Phylogenomic data exploration with increased sampling provides new insights into the higher-level relationships of butterflies and moths (Lepidoptera).

A robust and stable phylogenetic framework is a fundamental goal of evolutionary biology. As the third largest insect order in the world following Coleoptera and Diptera, Lepidoptera (butterflies and moths) play a central role in almost every terrestrial ecosystem as indicators of environmental change and serve as important models for biologists exploring questions related to ecology and evolutionary biology. However, for such a charismatic insect group, the higher-level phylogenetic relationships among its superfamilies are still poorly resolved. Compared to earlier phylogenomic studies, we increased taxon sampling among Lepidoptera (37 superfamilies and 68 families containing 263 taxa) and acquired a series of large amino-acid datasets from 69,680 to 400,330 for phylogenomic reconstructions. Using these datasets, we explored the effect of different taxon sampling with significant increases in the number of included genes on tree topology by considering a series of systematic errors using maximum-likelihood (ML) and Bayesian inference (BI) methods. Moreover, we also tested the effectiveness in topology robustness among the three ML-based models. The results showed that taxon sampling is an important determinant in tree robustness of accurate lepidopteran phylogenetic estimation. Long-branch attraction (LBA) caused by site-wise heterogeneity is a significant source of bias giving rise to unstable positions of ditrysian groups in phylogenomic reconstruction. Phylogenetic inference showed the most comprehensive framework to reveal the relationships among lepidopteran superfamilies, and presented some newly relationships with strong supports (Papilionoidea was sister to Gelechioidea and Immoidea was sister to Galacticoidea, respectively), but limited by taxon sampling, the relationships within the species-rich and relatively rapid radiation Ditrysia and especially Apoditrysia remain poorly resolved, which need to increase taxon sampling for further phylogenomic reconstruction. The present study demonstrates that taxon sampling is an important determinant for an accurate lepidopteran tree of life and provides some essential insights for future lepidopteran phylogenomic studies.

Interception Proximity Labeling for Interrogating Cell Efflux Microenvironment.

The difficulty in elucidating the microenvironment of extracellular H2O2 efflux has led to the lack of a critical extracellular link in studies of the mechanisms of redox signaling pathways. Herein, we mounted horseradish peroxidase (HRP) to glycans expressed globally on the living cell surface and constructed an interception proximity labeling (IPL) platform for H2O2 efflux. The release of endogenous H2O2 is used as a "physiological switch" for HRP to enable proximity labeling. Using this platform, we visualize the oxidative stress state of tumor cells under the condition of nutrient withdrawal, as well as that of macrophages exposed to nonparticulate stimuli. Furthermore, in combination with a proteomics technique, we identify candidate proteins at the invasion interface between fungal mimics (zymosan) and macrophages by interception labeling of locally accumulated H2O2 and confirm that Toll-like receptor 2 binds zymosan in a glycan-dependent manner. The IPL platform has great potential to elucidate the mechanisms underlying biological processes involving redox pathways.

Traceless Protein-Selective Glycan Labeling and Chemical Modification.

Executing glycan editing at a molecular level not only is pivotal for the elucidation of complicated mechanisms involved in glycan-relevant biological processes but also provides a promising solution to potentiate disease therapy. However, the precision control of glycan modification or glyco-editing on a selected glycoprotein is by far a grand challenge. Of note is to preserve the intact cellular glycan landscape, which is preserved after editing events are completed. We report herein a versatile, traceless glycan modification methodology for customizing the glycoforms of targeted proteins (subtypes), by orchestrating chemical- and photoregulation in a protein-selective glycoenzymatic system. This method relies on a three-module, ligand-photocleavable linker-glycoenzyme (L-P-G) conjugate. We demonstrated that RGD- or synthetic carbohydrate ligand-containing conjugates (RPG and SPG) would not activate until after the ligand-receptor interaction is accomplished (chemical regulation). RPG and SPG can both release the glycoenzyme upon photoillumination (photoregulation). The adjustable glycoenzyme activity, combined with ligand recognition selectivity, minimizes unnecessary glycan editing perturbation, and photolytic cleavage enables precise temporal control of editing events. An altered target protein turnover and dimerization were observed in our system, emphasizing the significance of preserving the native physiological niche of a particular protein when precise modification on the carbohydrate epitope occurs.

