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.

Smittia solominae sp. nov. (Diptera: Chironomidae: Orthocladiinae), living on ice of high mountain glaciers of the Elbrus Region (North Caucasus).

Illustrated morphological description with a study of DNA barcoding and biology of adult male, pupa and larva of Smittia solominae sp. nov., living on the ice surface of glaciers at an altitude of about 3000 m above sea level in the Elbrus region of the North Caucasus is given. DNA barcoding provided support that the new species unique within genus Smittia. The average interspecific distances between S. solominae sp. nov. and other Smittia from BOLD above 12% that correspond to species level.

Molecular and morphological characterisation of larvae of the genus Diamesa Meigen, 1835 (Diptera: Chironomidae) in Alpine streams (Ötztal Alps, Austria).

Diamesa species (Diptera, Chironomidae) are widely distributed in freshwater ecosystems, and their life cycles are closely linked to environmental variables such as temperature, water quality, and sediment composition. Their sensitivity to environmental changes, particularly in response to pollution and habitat alterations, makes them valuable indicators of ecosystem health. The challenges associated with the morphological identification of larvae invoke the use of DNA barcoding for species determination. The mitochondrial cytochrome oxidase subunit I (COI) gene is regularly used for species identification but faces limitations, such as similar sequences in closely related species. To overcome this, we explored the use of the internal transcribed spacers (ITS) region in addition to COI for Diamesa larvae identification. Therefore, this study employs a combination of molecular markers alongside traditional morphological identification to enhance species discrimination. In total, 129 specimens were analysed, of which 101 were sampled from a glacier-fed stream in Rotmoostal, and the remaining 28 from spring-fed streams in the neighbouring valleys of Königstal and Timmelstal. This study reveals the inadequacy of utilizing single COI or ITS genes for comprehensive species differentiation within the genus Diamesa. However, the combined application of COI and ITS markers significantly enhances species identification resolution, surpassing the limitations faced by traditional taxonomists. Notably, this is evident in cases involving morphologically indistinguishable species, such as Diamesa latitarsis and Diamesa modesta. It highlights the potential of employing a multi-marker approach for more accurate and reliable Diamesa species identification. This method can be a powerful tool for identifying Diamesa species, shedding light on their remarkable adaptations to extreme environments and the impacts of environmental changes on their populations.

Two chromosome-level genomes of Smittia aterrima and Smittia pratorum (Diptera, Chironomidae).

Chironomids are one of the most abundant aquatic insects and are widely distributed in various biological communities. However, the lack of high-quality genomes has hindered our ability to study the evolution and ecology of this group. Here, we used Nanopore long reads and Hi-C data to produce two chromosome-level genomes from mixed genomic data. The genomes of Smittia aterrima (SateA) and Smittia pratorum (SateB) were assembled into three chromosomes, with sizes of 78.45 Mb and 71.56 Mb, scaffold N50 lengths of 25.73 and 23.53 Mb, and BUSCO completeness of 98.5% and 97.8% (n = 1,367), 5.68 Mb (7.24%) and 1.94 Mb (2.72%) of repetitive elements, and predicted 12,330 (97.70% BUSCO completeness) and 11,250 (97.40%) protein-coding genes, respectively. These high-quality genomes will serve as valuable resources for comprehending the evolution and environmental adaptation of chironomids.

Environmentally Relevant Concentrations of the Insecticide Fipronil Modulated Molecular Response in Chironomus riparius.

