Nuclear organization.

This article discusses three reviews on the theme of nuclear organization.

The Balbiani Ring Story: Synthesis, Assembly, Processing, and Transport of Specific Messenger RNA-Protein Complexes.

Eukaryotic gene expression is the result of the integrated action of multimolecular machineries. These machineries associate with gene transcripts, often already nascent precursor messenger RNAs (pre-mRNAs). They rebuild the transcript and convey properties allowing the processed transcript, the mRNA, to be exported to the cytoplasm, quality controlled, stored, translated, and degraded. To understand these integrated processes, one must understand the temporal and spatial aspects of the fate of the gene transcripts in relation to interacting molecular machineries. Improved methodology is necessary to study gene expression in vivo for endogenous genes. A complementary approach is to study biological systems that provide exceptional experimental possibilities. We describe such a system, the Balbiani ring (BR) genes in polytene cells in the dipteran Chironomus tentans. The BR genes, along with their pre-mRNA-protein complexes (pre-mRNPs) and mRNA-protein complexes (mRNPs), allow the visualization of intact cell nuclei and enable analyses of where and when different molecular machineries associate with and act on the BR pre-mRNAs and mRNAs.

A sodium-dependent trehalose transporter contributes to anhydrobiosis in insect cell line, Pv11.

Pv11 is the only animal cell line that, when preconditioned with a high concentration of trehalose, can be preserved in the dry state at room temperature for more than one year while retaining the ability to resume proliferation. This extreme desiccation tolerance is referred to as anhydrobiosis. Here, we identified a transporter that contributes to the recovery of Pv11 cells from anhydrobiosis. In general, the solute carrier 5 (SLC5)-type secondary active transporters cotransport Na+ and carbohydrates including glucose. The heterologous expression systems showed that the transporter belonging to the SLC5 family, whose expression increases upon rehydration, exhibits Na+-dependent trehalose transport activity. Therefore, we named it STRT1 (sodium-ion trehalose transporter 1). We report an SLC5 family member that transports a naturally occurring disaccharide, such as trehalose. Knockout of the Strt1 gene significantly reduced the viability of Pv11 cells upon rehydration after desiccation. During rehydration, when intracellular trehalose is no longer needed, Strt1-knockout cells released the disaccharide more slowly than the parental cell line. During rehydration, Pv11 cells became roughly spherical due to osmotic pressure changes, but then returned to their original spindle shape after about 30 min. Strt1-knockout cells, however, required about 50 min to adopt their normal morphology. STRT1 probably regulates intracellular osmolality by releasing unwanted intracellular trehalose with Na+, thereby facilitating the recovery of normal cell morphology during rehydration. STRT1 likely improves the viability of dried Pv11 cells by rapidly alleviating the significant physical stresses that arise during rehydration.

New mitogenomes from the genus Cricotopus (Diptera: Chironomidae, Orthocladiinae): Characterization and phylogenetic implications.

Cricotopus is a large and diverse genus of non-biting midges composed of several subgenera. Complete mitogenome sequences are available for very few Cricotopus species. The subgenus Pseudocricotopus unites species with unusual morphological structures in adult male and pupal stages, however, molecular methods are needed to verify the placement of this subgenus within Cricotopus. We obtained mitogenomes of C. (Pseudocricotopus) cf. montanus and nine other Cricotopus species for phylogenetic analysis, coupled with two Rheocricotopus species and one Synorthocladius species as outgroups. The structure of the mitogenome was similar among these Cricotopus species, exhibiting A+T bias and retaining ancestral gene order. Mutation rate, estimated as Ka/Ks, varied among genes, and was highest for ATP8 and lowest for COI. The phylogenetic relationships among species of Cricotopus, Rheocricotopus and Synorthocladius was reconstructed using Bayesian inference and maximum likelihood estimation. The phylogenetic trees confirmed placement of subgenus Pseudocricotopus, represented by Cricotopus cf. montanus, within Cricotopus. Our study increases the library of chironomid mitogenomes and provides insight into the properties of their constituent genes.

DNA barcodes and morphology reveal new species within the Rheotanytarsus guineensis species group from China (Diptera: Chironomidae).