OCTN2 enhances PGC-1α-mediated fatty acid oxidation and OXPHOS to support stemness in hepatocellular carcinoma.

BACKGROUND: The Metabolic reprogramming of tumor cells plays a vital role in the progression of hepatocellular carcinoma. Organic cation/carnitine transporter 2 (OCTN2), a sodium-ion dependent carnitine transporter and a sodium-ion independent tetraethylammonium (TEA) transporter, has been reported to contribute tumor malignancies and metabolic dysregulation in renal and esophageal carcinoma. However, the role of lipid metabolism deregulation mediated by OCTN2 in HCC cells has not been clarified. METHODS: Bioinformatics analyses and immunohistochemistry assay were employed to identify OCTN2 expression in HCC tissues. The correlation between OCTN2 expression and prognosis was elucidated through K-M survival analysis. The expression and function of OCTN2 were examined via the assays of western blotting, sphere formation, cell proliferation, migration and invasion. The mechanism of OCTN2-mediated HCC malignancies was investigated through RNA-seq and metabolomic analyses. Furthermore, xenograft tumor models based on HCC cells with different OCTN2 expression levels were conducted to analyze the tumorigenic and targetable role of OCTN2 in vivo. RESULTS: We found that gradually focused OCTN2 was significantly upregulated in HCC and tightly associated with poor prognosis. Additionally, OCTN2 upregulation promoted HCC cells proliferation and migration in vitro and augmented the growth and metastasis of HCC. Moreover, OCTN2 promoted the cancer stem-like properties of HCC by increasing fatty acid oxidation and oxidative phosphorylation. Mechanistically, PGC-1α signaling participated in the HCC cancer stem-like properties mediated by OCTN2 overexpression, which is confirmed by in vitro and in vivo analyses. Furthermore, OCTN2 upregulation may be transcriptionally activated by YY1 in HCC. Particularly, treatment with mildronate, an inhibitor of OCTN2, showed a therapeutic influence on HCC in vitro and in vivo. CONCLUSIONS: Our findings demonstrate that OCTN2 plays a critical metabolic role in HCC cancer stemness maintenance and HCC progression, providing evidence for OCTN2 as a promising target for HCC therapy.

Mitogenomic phylogenetic analyses provide novel insights into the taxonomic problems of several hesperiid taxa (Lepidoptera: Hesperiidae).

Here, we present new molecular and morphological evidence that contributes towards clarifying the phylogenetic relations within the family Hesperiidae, and overcomes taxonomic problems regarding this family. First, nine new complete mitogenomes, comprising seven newly sequenced species and two samples of previously sequenced species collected from different localities, were obtained and assembled to analyze characteristics. The length of the mitogenomes ranges from 15,284 to 15,853 bp and encodes 13 protein-coding genes, two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and a control region. Two model-based methods (maximum likelihood and Bayesian inference) were used to infer the phylogenetic relationships. Based on the mitogenomic phylogenetic analyses and morphological evidence, we claim that the lineage that comprises two Asian genera, Apostictopterus Leech and Barca de Nicéville, should be a tribe Barcini stat. nov. of the subfamily Trapezitinae, Pseudocoladenia dea (Leech, 1894), P. festa (Evans, 1949), and Abraximorpha esta Evans, 1949 are considered distinct species. Finally, we suggest that Lotongus saralus chinensis Evans, 1932 should belong to the genus Acerbas de Nicéville, 1895, namely Acerbas saralus chinensis (Evans, 1932) comb. nov..

Protein-Targeted Glycan Editing on Living Cells Disrupts KRAS Signaling.

The frequent mutation of KRAS oncogene in some of the most lethal human cancers has spurred incredible efforts to develop KRAS inhibitors, yet only one covalent inhibitor for the KRASG12C mutant has been approved to date. New venues to interfere with KRAS signaling are desperately needed. Here, we report a "localized oxidation-coupling" strategy to achieve protein-specific glycan editing on living cells for disrupting KRAS signaling. This glycan remodeling method exhibits excellent protein and sugar specificity and is applicable to different donor sugars and cell types. Attachment of mannotriose to the terminal galactose/N-acetyl-D-galactosamine epitopes of integrin αv ß3 , a membrane receptor upstream of KRAS, blocks its binding to galectin-3, suppresses the activation of KRAS and downstream effectors, and mitigates KRAS-driven malignant phenotypes. Our work represents the first successful attempt to interfere with KRAS activity by manipulating membrane receptor glycosylation.