Pesticides employed worldwide for crop protection easily reach aquatic systems, which act as the main reservoirs, and become a risk factor for aquatic fauna. Fipronil is a broad-spectrum insecticide acting on the insect nervous system; however, other effects and systems unrelated to this mechanism could be affected in non-target organisms. Thus, the present study aimed to assess the impact of fipronil on the suborganismal response (gene expression and enzymatic activity) of Chironomus riparius larvae as a model organism in ecotoxicology. To this end, short-term toxicity tests were carried out with fourth-instar larvae exposed to 0.001, 0.01, and 0.1 µg L-1 of fipronil for 24 and 96 h. Messenger RNA levels of 42 genes related to diverse metabolic pathways were analyzed by real-time polymerase chain reaction, complemented with catalase (CAT), glutathione S-transferase (GST), and acetylcholinesterase (AChE) activities. Few effects were observed at 24 h; however, after longer exposure (96 h), genes involved in the endocrine, detoxification, stress, and immune response pathways were altered. Moreover, fipronil at 96 h increased CAT and GST activity at 0.01 µg L-1 and AChE at the highest concentrations. The results demonstrate that even low environmentally relevant fipronil concentrations can modulate the molecular response of several cellular pathways in C. riparius after short-term exposure. These results bring new information about the underlying response of fipronil and its mode of action on a key aquatic invertebrate. Despite no effects on mortality, strong modulation at the suborganismal level emphasizes the advantage of biomarkers as early damage responses and the harmful impact of this pesticide on freshwater organisms. Environ Toxicol Chem 2024;43:405-417. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

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.

Gene expression analysis of Chironomus riparius in response to acute exposure to tire rubber microparticles and leachates.

Tire rubber microparticles (TRPs) entering aquatic ecosystems through stormwater runoffs is a significant challenge. TRPs are formed by the abrasion of tires with the road surface and include chemical additives that are an additional cause for concern. Currently, information on the molecular effects of TRPs, or especially its additives, in freshwater organisms is scarce. To address this problem, an array covering different cellular processes has been designed for the freshwater midge Chironomus riparius. Fourth-instar larvae were exposed to two concentrations of TRPs (1 mg L-1, 10 mg L-1) and tire rubber leachates (TRLs) (0,0125 %, 5 %) to evaluate the transcriptional activity by Real-Time PCR. To assess acute toxicity, larvae were exposed for 24 h and genes related to the endocrine system, stress response, DNA repair mechanisms, immune system, oxidative stress, and detoxification mechanisms were evaluated. The activity of the enzymes: glutathione S-transferase (GST) and catalase was also examined. The main pathway affected was the stress response showing overexpression of HSPs (HSC70.3, HSC70.4, HSC70.5, HSP60). Moreover, there was a reduction of the GSTd3 and catalase disrupting the antioxidant system. The upregulation of InR indicates a potential disturbance in the insulin pathway and ABCB6 activation only in TRPs exposure suggests its potential implication in their transport. However, most of these alterations are caused by TRLs, showing higher toxicity than TRPs. The results obtained in this work provide the first approach at the molecular and cellular levels to elucidate the impact of TRLs in freshwater organisms. To perform a realistic evaluation of the TR effects, additional research is required to assess the TR's long-term effects at the molecular level.

DNA damage and molecular level effects induced by polystyrene (PS) nanoplastics (NPs) after Chironomus riparius (Diptera) larvae.

In this work, we analyzed the early molecular effects of polystyrene (PS) nanoplastics (NPs) on an aquatic primary consumer (larvae of Chironomus riparius, Diptera) to evaluate their potential DNA damage and the transcriptional response of different genes related to cellular and oxidative stress, endocrine response, developmental, oxygen transport, and immune response. After 24-h exposures of larvae to doses of PS NPs close to those currently found in the environment, the results revealed a large genotoxic effect. This end was evidenced after significant increases in DNA strand breaks of C. riparius larvae quantified by the comet assay, together with results obtained when analyzing the expression of four genes involved in DNA repair (xrrc1, ATM, DECAY and NLK) and which were reduced in the presence of these nanomaterials. Consequently, this reduction trend is likely to prevent the repair of DNA damage caused by PS NPs. In addition, the same tendency to reduce the expression of genes involved in cellular stress, oxidative stress, ecdysone pathway, development, and oxygen transport was observed. Taken together, these results suggest that PS NPs reduce the expression of hormonal target genes and a developmental gene. We show, for the first time, effects of PS NPs on the endocrine system of C. riparius and suggest a possible mechanism of blocking ecdysteroid hormones in insects. Moreover, the NPs were able to inhibit the expression of hemoglobin (Hb C), a protein involved in oxygen transport, and activate a gene of the humoral immune system. These data reveal for the first time the genomic effects of PS NPs in the aquatic invertebrate C. riparius, at the base of the food chain.