The Rheotanytarsus guineensis species group (Diptera: Chironomidae) is a species diverse and taxonomically difficult group. Using DNA barcodes, we found five new species within the R. guineensis species group and reviewed the species group based on adult males from China. Rheotanytarsus guoae Lin & Yao sp. n., Rheotanytarsus miaoae Lin & Yao sp. n., Rheotanytarsus qiangi Lin & Yao sp. n., Rheotanytarsus yueqingensis Lin & Yao sp. n., and Rheotanytarsus yui Lin & Yao sp. n. are all described and figured. A key to known adult males of the R. guineensis species group worldwide is provided for the first time.

New data on mitogenomes of Thienemanniella Kieffer, 1911 (Diptera: Chironomidae, Orthocladiinae).

The mitochondrial genome (mitogenome) has been widely used as a powerful marker in phylogenetic and evolutionary studies of various Dipteran groups. However, only a few mitogenomes from the Thienemanniella genus have been reported till now. Furthermore, there is still indeterminacy in the phylogenetic relationships of the genus Thienemanniella. In this study, mitogenomes of five Thienemanniella species were sequenced and analyzed newly. Combined with the published mitogenome of Thienemanniella nipponica, the obtained results showed that mitogenomes of Thienemanniella were conserved in structure, and all genes were observed to be arranged in the same gene order as the ancestral mitogenome. Nucleotide composition varied significantly among different genes, and the control region displayed the highest A + T content. All protein coding genes are subjected to purification selection, and the fastest evolving gene is ATP8. Maximum likelihood and Bayesian inference analyses showed the phylogeny of Thienemanniella which was supported in five topologies. Our present study provides valuable insight into the phylogenetic relationships of Thienemanniella species.

Characterization and expression of heat shock and immune genes in natural populations of Prodiamesa olivacea (Diptera) exposed to thermal stress.

This paper characterizes the heat stress response (HSR) and explores the impact of temperatures on the immune response of larvae from two chironomid species, Prodiamesa olivacea and Chironomus riparius. Genes involved in crucial metabolic pathways were de novo identified in P. olivacea: Hsp27, Hsp60, Hsp70, Hsc70, Cdc37, and HSF for the heat stress response (HSR) and TOLL, PGRP, C-type lectin, and JAK/hopscotch for the immune system response (ISR). Quantitative real-time PCR was used to evaluate the expression levels of the selected genes in short-term treatments (up to 120') at high temperatures (35 °C and 39 °C). Exposing P. olivacea to elevated temperatures resulted in HSR induction with increased expression of specific heat shock genes, suggesting the potential of HSPs as early indicators of acute thermal stress. Surprisingly, we found that heat shock represses multiple immune genes, revealing the antagonist relation between the heat shock response and the innate immune response in P. olivacea. Our results also showed species-dependent gene responses, with more significant effects in P. olivacea, for most of the biomarkers studied, demonstrating a higher sensitivity in this species to environmental stress conditions than that of C. riparius. This work shows a multi-species approach that enables a deeper understanding of the effects of heat stress at the molecular level in aquatic dipterans.

What does not kill it makes it stronger! The tolerance of the F1 larvae of Chironomus xanthus to a neonicotinoid insecticide formulation.

Thiamethoxam (TMX) is a systemic neonicotinoid that acts as a partial agonist of the nicotinic acetylcholine receptors (nAChRs). However, target species have shown resistance to formulations based on such neonicotinoids, which can also be expected for non-target insects. This research aimed to study the effects of a formulation based on TMX [Cruiser® 350 FS (CRZ)] on the life traits of Chironomus xanthus filial generation (F1) and compare it with the parental generation (P). Environmentally relevant concentrations of CRZ significantly decreased larvae growth P generation , also slowing and decreasing their emergence. Larvae of the F1 generation were less sensitive than their parents, suggesting that the progeny were able to thrive and perform basic physiological functions better than the parental generation. Our results highlight that insect resistance to neonicotinoids may be associated with the better performance of the filial generation, which is related to the change in affinities of the active ingredient for the sub-units constituting the nAChRs subtypes of F1 organisms, inherited from P organisms that were able to survive and reproduce. Moreover, further studies using biochemical and omics tools should be performed to disentangle the specific changes occurring at the nAChRs throughout insect development.