Cell-Selective Multifunctional Surface Covalent Reconfiguration Using Aptamer-Enabled Proximity Catalytic Labeling.

Cell surface engineering provides access to custom-made cell interfaces with desirable properties and functions. However, cell-selective covalent labeling methods that can simultaneously install multiple molecules with different functions are scarce. Herein, we report an aptamer-enabled proximity catalytic covalent labeling platform for multifunctional surface reconfiguration of target cells in mixed cell populations. By conjugating peroxidase with cell-selective aptamers, the probes formed can selectively bind target cells and catalyze target-cell-localized covalent labeling in situ. The universal applicability of the platform to different phenol-modified functional molecules allows us to perform a variety of manipulations on target cells, including labeling, tracking, assembly regulation, and surface remodeling. In particular, the platform has the ability of multiplexed covalent labeling, which can be used to install two mutually orthogonal click reactive molecules simultaneously on the surface of target cells. We thus achieve "multitasking" in complex multicellular systems: programming and tracking specific cell-cell interactions. We further extend the functional molecules to carbohydrates and perform ultrafast neoglycosylation on target living cells. These newly introduced sugars on the cell membrane can be recognized and remodeled by a glycan-modifying enzyme, thus providing a method package for cell-selective engineering of the glycocalyx.

First record of the family Neopseustidae from Yunnan, China with description of a new species (Lepidoptera, Neopseustidae).

The family Neopseustidae is reported from Yunnan Province, Southwest China, for the first time, with a new species, N. gaoligongensis Huang & Chen, sp. n., described from Mt. Gaoligong, West Yunnan, based on molecular and morphological evidence. The new species was found to belong to the N. bicornuta species-group and is closely related to N. fanjingshana Yang, 1988, but can be distinguished from it by the differences in morphology and a 3.2% COI distance. Adults and genitalia of the new species and N. fanjingshana are illustrated, and the molecular phylogeny based on COI barcoding of the genus Neopseustis is also updated.

A new species of the genus Ivela Swinhoe (Lepidoptera, Erebidae, Lymantriinae) from Guangdong, China.

Ivelayini sp. nov., is described from Guangdong, China based on morphological characters and molecular data. Adults, including genitalia and wing venation, and pupa are illustrated and compared to those of similar species. A key to Chinese Ivela species is provided. Assignment of the new species to Ivela Swinhoe is based primarily on a molecular phylogenetic analysis and is corroborated by morphology. Life histories of I.yini and Dendrophlepssemihyalina Hampson are discussed.

Mechanism of cancer stemness maintenance in human liver cancer.

Primary liver cancer mainly includes the following four types: hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), hepatoblastoma (HB), and combined hepatocellular carcinoma and cholangiocarcinoma (cHCC-CCA). Recent studies have indicated that there are differences in cancer stem cell (CSC) properties among different types of liver cancer. Liver cancer stem cells (LCSCs), also called liver tumor-initiating cells, have been viewed as drivers of tumor initiation and metastasis. Many mechanisms and factors, such as mitophagy, mitochondrial dynamics, epigenetic modifications, the tumor microenvironment, and tumor plasticity, are involved in the regulation of cancer stemness in liver cancer. In this review, we analyze cancer stemness in different liver cancer types. Moreover, we further evaluate the mechanism of cancer stemness maintenance of LCSCs and discuss promising treatments for eradicating LCSCs.

Downregulation of TRPC4 and TRPC5 Inhibits Smooth Muscle Cell Proliferation without Affecting Endothelial Cell Proliferation.

