Heteroplasmy is characterized by different mitochondrial haplotypes within the same organism. After evidence of heteroplasmy was found in mitochondrial sequences in Tetragonisca angustula—a bee species of great economic and ecological importance—the aim of this study was to confirm the presence of heteroplasmy in the species, analyse the frequency of synonymous and non-synonymous mutations, and classify them according to their impact on the protein’s tertiary structure. For this purpose, mitochondrial DNA was isolated from nuclear DNA in two T. angustula individuals, a portion of COI was amplified by PCR from pure mitochondrial DNA, and the different mitochondrial haplotypes were recovered through vector cloning in competent bacteria. Our results confirmed the presence of 11 and 17 different mitochondrial haplotypes in the first and second samples analysed, respectively. These different mitochondrial haplotypes are characterized by the predominance of synonymous mutations or mild and moderate non-synonymous mutations, meaning they have little impact on the protein’s tertiary structure. These results strongly support the presence of heteroplasmy in T. angustula and provide some insights into its characterization and inheritance.