Upregulation of TMEM40 is associated with the malignant behavior and promotes tumor progression in cervical cancer.

OBJECTIVE: Recent studies indicated that transmembrane protein 40 (TMEM40) is associated with several types of cancers but is not clear in cervical cancer (CC). The study aimed to examine the role of TMEM40 in CC and related mechanisms. METHODS: The expression of TMEM40 in CC tissues and cell lines was studied with western blot and real-time quantitative RT-PCR. The effect of TMEM40 on proliferation was evaluated by CCK-8, EdU and colony formation assay. The migration, invasion, cell cycle and apoptosis of CC cells were studied with wound healing, transwell assays and flow cytometry. Tumor growth was evaluated in vivo using a xenogenous subcutaneously implant model. RESULTS: The results revealed that the TMEM40 elevation in CC tissues and cell lines was closely correlated with tumor size and lymph node metastasis in clinical patients. Upregulation of TMEM40 with OE-TMEM40 vector promoted the invasion, migration and proliferation, inhibited the apoptosis and led to distinct S cell cycle arrest in CC cell lines. Silencing TMEM40 with shRNA inhibited the invasion, migration and proliferation, promoted apoptosis and led to a G0/G1 cell cycle arrest in CC cell lines. Silence of TMEM40 downregulated the expression of c-MYC, Cyclin D1, matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-9 (MMP-9), but in contrast, activated p53 and several apoptosis related proteins such as p53, Caspase-3, Caspase-9 and PARP1. In addition, TMEM40 silencing dramatically decreased tumor growth in mice models. CONCLUSION: The present study demonstrates that TMEM40 upregulation can be a potential prognostic biomarker and contribute to CC development.

Overexpression of the Arabidopsis MACPF Protein AtMACP2 Promotes Pathogen Resistance by Activating SA Signaling.

Immune response in plants is tightly regulated by the coordination of the cell surface and intracellular receptors. In animals, the membrane attack complex/perforin-like (MACPF) protein superfamily creates oligomeric pore structures on the cell surface during pathogen infection. However, the function and molecular mechanism of MACPF proteins in plant pathogen responses remain largely unclear. In this study, we identified an Arabidopsis MACP2 and investigated the responsiveness of this protein during both bacterial and fungal pathogens. We suggest that MACP2 induces programmed cell death, bacterial pathogen resistance, and necrotrophic fungal pathogen sensitivity by activating the biosynthesis of tryptophan-derived indole glucosinolates and the salicylic acid signaling pathway dependent on the activity of enhanced disease susceptibility 1 (EDS1). Moreover, the response of MACP2 mRNA isoforms upon pathogen attack is differentially regulated by a posttranscriptional mechanism: alternative splicing. In comparison to previously reported MACPFs in Arabidopsis, MACP2 shares a redundant but nonoverlapping role in plant immunity. Thus, our findings provide novel insights and genetic tools for the MACPF family in maintaining SA accumulation in response to pathogens in Arabidopsis.

Salivary protein 7 of the brown planthopper functions as an effector for mediating tricin metabolism in rice plants.

The brown planthopper (BPH), Nilaparvata lugens, is an important pest that affects rice (Oryza sativa) production in Asia. The flavone tricin (5,7,4'-trihydroxy-3',5'-dimethoxy flavone) is a valuable secondary metabolite commonly found in rice plants that can defend rice plants against infestation by BPH. BPH damage can reduce the metabolic level of tricin in rice. Our preliminary transcriptome research results showed that BPH salivary protein 7 (NlSP7), is highly responsive to tricin stimuli. However, the function of NlSP7 in mediating the interaction between the rice plant and the BPH is unknown. In this study, we cloned the NlSP7 gene in N. lugens and found that its mRNA level was greater in the presence of high tricin content than low tricin content, regardless of whether the BPHs were fed a rice plant diet or an artificial diet containing 100 mg/L tricin. Knocking down NlSP7 resulted in BPH individuals spending more time in the non-penetration and pathway phase, and less time feeding on the phloem of rice plants. These changes decreased BPH food intake, feeding behavior, and fitness, as well as the tricin content of the rice plants. These findings demonstrate that the salivary protein 7 of BPH functions as an effector for tricin metabolism in rice.

