Comparative mitogenomics of Drosophilidae and the evolution of the Zygothrica genus group (Diptera, Drosophilidae).

The Zygothrica genus group of Drosophilidae encompasses more than 437 species and five genera. Although knowledge regarding its diversity has increased, uncertainties about its monophyly and position within Drosophilidae remain. Genomic approaches have been widely used to address different phylogenetic questions and analyses involving the mitogenome have revealed a cost-efficient tool to these studies. Thus, this work aims to characterize mitogenomes of three species of the Zygothrica genus group (from the Hirtodrosophila, Paraliodrosophila and Zygothrica genera), while comparing them with orthologous sequences from other 23 Drosophilidae species and addressing their phylogenetic position. General content concerning gene order and overlap, nucleotide composition, start and stop codon, codon usage and tRNA structures were compared, and phylogenetic trees were constructed under different datasets. The complete mitogenomes characterized for H. subflavohalterata affinis H002 and P. antennta present the PanCrustacea gene order with 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, 13 protein coding genes and an A+T rich region with two T-stretched elements. Some peculiarities such as the almost complete overlap of genes tRNAH/ND4, tRNAF/ND5 and tRNAS2/ND1 are reported for different Drosophilidae species. Non-canonical secondary structures were encountered for tRNAS1 and tRNAY, revealing patterns that apply at different phylogenetic scales. According to the best depiction of the mitogenomes evolutionary history, the three Neotropical species of the Zygothrica genus group encompass a monophyletic lineage sister to Zaprionus, composing with this genus a clade that is sister to the Drosophila subgenus.

Insect fat body cell morphology and response to cold stress is modulated by acclimation.

Mechanistic understanding about the nature of cellular cryoinjury and mechanisms by which some animals survive freezing while others do not is currently lacking. Here, we exploited the broadly manipulable freeze tolerance of larval malt flies (Chymomyza costata) to uncover cell and tissue morphological changes associated with freeze mortality. Diapause induction, cold acclimation and dietary proline supplementation generate malt fly variants ranging from weakly to extremely freeze tolerant. Using confocal microscopy and immunostaining of the fat body, Malpighian tubules and anterior midgut, we described tissue and cytoskeletal (F-actin and α-tubulin) morphologies among these variants after exposure to various cold stresses (from chilling at -5°C to extreme freezing at -196°C), and upon recovery from cold exposure. Fat body tissue appeared to be the most susceptible to cryoinjury: freezing caused coalescence of lipid droplets, loss of α-tubulin structure and apparent aggregation of F-actin. A combination of diapause and cold acclimation substantially lowered the temperature at which these morphological disruptions occurred. Larvae that recovered from a freezing challenge repaired F-actin aggregation but not lipid droplet coalescence or α-tubulin structure. Our observations indicate that lipid coalescence and damage to α-tubulin are non-lethal forms of freeze injury, and suggest that repair or removal (rather than protection) of actin proteins is a potential mechanism of acquired freeze tolerance.

A role for nematocytes in the cellular immune response of the drosophilid Zaprionus indianus.

The melanotic encapsulation response mounted by Drosophila melanogaster against macroparasites, which is based on haemocyte binding to foreign objects, is poorly characterized relative to its humoral immune response against microbes, and appears to be variable across insect lineages. The genus Zaprionus is a diverse clade of flies embedded within the genus Drosophila. Here we characterize the immune response of Zaprionus indianus against endoparasitoid wasp eggs, which elicit the melanotic encapsulation response in D. melanogaster. We find that Z. indianus is highly resistant to diverse wasp species. Although Z. indianus mounts the canonical melanotic encapsulation response against some wasps, it can also potentially fight off wasp infection using two other mechanisms: encapsulation without melanization and a non-cellular form of wasp killing. Zaprionus indianus produces a large number of haemocytes including nematocytes, which are large fusiform haemocytes absent in D. melanogaster, but which we found in several other species in the subgenus Drosophila. Several lines of evidence suggest these nematocytes are involved in anti-wasp immunity in Z. indianus and in particular in the encapsulation of wasp eggs. Altogether, our data show that the canonical anti-wasp immune response and haemocyte make-up of the model organism D. melanogaster vary across the genus Drosophila.

Variation in mesoderm specification across Drosophilids is compensated by different rates of myoblast fusion during body wall musculature development.