Species-specific Microorganisms in Acid-tolerant Chironomus Larvae Reared in a Neutral pH Range under Laboratory Conditions: Single Dataset Analysis.

To obtain a more detailed understanding of organismal acid tolerance, the larval microbiomes of 11 Chironomus species collected from acidic or neutral pH areas in Japan and reared at pH 7-8 under laboratory conditions were systematically compared using an amplicon sequencing ana-lysis. Evenness values were lower for the larval microbiomes of acid-tolerant Chironomus cf. riparius, Chironomus fusciceps, and Chironomus sulfurosus than for eight acid-sensitive species based on an alpha diversity ana-lysis. The lower evenness observed suggested a biased abundance of microorganisms, which was consistent with the identification of Chironomus species-specific microorganisms (such as Agromyces mediolanus and Comamonas odontotermitis related bacteria) with high abundance in acid-tolerant larvae. The abundance of specific microorganisms was also high in the microbiome of acid-tolerant larvae of Chironomus acerbiphilus reared at pH 4, but not in that of acid-sensitive larvae. Based on a PICRUSt2 ana-lysis, genes involved in saccharide transport were less abundant in the microbiome of acid-tolerant larvae than in that of acid-sensitive larvae, indicating nutrient-poor acidic environments. Although these results were obtained from single datasets, acid-tolerant larvae appeared to establish Chironomus species-specific interactions with microorganisms independent of saccharides, in contrast to acid-sensitive larvae. The present study is the first step towards understanding organismal acid tolerance.

Oxidative stress is an essential factor for the induction of anhydrobiosis in the desiccation-tolerant midge, Polypedilum vanderplanki (Diptera, Chironomidae).

The sleeping chironomid (Polypedilum vanderplanki) is the only insect capable of surviving complete desiccation in an ametabolic state called anhydrobiosis. Here, we focused on the role of oxidative stress and we observed the production of reactive oxygen species (ROS) in desiccating larvae and in those exposed to salinity stress. Oxidative stress occurs to some extent in desiccating larvae, inducing carbonylation of proteins. Oxidative stress overcomes the antioxidant defenses of the larvae during the first hour following rehydration of anhydrobiotic larvae. It facilitates the oxidation of DNA and cell membrane lipids; however, these damages are quickly repaired after a few hours. In addition to its deleterious effects, we demonstrated that artificial exposure to oxidative stress could induce a response similar to desiccation stress, at the transcriptome and protein levels. Furthermore, the response of anhydrobiosis-related genes to desiccation and salinity stress was inhibited by antioxidant treatment. Thus, we conclude that oxidative stress is an essential trigger for inducing the expression of protective genes during the onset of anhydrobiosis in desiccating of P. vanderplanki larvae.

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.

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.

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.

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.

Surveillance spilled Chironomidae (Diptera) larvae from drinking water treatment plants in South Korea using morphogenetic species analysis and eDNA metabarcoding.

Chironomid larvae (Diptera: Chironomidae) are tremendous indicator species that can tolerate a broad range of environmental conditions, from polluted to unimpaired water ecosystems. These species are ubiquitously observed in all bioregions and can even be found in drinking water treatment plants (DWTPs). Detection of chironomid larvae in DWTPs is a critical issue because their presence may be indicative of the water quality in the supply of tap water for human consumption. Therefore, the aim of the present study was to identify the chironomid communities that reflect the water quality of DWTPs and develop a biomonitoring tool to detect biological contamination of the chironomids in DWTPs. To do so, we investigated the identity and distribution of chironomid larvae in seven DWTP areas using morphological identification, DNA barcoding, and sediment environmental DNA (eDNA) analysis. A total of 7924 chironomid individuals encompassing three subfamilies and 25 species of 19 genera were identified in 33 sites within the DWTPs. The Gongchon and Bupyeong DWTPs were dominated by Chironomus spp. larvae, which were correlated with low levels of dissolved oxygen in the water. In the Samgye DWTP and Hwajeong DWTP, Chironomus spp. were almost absent, and instead, Tanytarsus spp. were abundant. Additionally, the Gangjeong DWTP was dominated by a Microtendipes sp., and two species of Orthocladiinae (a Parametriocnemus sp. and a Paratrichocladius sp.) were found only in the Jeju DWTP. We also identified the eight most abundant Chironomidae larvae found in the DWTPs. Furthermore, eDNA metabarcoding of DWTP sediment indicated the presence of different eukaryotic fauna and confirmed the presence of chironomids in DWTPs. These data provide useful morphological and genetic information regarding chironomid larvae that can be used for the water quality biomonitoring of DWTPs to support the supply of clean drinking water.