Aims: The main treatment for coronary heart disease is percutaneous coronary intervention (PCI), and drug-eluting stents are designed to inhibit vascular smooth muscle cell (VSMCs) proliferation and migration causing restenosis by releasing pharmacological agents into the vessel wall. Once drug-eluting stents are deployed, these pharmacological agents exert many biological effects in the coronary circulation, not only inhibition of VSMCs but also extension to vascular endothelial cells (VECs). The purpose of this study was to explore target molecules that inhibit VSMCs proliferation without affecting VECs. Methods: mRNA and protein expressions of transient receptor potential channels (TRPCs) in cultured VSMCs and VECs were determined by western blotting and RT-qPCR. VSMCs and VECs proliferation was evaluated using CCK-8 assays and western blotting of proliferating cell nuclear antigen (PCNA). Calcium backfilling assays were performed to detect intracellular calcium ion concentration in cultured VSMCs and VECs. Results: The TRPC6 expression was more abundant in VECs than VSMCs, while TRPC4 and TRPC5 expressions were more abundant in VSMCs than VECs. Knockdown of TRPC4 or TRPC5 alone had no remarkable inhibitory effect on VSMC proliferation. Synergistic knockdown of TRPC4 and TRPC5 inhibited the proliferation of VSMCs, declined the expression of the PCNA, and reduced the intracellular calcium ion concentration but not VECs. Conclusion: These data suggest that concurrent inhibition of TRPC4 and TRPC5 inhibits VSMCs proliferation without affecting VECs, thus providing novel targets for developing pharmacological agents for drug-eluting stents.

68Ga-Labeled GX1 Dimer: A Novel Probe for PET/Cerenkov Imaging Targeting Gastric Cancer.

PURPOSE: To synthesize the dimer of GX1 and identify whether its affinity and targeting are better than those of GX1. To prepare 68Ga-DOTA-KEK-(GX1)2 and to apply it to PET and Cerenkov imaging of gastric cancer. METHODS: 68Ga-DOTA-KEK-(GX1)2 was prepared, and the labeling yield and stability were determined. Its specificity and affinity were verified using an in vitro cell binding assay and competitive inhibition test, cell immunofluorescence, and cell uptake and efflux study. Its tumor-targeting ability was determined by nano PET/CT and Cerenkov imaging, standardized uptake value (SUV), signal-to-background ratio (SBR) quantification, and a biodistribution study in tumor-bearing nude mice. RESULTS: 68Ga-DOTA-KEK-(GX1)2 was successfully prepared, and the labeling yield was more than 97%. It existed stably for 90 min in serum. The binding of 68Ga-DOTA-KEK-(GX1)2 to cocultured HUVECs (Co-HUVECs) was higher than that to human umbilical vein endothelial cells (HUVECs), BGC823 cells, and GES cells. It was also higher than that of 68Ga-DOTA-GX1, indicating that the dimer did improve the specificity and affinity of GX1. The binding of KEK-(GX1)2 to Co-HUVECs was significantly higher than that of GX1. Additionally, the uptake of 68Ga-DOTA-KEK-(GX1)2 by Co-HUVECs was higher than that of 68Ga-DOTA-GX1 and reached a maximum at 60 min. Nano PET/CT and Cerenkov imaging showed that the tumor imaging of the nude mice injected with 68Ga-DOTA-KEK-(GX1)2 was clear, and the SUV and SBR value of the tumor sites were significantly higher than those of the nude mice injected with 68Ga-DOTA-GX1, indicating that the probe had better targeting in vivo. Finally, the biodistribution showed quantitatively that when organs such as the kidney and liver metabolized rapidly, the radioactivity of the tumor site of the nude mice injected with 68Ga-DOTA-KEK-(GX1)2 decreased relatively slowly. At the same time, the percentage of injected dose per gram (%ID/g) of the tumor site was higher than that of other normal organs except the liver and kidney at 60 min, which indicated that the tumor had good absorption of the probe. CONCLUSION: GX1 was modified successfully, and the in vivo and in vitro properties of the GX1 dimer were significantly better than those of GX1. The imaging probe, 68Ga-DOTA-KEK-(GX1)2, was successfully prepared, which provides a candidate probe for PET and Cerenkov diagnosis of gastric cancer.

Hierarchical Fluorescence Imaging Strategy for Assessment of the Sialylation Level of Lipid Rafts on the Cell Membrane.