UDP-glucuronosyltransferase is involved in susceptibility of Chironomus kiiensis Tokunaga, 1936 (Diptera: Chironomidae) to insecticides.

UDP-glucuronosyltransferases (UGTs) could transform various exogenous and endogenous compounds, which help detoxification of pesticides in insects. To investigate the role of UGTs in the detoxification metabolism of insecticides in Chironomus kiiensis, CkUGT302M1, CkUGT302N1, CkUGT308N1 and CkUGT36J1 genes were identified with 1449-1599 bp encoding 482-532 amino acids. Four UGT genes shared 40.86∼53.36% identity with other homologous insect species, and expressed in all developmental stages, notably in the larval and adult stages. Expression of CkUGTs was higher in the gastric caecum, midgut and head. Moreover, CkUGTs expression and activity were significantly increased in C. kiiensis larvae in exposure to sublethal concentrations of carbaryl, deltamethrin and phoxim, respectively. To further explore the functions of UGT genes, the CkUGT308N1 was effectively silenced in 4th instar C. kiiensis larvae by RNA interference, which resulted in the mortality of dsCkUGT308N1 treated larvae increased by 71.43%, 111.11% and 62.50% under sublethal doses of carbaryl, deltamethrin and phoxim at the 24-h time point, respectively. The study revealed that the CkUGT308N1 gene in C. kiiensis could contribute to the metabolism of pesticides and provide a scientific basis for evaluating the water pollution of pesticides.

Transcriptome and gut microbiota analyses reveal a possible mechanism underlying rifampin-mediated interruption of the larval development of chironomid Propsilocerus akamusi (Diptera: Chironomidae).

Chironomids, the most abundant insect group found in freshwater habitats, are known to be pollution tolerate and serve as important bioindicators of contaminant stress. Gut microbiota has recently been shown to potentially provide a number of beneficial services to insect hosts. However, the antibiotic-mediated interruption of chironomid gut microbial community and its subsequent influence on host body are still unclear. In the present study, the effects of rifampin on chironomid larvae were investigated at both transcriptome and microbiome level to assess the relationship between gut bacteria and associated genes. Our data indicated that the rifampin-induced imbalance of gut ecosystem could inhibit the development of chironomid larvae via decreasing the body weight, body length and larval eclosion rate during 96-h treatment. Both the community structure and taxonomic composition were significantly altered due to the invasion of rifampin in digestive tracts. The relative abundance of phylum Deferribacterota and Bacteroidota were dramatically increased with rifampin exposure. A set of genes involved in amino acid synthesis as well as xenobiotic metabolism pathways were greatly changed and proved to have tight correlation with certain genus. Bacterial genus Tyzzerella was positively correlated with detoxifying PaCYP6GF1 and PaCYP9HL1 genes. This study provides a reference for understanding the environmental risks of antibiotic and aims to accelerate new biological insights into the effects of antibiotic on the fitness of chironomids and into the microbe mediated-regulatory mechanism of aquatic insects.

A new molecular phylogeny for the Tanypodinae (Diptera: Chironomidae) places the Australian diversity in a global context.

The non-biting midge subfamily Tanypodinae (Diptera: Chironomidae) is species-rich, ecologically diverse, and near-globally distributed. Within the subfamily, aspects of generic and species-level taxonomy remain poorly understood, in particular the validity of assignment of Australian and New Zealand taxa to genera erected for northern hemisphere (Holarctic) fauna. Here, we place the austral diversity within this global context by extensive geographical and taxonomic sampling in concert with a multilocus phylogenetic approach. We incorporated sequence data for mitochondrial COI, and nuclear 28S and CAD, and conducted Bayesian and maximum likelihood phylogenetic inferences and Bayesian divergence time estimation. The resolved phylogeny supported many associations of Australian taxa with their proposed Holarctic congeners, with the exception of Apsectrotanypus Fittkau, and validates several taxa as endemic. Three of four New Zealand sampled taxa had their sister groups in Australia; New Zealand Monopelopia Fittkau was sister to a German congener. This included the first record of Procladius Kieffer from New Zealand. Most nodes connecting austral and Holarctic taxa clustered around the Cretaceous-Tertiary boundary (60-80 mya), whereas New Zealand-Australia nodes were generally slightly younger (53-57 mya). Together, these data contribute substantially to our understanding of the taxonomy, systematics and biogeography of the Australian Tanypodinae and more broadly to knowledge of Australia's aquatic insect biodiversity.