Fauna and taxonomy of Diamesinae (Diptera, Chironomidae) from the Caucasus, with a morphological description and DNA barcoding of new taxa and a discussion of diagnostic problems for Diamesa Meigen and Pseudodiamesa Goetghebuer.

As a result of the revision of adult males as well as available literature data, 26 species of the subfamily Diamesinae are registered for the Caucasus, belonging to 5 genera. Four species are recorded for the first time for this region, one species, D. elbrusica sp. nov., and one subspecies, D. sakartvella gidanica subsp. nov., are new to science and are described. Six species are classified as endemics of the Caucasus. Distribution of other species of Caucasian Diamesinae is discussed. DNA barcodes of 102 specimens and 20 species of four genera, Boreoheptagyia Brundin, Diamesa Meigen, Pseudodiamesa Goetghebuer and Syndiamesa Kieffer were obtained in this study. Of these, 12 species were deposed in the GenBank and BOLD systems for the first time. We have established that D. cinerella group includes D. kasymovi and probably D. lavillei whereas D. zernyi group includes D. vaillanti and D. valentinae. Highly supported phylogeny and results of species delimitation suggest the description of D. elbrusica sp. nov. and D. sakartvella gidanica subsp. nov. Ps. aff. branickii and Ps. aff. nivosa are new species based on DNA barcoding. The results of species delimitation show that genus Pseudodiamesa includes 10 (ASAP, GMYC), 14 (mPTP) or 21 (BOLD) distinct molecular taxonomic units (mOTUs) among which only Ps. stackelbergi have an undoubted species status that requires a large revision using both morphological and molecular approaches.

DNA Barcode of symbiotic chironomids: Findings in the genus Symbiocladius (Diptera: Chironomidae).

Chironomidae of symbiotic habits have been recorded in different parts of the world, among commensals and parasites. There are different genera reported at the moment, however questions such as the origin of commensal or parasitic life, which occurred first or what are their benefits remain debatable. In order to contribute with information to elucidate the above mentioned issues, the present study reports the finding of immature stages of Symbiocladius (Acletus) wygodzinskyi Roback, 1965 in the Churup stream located in the Andes Cordillera (Peru), living on nymphs of Leptophlebiidae (Ephemeroptera). We present a morphological description of immature stages of this species and for the first time the sequence of COX1 gene S. (A.) wygodzinskyi. The genetic result also supports differences between the morphospecies of Symbiocladius (Symbiocladius) rhithrogenae Zavrel, 1924 and S. (A.) wygodzinskyi in 23%.

Comparative mitogenome analyses of twelve non-biting flies and provide insights into the phylogeny of Chironomidae (Diptera: Culicomorpha).

The family Chironomidae is represented by seven subfamilies in China, among which Chironominae and Orthocladiinae are the most diverse. To gain a better understanding of the architecture and evolution of the mitogenomes of Chironomidae, we sequenced mitogenomes of twelve species (including two published species) of the two subfamilies Chironominae and Orthocladiinae, and comparative mitogenomic analyses were performed. Thus, we identified highly conserved genome organization of twelve species with regard to genome content, nucleotide and amino acid composition, codon usage, and gene characteristics. The Ka/Ks values of most protein-coding genes were far smaller than 1, indicating that these genes were evolving under purifying selection. Phylogenetic relationships between the family Chironomidae were reconstructed using 23 species representing six subfamilies, based on protein-coding genes and rRNAs using Bayesian Inference and Maximum Likelihood. Our results suggested the following relationship within the Chironomidae: (Podonominae + Tanypodinae) + (Diamesinae + (Prodiamesinae + (Orthocladiinae + Chironominae))). This study contributes to the mitogenomic database of Chironomidae, which will be significant for studing the mitogenome evolution of Chironomidae.