Genome-wide annotation and comparative analysis revealed conserved cuticular protein evolution among non-biting midges with varied environmental adaptability.

Chironomidae, non-biting midges, a diverse and abundant insect group in global aquatic ecosystems, represent an exceptional model for investigating genetic adaptability mechanisms in aquatic insects due to their extensive species diversity and resilience to various environmental conditions. The cuticle in insects acts as the primary defense against ecological pressures. Cuticular Proteins (CPs) determine cuticle characteristics, facilitating adaptation to diverse challenges. However, systematic annotation of CP genes has only been conducted for one Chironomidae species, Propsilocerus akamusi, by our team. In this study, we expanded this annotation by identifying CP genes in eight additional Chironomidae species, covering all Chironomidae species with available genome data. We identified a total of 889 CP genes, neatly categorized into nine CP families: 215 CPR RR1 genes, 272 CPR RR2 genes, 23 CPR RR3 genes, 21 CPF genes, 16 CPLCA genes, 19 CPLCG genes, 28 CPLCP genes, 77 CPAP genes, and 37 Tweedle genes. Subsequently, we conducted a comprehensive phylogenetic analysis of CPs within the Chironomidae family. This expanded annotation of CP genes across diverse Chironomidae species significantly contributes to our understanding of their remarkable adaptability.

Six new species of Cryptochironomus Kieffer (Diptera, Chironomidae) from the Nearctic region.

Six new species of Cryptochironomus Kieffer, 1918, C.absum Liu, sp. nov., C.beardi Liu, sp. nov., C.dentatus Liu, sp. nov., C.ferringtoni Liu, sp. nov., C.parallelus Liu, sp. nov. and C.taylorensis Liu, sp. nov., are described and illustrated based on adult males. The specimens were collected from various water systems in the United States and preserved by Dr. Leonard Charles Ferrington Jr. An updated key to adult males of all known Cryptochironomus species in the Nearctic region is also provided.

New descriptions of the larval and pupal stages of Orthocladiusnitidoscutellatus and Psectrocladiusnevalis from Xizang, China (Diptera, Chironomidae).

Background: Tibetan Plateau is one of the most typical areas of biodiversity in the world because of its unique environmental and regional units, which breed unique biological communities and concentrate on many unique and rare wild animals and plants. Research on Chironomidae in the Tibetan Plateau is relatively weak. At present, the identification of Chironomidae species mainly depends on male adults, while identification of larvae and pupae is relatively difficult and there is less research on them. New information: During the investigations of insect diversity in the Tibetan Plateau, larval and pupal stages of Orthocladiusnitidoscutellatus Lundström, 1915 and Psectrocladiusnevalis Akhrorov, 1977 were described and illustrated. Matching and identification of larval and pupal stages were based on DNA barcodes. Neighbour-joining trees were reconstructed, based on known Orthocladius and Psectrocladius COI DNA barcodes, respectively.

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.

Genome-wide analyses of Glutathione S-transferase gene family and expression profiling under deltamethrin exposure in non-biting midge Propsilocerus akamusi.

Glutathione S-transferases (GSTs) are major enzymes in detoxification phase II, and have been functioned in resistance to various insecticides or oxidative stress. Herein, we selected the non-biting midge, Propsilocerus akamusi, widespread in Asian aquatic ecosystems, to uncover the gene location, structure, and phylogenetics relationship of GSTs at genome scale first time. Thirty-three cytosolic and four microsomal GST genes were identified and located on the four chromosomes. The cytosolic GSTs involved in the eight subclasses and five GST genes were unclassified. The expansion of GST genes in P. akamusi experienced duplication events on the delta, theta, xi, iota, and unclassified subclasses. The RNA-Seq analyses and RT-qPCR validation showed that the expression of PaGSTt2 gene is significantly elevated, with deltamethrin concentration increasing. The tertiary structure of PaGSTt2 enzyme was reconstructed, which was different from the other theta gene in the active site. In addition, the GST genes of six chironomids were first described based on the assembled genomes to explore the difference of those in the adaptation to kinds of environments. The GST frame for P. akmusi and its expression profiles provide valuable resources to understand their role in insecticide resistance of this species, as well as those of other biting midges.