Molecular phylogeny of Japanese marine Tanytarsini chironomids (Chironomidae: Chironominae).

Tanytarsini is a large tribe of Chironomidae with at least 11 recorded marine species grouped in three genera. In this study, we performed a phylogenic analysis using molecular data from 13 Tanytarsini genera, including all Japanese marine species in a large tribe of Chironominae, to estimate their phylogenetic positions and evolutionary history. The phylogenetic reconstruction grouped the marine species in two clades. One clade was composed of five marine Tanytarsus and two marine Pontomyia species within a larger clade of Tanytarsus. Pontomyia is considered to have morphologically specialized and adapted to marine environments by rapid evolution, although it formed a clade with Tanytarsus. The only one species of Tanytarsus, T. pelagicus, clustered as a member of the second clade, which was mainly composed by species of the genus Paratanytarsus. Thus, we redescribe T. pelagicus as Paratanytarsus pelagicus.

The genomic basis of circadian and circalunar timing adaptations in a midge.

Organisms use endogenous clocks to anticipate regular environmental cycles, such as days and tides. Natural variants resulting in differently timed behaviour or physiology, known as chronotypes in humans, have not been well characterized at the molecular level. We sequenced the genome of Clunio marinus, a marine midge whose reproduction is timed by circadian and circalunar clocks. Midges from different locations show strain-specific genetic timing adaptations. We examined genetic variation in five C. marinus strains from different locations and mapped quantitative trait loci for circalunar and circadian chronotypes. The region most strongly associated with circadian chronotypes generates strain-specific differences in the abundance of calcium/calmodulin-dependent kinase II.1 (CaMKII.1) splice variants. As equivalent variants were shown to alter CaMKII activity in Drosophila melanogaster, and C. marinus (Cma)-CaMKII.1 increases the transcriptional activity of the dimer of the circadian proteins Cma-CLOCK and Cma-CYCLE, we suggest that modulation of alternative splicing is a mechanism for natural adaptation in circadian timing.

Copper and chlorpyrifos stress affect the gut microbiota of chironomid larvae (Propsilocerus akamusi).

Chironomids are characterized by their ubiquitous distribution, global diversity and tolerant ability to deal with environmental stressors. To our knowledge, this is the first study presenting the gut microbial structure of chironomid larvae and examining the microbial alteration induced by invading chlorpyrifos and copper with different dosages. Lethal bioassay displayed a significantly decreased percentage survival of Propsilocerus akamusi larvae exposed to 800 mg/L copper and 50 µg/L chlorpyrifos at 96 h. Larvae with deficient gut microbiota exhibited a depressed level of glutathione S-transferase activity after stressful exposure. The high-throughput 16S rRNA gene sequencing was adopted to investigate the community structure and it turned out that both copper and chlorpyrifos were able to generate distinguished variations of gut microbiota in the stressor-specific and concentration-dependent manner. Of note, the relative abundance of Comamonas, Stenotrophomonas, and Yersinia remarkably elevated in the presence of copper while chlorpyrifos exposure upregulated the prevalence of certain genera (e.g. Serratia). Flavobacterium was greatly attenuated in chlorpyrifos group with lethal dosage exhibiting more severe impacts. The predicted gene functions of the gut commensals differed between normal samples and those subjected to distinct toxins. Besides, more positive associations and limited modularity of microbial interactions were observed in stressor-challenged larvae, presenting a network with impaired complexity and stability. The appearance of either copper or chlorpyrifos exhibited strong positive correlations with genera belonging to Proteobacteria and Firmicutes. Collectively, this investigation introduces a general outline of gut microbiota obtained from chironomid individuals with latent adaptive tactics to nocuous factors (heavy metal and pesticide), which could build a fundamental basis for us to further explore the protective roles of chironomid gut bacterial colonizers in defending against aquatic contaminants.