Network pharmacology-based strategy to investigate the active ingredients and molecular mechanisms of Scutellaria Barbata D. Don against radiation pneumonitis.

INTRODUCTION: Herbal medicines combined with radiotherapy significantly reduced the incidence of radiation pneumonitis (RP), and the Scutellaria barbata D. Don (SBD) is a perennial herb that has been reported to protect against radiation-induced pneumonitis. However, the exact molecular mechanism is not known. The objective of this research was to investigate the against radiation pneumonitis ingredients and their functional mechanisms in SBD. METHODS: Based on the network pharmacology approaches, we collected active ingredients and target genes in SBD against RP through Traditional Chinese Medicine System Pharmacology (TCMSP) Database, and the "Herb-Ingredients-Target Genes-Disease" Network was constructed by using of Cytoscape. STRING analysis was performed to reveal the protein-protein interactions, and then we applied enrichment analysis on these target proteins, gene function, and pathways. RESULTS: A total of 18 ingredients in SBD regulate 65 RP related target proteins, which show that quercetin, luteolin, baicalein, wogonin may be the key active ingredients, while IL6, AKT1, VEGFA, MMP9, CCL2, prostaglandin-endoperoxide synthase 2 (PTGS2) (cyclooxygenase-2 [COX-2]), CXCL8, IL1B, mitogen-activated protein kinase (MAPK1), and IL10 were identified as critical targets. Besides, the results of Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that predicted targets of SBD are mostly associated with the pathological process of oxidative stress and inflammation. AGE- Receptor of Advanced Glycation Endproducts (RAGE) signaling pathway in diabetic complications, IL-17 signaling pathway, hypoxia-inducible factor-1 (HIF-1) signaling pathway, NF-kappa B signaling pathway might serve as the principal pathways for RP treatment. CONCLUSION: In our study, the pharmacological and molecular mechanism of SBD against RP was predicted from a holistic perspective, and the results provided theoretical guidance for researchers to explore the mechanism in further research.

Source Regions of the First Immigration of Fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) Invading Australia.

Fall armyworm is recognized as one of most highly destructive global agricultural pests. In January 2020, it had first invaded Australia, posing a significant risk to its biosecurity, food security, and agricultural productivity. In this study, the migration paths and wind systems for the case of fall armyworm invading Australia were analyzed using a three-dimensional trajectory simulation approach, combined with its flight behavior and NCEP meteorological reanalysis data. The analysis showed that fall armyworm in Torres Strait most likely came from surrounding islands of central Indonesia on two occasions via wind migration. Specifically, fall armyworm moths detected on Saibai and Erub Islands might have arrived from southern Sulawesi Island, Indonesia, between January 15 and 16. The fall armyworm in Bamaga most likely arrived from the islands around Arafura Sea and Sulawesi Island of Indonesia, between January 26 and 27. The high risk period for the invasion of fall armyworm is only likely to have occurred in January-February due to monsoon winds, which were conducive to flight across the Timor Sea towards Australia. This case study is the first to confirm the immigration paths and timing of fall armyworm from Indonesia to Australia via its surrounding islands.

[Pollution Characteristics and Risk Assessment of Organophosphate Esters in Rivers and Water Body Around Taihu Lake].