New species, a new combination, and DNA barcodes of Parachironomus Lenz, 1921 (Diptera, Chironomidae).

The genus Parachironomus has a cosmopolitan distribution including 85 valid described species worldwide. Species records and studies of the genus in the Tibetan Plateau are scarce. In this study, the genus Parachironomus from China is revised and two new species, Parachironomuswangi Liu & Lin, sp. nov. and Parachironomusnankaiensis Liu & Lin, sp. nov., are described based on adult morphology and molecular data. Paracladopelmademissum Yan, Wang & Bu is placed in the genus Parachironomus as a new combination. A neighbor-joining tree was reconstructed based on all known ParachironomusCOI DNA barcodes. A key to adult males of the genus Parachironomus from China is also provided.

Genome-wide annotation of cuticular protein genes in non-biting midge Propsilocerus akamusi and transcriptome analysis of their response to heavy metal pollution.

The insect cuticle is a sophisticated chitin-protein extracellular structure for mutable functions. The cuticles varied their structures and properties in different species, and the same species but in different regions or at different stages, to fill the requirements of different functions. The alteration of cuticle structures may also be induced due to challenges by some environmental crises, such as pollution exposures. The physical properties of the cuticle were determined by the cuticle proteins (CPs) they contain. The cuticle proteins are large protein groups in all insects, which are commonly divided into different families according to their conserved protein sequence motifs. Although Chironomidae is an abundant and universal insect in global aquatic ecosystems and a popular model for aquatic toxicology, no systematic annotation of CPs was done for any species in Chironomidae before. In this work, we annotated the CP genes of Propsilocerus akamusi, the most abundant Chironomidae species in Asia. A total of 160 CP genes were identified, and 97 of them could be well classified into eight CP families: 76 CPR genes can be subdivided into three groups (further divided into three subgroups: 36 RR1 genes, 37 RR2 genes, and 3 RR3 genes), 2 CPF genes, 3 CPLCA genes, 1 CPLCG gene, 8 CPAP genes, and 3 Tweedle genes. Additionally, we analyzed the response of P. akamusi CP genes at expression level to Cu exposure, which is related to the high heavy metal tolerance and the earlier onset of pupariation in heavy metal polluted water.

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.

Genome-Wide Identification of P450 Genes in Chironomid Propsilocerus akamusi Reveals Candidate Genes Involved in Gut Microbiota-Mediated Detoxification of Chlorpyrifos.

Chironomids commonly dominate macroinvertebrate assemblages in aquatic habitats and these non-biting midges are known for their ability to tolerate contaminants. Studies regarding the interplay between gut microbiota and host detoxification ability is currently a point of interest. Cytochrome P450s (P450s) are critical metabolic enzymes in which a subset is involved in xenobiotic detoxification. In this study, we first conducted an integrated global investigation of P450s based on the whole genomic sequence of Propsilocerus akamusi and retrieved a series of 64 P450 genes which were further classified into 4 clans and 25 families on the basis of phylogenetic relationships. With assistance of RNA-Seq and RT-qPCR validation, the expression profile of screened PaP450s in guts was compared between chlorpyrifos-challenged larvae with deficient gut microbiota (GD) and those with a conventional gut community (CV). An increasing prevalence of chlorpyrifos from sublethal to lethal dosages induced a greater mortality rate of individuals coupled with remarkable downregulation of 14 P450s in GD larval guts when compared to CV ones. Moreover, it turned out that the decreased level of PaCYP3998B1 and PaCYP3987D1 might imply impaired host endogenous detoxification capability potentiated by gut dysbiosis, reflected by a remarkably severe mortality in GD larvae treated with lethal chlorpyrifos. Collectively, our study unveiled candidate P450 genes that might be mediated by gut symbionts in chlorpyrifos-challenged P. akamusi larvae, possibly facilitating further understanding of the detoxified mechanism that chironomids might employ to alleviate poisonousness.

Contribution to the knowledge of Rheotanytarsus pellucidus species group from China (Diptera, Chironomidae): three new and one newly recorded species.