The importance of DNA barcode choice in biogeographic analyses - a case study on marine midges of the genus Clunio.

DNA barcodes are widely used for species identification and biogeographic studies. Here, we compare the use of full mitochondrial genomes versus DNA barcodes and other mitochondrial DNA fragments for biogeographic and ecological analyses. Our dataset comprised 120 mitochondrial genomes from the genus Clunio (Diptera: Chironomidae), comprising five populations from two closely related species (Clunio marinus and Clunio balticus) and three ecotypes. We extracted cytochrome oxidase c subunit I (COI) barcodes and partitioned the mitochondrial genomes into non-overlapping windows of 750 or 1500 bp. Haplotype networks and diversity indices were compared for these windows and full mitochondrial genomes (15.4 kb). Full mitochondrial genomes indicate complete geographic isolation between populations, but do not allow for conclusions on the separation of ecotypes or species. COI barcodes have comparatively few polymorphisms, ideal for species identification, but do not resolve geographic isolation. Many of the similarly sized 750 bp windows have higher nucleotide and haplotype diversity than COI barcodes, but still do not resolve biogeography. Only when increasing the window size to 1500 bp, two windows resolve biogeography reasonably well. Our results suggest that the design and use of DNA barcodes in biogeographic studies must be carefully evaluated for each investigated species.

Multi-Level Gene Expression in Response to Environmental Stress in Aquatic Invertebrate Chironomids: Potential Applications in Water Quality Monitoring.

In freshwater ecosystems, aquatic invertebrates are influenced continuously by both physical stress and xenobiotics. Chironomids (Diptera; Chironomidae), or non-biting midges, are the most diverse and abundant invertebrates in freshwater habitats. They are a fundamental link in food chains of aquatic ecosystems. Chironomid larvae tolerate stress factors in their environments via various physiological processes. At the molecular level, environmental pollutants induce multi-level gene responses in Chironomus that regulate cellular protection through the activation of defense processes. This paper reviews literature on the transcriptional responses of biomarker genes to environmental stress in chironomids at the molecular level, in studies conducted from 1991 to 2020 (120 selected literatures of 374 results with the keywords "Chironomus and gene expression" by PubMed search tool). According to these studies, transcriptional responses in chironomids vary depending on the type of stress factor and defensive responses associated with antioxidant activity, the endocrine system, detoxification, homeostasis and stress response, energy metabolism, ribosomal machinery, apoptosis, DNA repair, and epigenetics. These data could provide a comprehensive overview of how Chironomus species respond to pollutants in aquatic environments. Furthermore, the transcriptomic data could facilitate the development of genetic tools for water quality and environmental monitoring based on resident chironomid species.

Cooption of heat shock regulatory system for anhydrobiosis in the sleeping chironomid Polypedilum vanderplanki.

Polypedilum vanderplanki is a striking and unique example of an insect that can survive almost complete desiccation. Its genome and a set of dehydration-rehydration transcriptomes, together with the genome of Polypedilum nubifer (a congeneric desiccation-sensitive midge), were recently released. Here, using published and newly generated datasets reflecting detailed transcriptome changes during anhydrobiosis, as well as a developmental series, we show that the TCTAGAA DNA motif, which closely resembles the binding motif of the Drosophila melanogaster heat shock transcription activator (Hsf), is significantly enriched in the promoter regions of desiccation-induced genes in P. vanderplanki, such as genes encoding late embryogenesis abundant (LEA) proteins, thioredoxins, or trehalose metabolism-related genes, but not in P. nubifer Unlike P. nubifer, P. vanderplanki has double TCTAGAA sites upstream of the Hsf gene itself, which is probably responsible for the stronger activation of Hsf in P. vanderplanki during desiccation compared with P. nubifer To confirm the role of Hsf in desiccation-induced gene activation, we used the Pv11 cell line, derived from P. vanderplanki embryo. After preincubation with trehalose, Pv11 cells can enter anhydrobiosis and survive desiccation. We showed that Hsf knockdown suppresses trehalose-induced activation of multiple predicted Hsf targets (including P. vanderplanki-specific LEA protein genes) and reduces the desiccation survival rate of Pv11 cells fivefold. Thus, cooption of the heat shock regulatory system has been an important evolutionary mechanism for adaptation to desiccation in P. vanderplanki.