To explore the pollution characteristics, potential sources, and ecological and health risk of organophosphate eaters (OPEs) in the surface water of Taihu Lake, water samples from 18 surrounding rivers were collected, as well as 11 water samples from Taihu Lake. The concentrations of 13 OPEs in the water were determined using UPLC-MS/MS, and the spatial distribution of the OPEs in surface water of Taihu Lake basin was further analyzed. The results indicate that, in addition to tripropyl phosphate (TPrP), 2-ethylhexyl diphenyl phosphate (EHDPP), and tricresyl phosphate (TCrP), ten OPEs were detected in all the water samples, the total concentration (ΣOPEs) ranged from 152.5 ng·L-1 to 2524 ng·L-1, and the concentration median value was 519.2 ng·L-1. Tri(chloropropyl) phosphate (TCPP) and tri(2-chloroethyl) phosphate (TCEP) were the dominant OPEs, with the concentration ranges of 73.7-1753.9 ng·L-1 (medium value:204.6 ng·L-1) and 43.9-313.5 ng·L-1 (medium value:131.3 ng·L-1), respectively. The ΣOPEs decreased from the northwest region to the southeast, which corresponds to the economic and industrial development. The results of the source identification reveal that the wastewater discharge from electronics and textile enterprises, construction materials, and vehicular and marine traffic emissions may be the principal sources of the OPEs in Taihu Lake. The ecological risk assessment results indicate that only TCPP, tri(dichloropropyl) phosphate (TDCP), and triphenyl phosphate (TPhP) in some sites had a low risk. The health risk assessment reveals that there were no risks based on water intake, but the long-term risk of OPEs to the aquatic ecosystem and surrounding residents still need attention.

[Effects of different corn varieties on development and reproduction of Spodoptera frugiperda]. / 不同品种玉米对草地贪夜蛾生长发育及繁殖的影响.

To understand the damage capability of the invasive pest Spodoptera frugiperda on diffe-rent corn varieties in China, we evaluated survival fitness of S. frugiperda on three varieties of sweet corn (i.e., Suitian No.1, Zhengtian 68, and Huajintian No.1) and three varieties of waxy corn (i.e., Jingkenuo 2000, Guangheitiannuo, and Guangnuo No.1) by constructing life table for all populations. The correlation of biological parameters of S. frugiperda to the main nutrients and crude fiber content in corn leaves were analyzed. Results showed that the larva survival rate, pupal weight, and production of S. frugiperda on sweet corns were significantly higher than those on waxy corns. The intrinsic rate of increase (rm) of S. frugiperda on the sweet corns ranged from 0.1566 to 0.1843, and the net reproductive rate (R0) ranged from 187.97 to 353.35, being higher than those reared on the waxy corns (rm was from 0.0998 to 0.1465, and R0 was from 25.89 to 95.34). S. frugiperda reared on the sweet corns had higher population growth ability than those reared on the waxy corns. The content of main nutrients such as vitamin C, starch, soluble sugar, protein, fat, total amino acids, and crude fiber in the sweet corn leaves were higher than those in the waxy corn leaves. There were positive correlations between R0 of S. frugiperda to the contenst of vitamin C, starch, soluble sugar, protein, and crude fiber in corn leaves. The results indicated that S. frugiperda reared on the sweet corn leaves had higher survival fitness than those reared on the waxy corn leaves.

[Distribution Characteristics and Source Identification of Organophosphate Esters in Key Waters Areas of Taihu Lake].

Organophosphate esters (OPEs) are ubiquitous in the environment and pose potential risks to ecosystems in that they produce cytotoxicity, genetic toxicity, developmental toxicity, neurotoxicity, and the endocrine disrupting effect. This study aimed to investigate the distribution, sources, and ecological risk of thirteen OPEs in industrial wastewater, influents and effluents of wastewater treatment plants, and the surface waters of key areas (Changzhou City) in Taihu Lake. The results showed that ∑OPEs in industrial wastewater ranged from 91.70-840.52 ng·L-1. The profiles varied from different industries; however, tris (1-chloro-2-propyl) phosphate (TCPP), triethyl phosphate (TEP), and tris (2-chloroethyl) phosphate (TCEP) were the dominant compounds. The ∑OPEs from the wastewater treatment plants were relatively higher, ranging from 1859.59-2778.57 ng·L-1. They are rather resistant to traditional wastewater treatment techniques. The removal rate of ∑OPEs, aryl OPEs, and chlorinated OPEs was 14.97%, 97.91%, and 4.37% (for W1) and 17.32%, 90.83%, and 5.40% (for W2), respectively. The concentration of OPEs in the surface water was in the range of 219.47-689.85 ng·L-1, which was lower than that in wastewater. In addition, TEP, TCPP and TCEP predominated in the surface water samples. A principle component analysis (PCA) indicated that the industrial wastewater, emissions of vehicles, and the release of building materials may be the main sources of OPEs in the study area. A risk quotient was used to assess the ecological risk of OPEs to aquatic organisms in the surface water. Most of the OPEs showed low ecological risk, but 2-ethylhexyl diphenyl phosphate (EHDPP) posed a moderate risk in some sites, and the possible ecological effects caused by the OPEs should not be ignored in the key areas of Taihu Lake.