Glycosylation is one of the most ubiquitous and complicated modifications of proteins and lipids. The revelation of glycosylation-mediated regulation mechanisms of biological processes relies critically on the tools that can reflect the spatial heterogeneity of cell surface glycans, for example, distinguishing glycans exhibited in lipid raft or nonraft domains. To achieve simultaneous visualization of raft and raft-harbored glycans on the cell surface, we combine specific raft recognition, glycan chemoselective labeling, and DNA dynamic hybridization techniques to develop a hierarchical fluorescence imaging strategy using N-acetyl-neuraminic acid (Sia) as the model sugar. We fabricate a raft probe and Sia probe for rafts and Sia, respectively. After specifically anchoring the two probes on the cell surface, the raft probe can be cyclically utilized to turn on the fluorescence of the Sia probe, only residing in rafts, via a proximity cascade DNA reaction. The duplex imaging capability for spatially relevant levels of biological structures enables the revelation of the reason for raft-confined Sia variation in different biological processes. Thus, this work provides an elegant and powerful tool for interrogation of the glycan regulation mechanisms on raft composition, organization, and functions and also contributes to the development of raft-carried glycoconjugate-based theranostic techniques.

Notes on the genus Neopseustis Meyrick with description of a new species from China (Lepidoptera: Neopseustidae).

A new species, Neopseustis rectagnatha Liao, Chen Huang sp. nov., is described from the Nanling Mountains, China. The photos of the adults, male and female genitalia, and abdominal special structures are presented. The new species is similar to N. meyricki Hering, 1925 and N. sinensis Davis, 1975, but it can be distinguished easily by characters in the male genitalia as the straight gnathos and the lateroposterior process of anellus h-type bifurcated. Key to males of all species of Neopseustis Meyrick, 1909 and some biological information are also given. Molecular analysis based on COI sequences is used to infer the relationship of the new species within Neopseustis.

?Description of a new species of the genus Neopseustis Meyrick, 1909 from China, with a new classification of the genus (Lepidoptera, Neopseustoidea, Neopseustidae).

A new species of the genus Neopseustis Meyrick, 1909, Neopseustischentangensis S.Y. Huang & Chen sp. nov., which was confirmed by both morphological and molecular methods, is described from Xizang, China. This is currently the westernmost species in Asia of the primitive lepidopteran family Neopseustidae. The new species is externally reminiscent of N.moxiensis Chen & Owada, 2009; however, it can be easily distinguished from the latter by comparison of the male genitalia and is further distinguished by the large genetic distance in DNA barcodes (COI). The adult and genitalia of the new and similar species have been illustrated. Utilizing our new data, a new classification of the genus is provided, with its members subdivided into four species groups: the meyricki-group, the moxiensis-group, the bicornuta-group, and the chentangensis-group, which are supported by both molecular and morphological evidence. A checklist of the genus and a key to the species groups are also provided.

Intermittent versus continuous enteral nutrition on feeding intolerance in critically ill adults: A meta-analysis of randomized controlled trials.

OBJECTIVES: Enteral formula delivery strategy is an important part of enteral nutrition. We aimed to synthesize up-to-date studies to clarify the effects of intermittent versus continuous feeding on feeding intolerance during enteral nutrition in critically ill adults. DESIGN: A meta-analysis of randomized controlled trials. DATA SOURCES: Embase, PubMed, Information Sciences Institute Web of Science, CINAHL EBSCO, Cochrane Central Register of Controlled Trials, Chinese Biomedical Literature Database, China National Knowledge Infrastructure databases were searched from inception to 17th of June 2020. REVIEW METHODS: The Cochrane "risk of bias" tool was used to assess the quality of individual studies, and the quality of each outcome was assessed by GRADE approach. Fixed or random effect meta-analysis was used pending the presence of heterogeneity. Dichotomous data synthesis was presented as risk ratio and 95% confidence interval, and quantitative data synthesis was shown as mean difference and 95% confidence interval. RESULTS: Fourteen trials with 1025 critically ill adults were included in the meta-analysis. We found that intermittent feeding could significantly increase the occurrence of feeding intolerance (risk ratio = 1.64, 95% confidence interval = 1.23 to 2.18, P < 0.001) compared with continuous feeding, as well as the incidence of high gastric volume (risk ratio = 3.62, 95% confidence interval = 1.43-9.12, P = 0.006) and aspiration (risk ratio = 3.29, 95% confidence interval = 1.18-9.16, P = 0.02) in > 1-week trial duration, while constipation rate was reduced in intermittent feeding group (risk ratio = 0.66, 95% confidence interval = 0.45-0.98, P = 0.04). Patients in intermittent feeding group received more calories compared with continuous feeding group (mean difference = 184.81, 95% confidence interval = 56.61-313.01, P = 0.005). The quality of all evidence synthesis was "low" or "very low". CONCLUSIONS: In critically ill adults, continuous feeding was associated with lower overall incidence of feeding intolerance, especially in high gastric volume and aspiration. However, decreased constipation incidence and more calorie intake were observed in intermittent feeding group. Because quality of the synthesized evidence was "low" or "very low", there is considerable uncertainty about this estimate.