Generational sensitivity alteration in Chironomus yoshimatsui to carbamate and pharmaceutical chemicals and the effect on Catalase, CYP450, and hemoglobin gene transcription.

To ascertain the tolerance mechanisms of aquatic organisms to artificial chemicals, intergenerational sensitivity changes of Chironomus yoshimatsui to a carbamate pesticide (pirimicarb) and pharmaceutical chemical (diazepam) were investigated. The larvae (<48-h-old) in each generation were exposed to both chemicals for 48 h and then the surviving chironomids were cultured until the fifth generation (F0-F4) without chemical addition. The 48-h 50% effective concentration (EC50) value of chironomids was determined for each generation. In the pirimicarb treatment group, the EC50 values significantly increased in F3 and F4, and those in the diazepam treatment group slightly increased. Catalase, Cytochrome P450 and hemoglobin (Hb) mRNA levels were monitored to see whether these were related to the trans-generational sensitivity. Although the generalized linear model results showed that the sensitivity to diazepam was slightly increased in the diazepam treatment, we could not find any mRNA levels related to sensitivity alteration. In contrast, the model approach showed that the chironomids exposed to pirimicarb trans-generationally became tolerant with increasing Hb mRNA levels. Therefore, they might decrease their chemical stress by modifying Hb gene transcription.

CriTER-A: A Novel Temperature-Dependent Noncoding RNA Switch in the Telomeric Transcriptome of Chironomus riparius.

The telomeric transcriptome of Chironomus riparius has been involved in thermal stress response. One of the telomeric transcripts, the so-called CriTER-A variant, is highly overexpressed upon heat shock. On the other hand, its homologous variant CriTER-B, which is the most frequently encoded noncoding RNA in the telomeres of C. riparius, is only slightly affected by thermal stress. Interestingly, both transcripts show high sequence homology, but less is known about their folding and how this could influence their differential behaviour. Our study suggests that CriTER-A folds as two different conformers, whose relative proportion is influenced by temperature conditions. Meanwhile, the CriTER-B variant shows only one dominant conformer. Thus, a temperature-dependent conformational equilibrium can be established for CriTER-A, suggesting a putative functional role of the telomeric transcriptome in relation to thermal stress that could rely on the structure-function relationship of the CriTER-A transcripts.

Genome-Wide Role of HSF1 in Transcriptional Regulation of Desiccation Tolerance in the Anhydrobiotic Cell Line, Pv11.

The Pv11, an insect cell line established from the midge Polypedilum vanderplanki, is capable of extreme hypometabolic desiccation tolerance, so-called anhydrobiosis. We previously discovered that heat shock factor 1 (HSF1) contributes to the acquisition of desiccation tolerance by Pv11 cells, but the mechanistic details have yet to be elucidated. Here, by analyzing the gene expression profiles of newly established HSF1-knockout and -rescue cell lines, we show that HSF1 has a genome-wide effect on gene regulation in Pv11. The HSF1-knockout cells exhibit a reduced desiccation survival rate, but this is completely restored in HSF1-rescue cells. By comparing mRNA profiles of the two cell lines, we reveal that HSF1 induces anhydrobiosis-related genes, especially genes encoding late embryogenesis abundant proteins and thioredoxins, but represses a group of genes involved in basal cellular processes, thus promoting an extreme hypometabolism state in the cell. In addition, HSF1 binding motifs are enriched in the promoters of anhydrobiosis-related genes and we demonstrate binding of HSF1 to these promoters by ChIP-qPCR. Thus, HSF1 directly regulates the transcription of anhydrobiosis-related genes and consequently plays a pivotal role in the induction of anhydrobiotic ability in Pv11 cells.