Three new species, Rheotanytarsus ashei Yao & Liu sp. n., Rheotanytarsus pinderi Yao & Liu sp. n. and Rheotanytarsus vallenduuki Yao & Liu sp. n. are described and illustrated as adult males. Rheotanytarsus oss Cranston, 1997 is newly recorded and re-described from China. An updated key to males of Rheotanytarsus pellucidus species group is presented.

Compterosmittia wangi sp. nov. from Oriental China (Diptera, Chironomidae).

A new species, Compterosmittia wangi Liu sp. nov. is described and illustrated as adult males. An updated key to males of Compterosmittia worldwide is presented.

Genome-wide analyses of ATP-Binding Cassette (ABC) transporter gene family and its expression profile related to deltamethrin tolerance in non-biting midge Propsilocerus akamusi.

Non-biting midges are dominant species in aquatic systems and often used for studying the toxicological researches of insecticides. ATP-binding cassette (ABC) transporters represent the largest known members in detoxification genes but is little known about their function in non-biting midges. Here, we selected Propsilocerus akamusi, widespread in urban streams, to first uncover the gene structure, location, characteristics, and phylogenetics of chironomid ABC transporters at genome-scale. Fifty-seven ABC transporter genes are located on four chromosomes, including eight subfamilies (ABCA-H). The ABCC, ABCG, and ABCH subfamilies experienced the duplication events to different degrees. The study showed that expression of the PaABCG17 gene is uniquely significantly elevated, with deltamethrin concentration increasing (1, 4, and 20 ug/L) both in RNA-seq and qPCR results. Additionally, the ABC transporter members of other six chironomids with assembled genomes are first described and used to investigate the characteristic of those living in the different adverse habitats. The ABC transporter frame for Propsilocerus akamusi and its transcriptomic results lay an important foundation for providing valuable resources for understanding the ABC transporter function in insecticide toxification of this species as well as those of other non-biting midges. The PaABCG17 gene is shown to play an important role in deltamethrin detoxification, and it functions need to be further investigated and might be used in the management of insecticide-resistance in chironomid adults.

New or little-known Boreoheptagyia (Diptera, Chironomidae) in China inferred from morphology and DNA barcodes.

The male adult of Boreoheptagyia zhengi Lin & Liu, sp. nov. is described and illustrated based on material collected in China. Associated morphological characteristics and reference to its DNA barcode are provided. Boreoheptagyia kurobebrevis (Sasa & Okazawa, 1992) is newly recorded from China based on both a male and female, with additional associated data on the DNA barcode of the male adult. A neighbor-joining tree based on available Boreoheptagyia DNA barcodes and a key to the adults of Boreoheptagyia from China are given.

Stenochironomus zhengi sp. nov. from Dawei Mountain National Park, Yunnan, China (Diptera: Chironomidae).

A new species, Stenochironomus zhengi Lin Liu sp. nov., is described and illustrated as adult males. An updated key to males of Stenochironomus from China is presented.

Molecular characterization of a novel p38 MAPK cDNA from Cyclina sinensis and its potential immune-related function under the threat of Vibrio anguillarum.

The p38 mitogen-activated protein kinase (MAPK) is one important member of MAPK family and reported to serve a predominant function in regulating innate immunity after the occurrence of certain infection. In the present study, one novel p38 MAPK gene was acquired from Cyclina sinensis based on the RNA-seq analysis and designated as Csp38 MAPK. This novel gene contained a full length of 1781 bp, 1104 bp of which was deemed as open reading frames and gave rise to a peptide of 367 amino acids with a predicted molecular weight of 42.31 KDa. A conserved serine/threonine protein kinase (S_Tkc) region along with a Thr-Gly-Tyr motif was discovered in the deduced sequence. According to the phylogenetic analysis, there was a close relationship between this kinase and Meretrix petechialis p38 MAPK. As for the expression pattern, this newly-identified Csp38 MAPK was ubiquitously distributed in several tissues throughout the body but with varied abundance. After the challenge of Vibrio anguillarum, both the transcription and phosphorylation level of Csp38 MAPK in hemolymph were coordinately altered with a time-dependent manner. Besides, with the application of double strand RNA homologous to myeloid differentiation factor 88 (MyD88) of C. sinensis, the activation of Csp38 MAPK was found to obviously decrease in hemolymph after the pathogen stimulation. Hence, our experimental data presented evidence for the potential involvement of p38 MAPK in response to bacterial invaders in C. sinensis, possibly facilitating the understanding for pathogen-induced innate immunity in clams.