Endovascular treatment of cerebrovascular stenosis with stent for patients with ischemic cerebrovascular disease.

This study aimed to investigate the therapeutic effect of cerebrovascular stent implantation in southwest Chinese patients with ischemic cerebrovascular disease and underlying risk factors for stent restenosis.We made a retrospectively analysis of occurring risk, cerebrovascular lesion, stent implantation, complication treatment, and prognosis of 54 patients with ischemic cerebrovascular disease in our department.A total of 85 stents were implanted into 54 patients, involving 44 of the internal carotid artery system, 34 of the vertebral-basal artery system and 7 of the subclavian artery system. All patients with stenosis were reduced by >70%, with all stenosis complete reduction in 5 (9%) patients and reduction of over 90% in 25 (46%) patients. A total of 50 patients were followed up for 28.5 (21-35) months. The stents in 42 patients exhibited satisfactory shape and location while restenosis occurred in 8 patients. Univariate analysis revealed that hyperlipidemia, hyperuricemia, surgery duration, and total length of hospital stay are significantly correlated with stent restenosis, and hyperlipidemia and hyperuricemia were proven to be independent risk factors for restenosis using logistic regression analysis.Cerebrovascular stent implantation and balloon inflation surgery can assist in abating angiostenosis and improving blood supplement effectively in patients with ischemic cerebrovascular disease. Besides, an overall evaluation, strict care, and regular check-up in perioperative period may reduce the occurrence of complications. Finally, several clinical parameters may need to be highly focused on in surgery for better prognosis.

Characterization of Response Frequency for the Pressure-Sensitive Paint Based on Fluorine-Containing Polymer Matrixes Using Oscillating Sound Wave Technique.

Five kinds of new homo-polymer and copolymers of methacrylate containing a fluorine ester group were synthesized and used for the binder of pressure-sensitive paint (PSP)to ensure the good compatibility between luminophore (Pt(II) meso-tetra (pentafluorophenyl) porphine (PtTFPP)) and polymer binder. In the work, we were concerned with how the structure of thesepolymers containing fluorine, especially the various ester group structure, affects the response frequency of PSP using oscillating sound wave technique. The results showed that the pressure sensitivities (Sp) of these PSP samples containing different polymers, exhibit some difference. The length of ester chain on the methacrylatepolymer affects the response frequency of PSP sensor layer composed of the polymer. The longer the chain length of the ester group, the higher the response frequency of the PSP sensor layer quenching by oxygen.

Taxonomic Studies of the genus Decticus Serville, 1831 from China (Orthoptera: Tettigoniidae: Tettigoniinae), based on Morphology and Songs.

The genus Decticus Serville contains sixteen known species and subspecies, those widely distributed in the Palaearctic, among which three species and subspecies have been recorded in China. Morphological features in specimens of Decticus are variable. Stable characteristics and reliable methods for distict the species and subspcies are required. The geometric morphometrics was conducted to explore morphological variance on structure of right tegmen of Decticus specimens based on 30 landmarks, as well as qualitative analysis on variances of body size, male stridualtory apparatus and genital characters. The male calling songs structures, if available, can be compared and identified species usefully. The results indicate that the analyzed samples suggest two species, D. albifrons (Fabricius, 1775) and D. verrucivorus (Linnaeus, 1758). D. verrucivorus includes four subspecies, D. v. verrucivorus (Linnaeus, 1758), D. v. crassus Götz, 1970 (a newly recorded subspecies from China), and two new subspecies, D. v. latipennis ssp. n. and D. v. sayram ssp. n.. The songs of D. albifrons and D. v. verrucivorus, D. v. crassus are composed of numerous single chirps. One chirp of D. albifrons consists of one low-amplitude and short-duration syllable, and one high-amplitude and long-duration syllable. One chirp of D. v. verrucivorus consists of five syllables with different amplitudes. One chirp of D. v. crassus consists of five or six syllables with different amplitudes. Illustrations of necessary morphological and bioacoustical characters are presented.