BACKGROUND: It has been shown that species separated by relatively short evolutionary distances may have extreme variations in egg size and shape. Those variations are expected to modify the polarized morphogenetic gradients that pattern the dorso-ventral axis of embryos. Currently, little is known about the effects of scaling over the embryonic architecture of organisms. We began examining this problem by asking if changes in embryo size in closely related species of Drosophila modify all three dorso-ventral germ layers or only particular layers, and whether or not tissue patterning would be affected at later stages. PRINCIPAL FINDINGS: Here we report that changes in scale affect predominantly the mesodermal layer at early stages, while the neuroectoderm remains constant across the species studied. Next, we examined the fate of somatic myoblast precursor cells that derive from the mesoderm to test whether the assembly of the larval body wall musculature would be affected by the variation in mesoderm specification. Our results show that in all four species analyzed, the stereotyped organization of the body wall musculature is not disrupted and remains the same as in D. melanogaster. Instead, the excess or shortage of myoblast precursors is compensated by the formation of individual muscle fibers containing more or less fused myoblasts. CONCLUSIONS: Our data suggest that changes in embryonic scaling often lead to expansions or retractions of the mesodermal domain across Drosophila species. At later stages, two compensatory cellular mechanisms assure the formation of a highly stereotyped larval somatic musculature: an invariable selection of 30 muscle founder cells per hemisegment, which seed the formation of a complete array of muscle fibers, and a variable rate in myoblast fusion that modifies the number of myoblasts that fuse to individual muscle fibers.

Cell cycle arrest as a hallmark of insect diapause: changes in gene transcription during diapause induction in the drosophilid fly, Chymomyza costata.

The division cycle of CNS cells was arrested in G0/G1 (86.6%) and G2 (12.8%) phases in diapausing larvae of Chymomyza costata. A two-step response was observed when the diapause was induced by transferring the 3rd instar larvae from long-day to short-day conditions: first, the proportion of G2-arrested cells increased rapidly within a single day after transfer; and second, the increase of G0/G1-arrested cells started with a delay of 5 days after transfer. The changes of relative mRNA levels of seven different genes, which code for important cell cycle regulatory factors [Cyclins D and E, kinases Wee1 and Myt1, phosphatase Cdc25 (String), Dacapo (p27), and PCNA] were followed using qRT-PCR technique. Two reference genes (Rp49 and ss-tubulin) served as a background. Significant transcriptional responses to photoperiodic transfer were observed for two genes: while the relative levels of dacapo mRNA increased during the rapid entry into the G2 arrest, the pcna expression was significantly downregulated during the delayed onset of G0/G1 arrest. In addition, moderate transcriptional upregulations of the genes coding for two inhibitory kinases, wee1 and myt1 accompanied the entry into diapause. The other genes were expressed equally in all photoperiodic conditions.

Differences in sensory projections between macro- and microchaetes in Drosophilid flies.

From examination of the central axonal projections of sensory bristles on the notum of several species of Drosophilidae, we demonstrate different features that may indicate different functions for macro- and microchaetes. The large macrochaetes have conserved arborizations that correlate with their conserved position. Nevertheless, we find evidence for only two discrete projection patterns for bristles in the dorsocentral (DC) row, even when there may be four or five bristles present. We show that the small microchaetes of Drosophila melanogaster display regional specificity and subsets of contiguous bristles project to a common region in the thoracic ganglion. Interestingly, the axons of each of these subsets also form a specific fasciculation group on the scutum before joining the axon of a particular macrochaete. The positions of microchaetes on the scutum and the shape of the fasciculation groups vary between closely related species. There is no correlation between body size, bristle patterns, and fasciculation patterns. Furthermore, none of these traits correlate with the phylogenetic relationships between the species studied. We discuss the possibility that macro- and microchaetes may have different functions and that these have implications for evolutionary constraints on bristle patterns.

Remodelling of membrane phospholipids during transition to diapause and cold-acclimation in the larvae of Chymomyza costata (Drosophilidae).

The composition of molecular species of phosphatidylethanolamines (PEs) and phosphatidylcholines (PCs) was analysed in fat body and muscle tissues of Chymomyza costata larvae of different physiological states that markedly differed in their level of freeze-tolerance. Actively moving and feeding 3rd instar larvae had low (zero) capacity of freeze-tolerance and similar phospholipid (PL) compositions irrespective of their developmental destiny (non-diapause vs. diapause). Extensive remodelling of PL composition was found in these larvae in response to: (a) chilling of non-diapause larvae at 5 degrees C for 1 month; (b) developmental transition to diapause; and (c) chilling of diapause larvae. Transition to diapause and chilling led to an increase in freeze-tolerance. The increase in molar proportion of molecular species containing palmitic/linoleic (16:0/18:2) fatty acyls (FAs) esterified to sn-1/sn-2 positions of glycerol was the most prominent change, which was tightly statistically correlated with increasing freeze-tolerance. The increase of PLs with combination of 16:0/18:2 FAs was registered consistently in PEs and PCs in fat body and muscle tissues in response to chilling and to diapause onset. This increase was countered by a decreases of various molecular species, depending on tissue and lipid class. Most decreasing species shared one common theme: they had a saturated FA (palmityl, margaryl, stearyl) esterified at sn-1 position and a monounsaturate (palmitoleyl, oleyl) esterified at sn-2 position of glycerol. Possible adaptive meaning of PL molecular species remodelling is discussed.