A chromosome level genome assembly of Propsilocerus akamusi to understand its response to heavy metal exposure.

Chironomidae species are universally used for studying the impact of pollutants in aquatic systems. The nonbiting midge Propsilocerus akamusi is often found in urban streams and is suitable for use as a toxicological bioindicator. However, few studies have previously examined metal stress in this species. We sequenced the genome of this urban midge to address this question. Here, we present the first chromosome-level genome of P. akamusi, obtained from Illumina short-read and PacBio long-read sequences with Hi-C technology. The size of the very small assembled genome was 85.83 Mb with a contig N50 of 6.2 Mb and a scaffold N50 of 26.1 Mb. This assembly revealed significant expansion of haemoglobin (Hb) genes, some of which formed large tandem repeats. Transcriptomic studies for copper tolerance identified four genes in the tandem array that were highly expressed, all of which presented intron loss. This characteristic might highlight the potential role of Hb genes in copper tolerance. Additionally, detoxification genes, chemosensory genes and heat shock protein genes of this midge were identified, some of which are associated with metal stress. The high-quality assembled genome of P. akamusi and the transcriptomic analyses provide new insight into the molecular mechanisms of heavy metal stress. Our comparison of the P. akamusi genome with other dipteran genomes provides valuable resources for understanding the evolutionary history, genetics, and ecology of this species as well as those of other midges.

New species of Limnophyes Eaton (Diptera, Chironomidae) from China and synonymy of L. fuscipygmus Tokunaga, 1940.

Two new species, L. minerus Liu & Yan, sp. nov. and L. subtilus Liu & Yan, sp. nov. are described and illustrated as adult males. Limnophyes minimus (Meigen, 1818) is assigned as a senior synonym of L. minerus Tokunaga, 1940. A key to males of Limnophyes from China is presented.

A new species of Polypedilum (Cerobregma) (Diptera, Chironomidae) from Oriental China.

Polypedilum (Cerobregma) huapingensis Liu & Lin, sp. nov. is described and illustrated based on an adult male from Huaping National Nature Reserve, Guangxi, China. A DNA barcode analysis, including the known partial COI sequences of species in the Cerobregma subgenus, was conducted. An updated key to adult males of the subgenus Cerobregma is provided.

Identification and expression pattern of chemosensory genes in the transcriptome of Propsilocerus akamusi.

Chironomidae is the most ecologically diverse insects in aquatic and semi-aquatic habitats. Propsilocerus akamusi (Tokunaga) is a dominant and ubiquitous chironomid species in Eastern Asia and its morphologically unique larvae are also considered as indicator organisms to detect water contamination, potential toxicity and waterborne pathogens. Since few studies to date have focused on the olfactory system of P. akamusi, our study aims to elucidate the potential functions of chemosensory genes in P. akamusi. In our study, we found that although signals released from male groups might attract female swarmers, it was a completely male-dominated mating process. Sequencing the transcriptome of P. akamusi on an Illumina HiSeq platform generated 4.42, 4.46 and 4.53 Gb of clean reads for heads, legs, and antennae, respectively. 27,609 unigenes, 20,379 coding sequences (CDSs), and 8,073 simple sequence repeats were finally obtained. The gene-level differential expression analysis demonstrated variants among three different tissues, including 2,019 genes specifically expressed in heads, 1,540 genes in legs, and 2,071 genes in antennae. Additionally, we identified an assortment of putative olfactory genes consisting of 34 odorant binding proteins, 17 odorant receptors, 32 gustatory receptors, 22 ionotropic receptors, six chemosensory proteins as well as 3 sensory neuron membrane proteins; their relative abundances in the above three tissues were also determined by RT-qPCR. Our finding could allow a more plausible understanding of certain olfaction-mediated behaviors in groups of this macroinvertebrate.