[Pollution Characteristics and Risk Assessment of Typical Organophosphate Esters in Beijing Municipal Wastewater Treatment Plant and the Receiving Water].

Organophosphate esters (OPEs) are ubiquitous in the environment and pose a potential threat to ecosystems and human health. A method for the determination of eight OPEs by ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) was established. The recovery rates of eight target compounds with different solid-phase extraction columns, different eluents, and different eluent volumes were compared. The results showed that using ENVI-18 column enrichment, OPEs were eluted with 8 mL acetonitrile containing 25% (volume fraction) dichloromethane, and the labeled recovery rate of the target compound was 92.5%-102.2%. The recoveries of different matrix samples were 88.5%-116.1% and relative standard deviation was 1.7%-9.9%. The concentration range of 8 different detectable organophosphate esters in the effluent of sewage treatment plant is 85.9-235.4 ng·L-1 during the six-day sampling process, permissive river downstream of the six-day ΣOPEs average total concentration was 130.3 ng·L-1, higher than the 119.4 ng·L-1 upstream water concentration, but lower than the sewage treatment plant effluent concentration of total 162.5ng·L-1. The study shows that the sewage treatment plant cannot completely remove OPEs; for triethyl phosphate (TEP) and 3 (2-ethyl hexyl) phosphate ester (TEHP) there exists a negative removal phenomenon, whereas for other OPEs the removal rate was between 14.1% and 84.9%, and the total ΣOPEs removal rate by the sewage plant was 50.0%. The TPhP in the effluent of the sewage treatment plant has medium environmental risk (RQ>0.1), and other organophosphates have low environmental risk (RQ<0.1); however, the long-term mixing effects of organophosphate esters on the ecosystem of the receiving river should not be ignored.

Human papillomavirus 16 E7 oncoprotein alters the expression profiles of circular RNAs in Caski cells.

Cervical cancer is one of the most common cancer in female worldwide. The expression of high-risk human papillomavirus E7 oncogene is necessary for the maintenance of malignant phenotypes and transformation. Accumulating studies of this protein has been explored in cervical cancer, however, there are fewer studies on how E7 expression affects the expression of global circular RNA. CircRNA, a promising biomarker and even therapeutic target, has become a star molecular in research after miRNA and long non-coding RNA. Our aim of this study was to investigate the global circRNA levels modulated by HPV E7 expression and identified the potential consequences for mechanism studies. Here we investigated the expression profiles of circRNAs by transfecting E7 siRNA in Caski cells with high-throughput microarray technology. In total, we identified 526 dysregulated circRNAs with fold change ≥2 or≤0.5, and p< 0.05. Among them, 352 were up-regulated and 174 were down-regulated. In addition, 8 selected circRNAs confirmed using qRT-PCR was in line with the results of microarray analysis. Furthermore, bioinformatic analyses indicated that differently expressed circRNAs might implicate in the mTOR signaling pathway, proline metabolism and glutathione metabolism. In conclusion, this study showed the expression profiles of circRNAs regulated by HPV16 E7 in cervical cancer cells and provides novel insights into the new potential candidates for future mechanism studies.

The structure and morphologic changes of antennae of Cyrtorhinus lividipennis (Hemiptera: Miridae: Orthotylinae) in different instars.

Cyrtorhinus lividipennis Reuter (Hemiptera: Miridae: Orthotylinae), including nymphs and adults, are one of the dominant predators and have a significant role in the biological control of leafhoppers and planthoppers in irrigated rice. In this study, we investigated the antennal morphology, structure and sensilla distribution of C. lividipennis in different instars using scanning electron microscopy. The antennae of both five different nymphal stages and adults were filiform in shape, which consisted of the scape, pedicel and flagellum with two flagellomeres. There were significant differences found in the types of antennal sensilla between nymphs and adults. The multiporous placodea sensilla (MPLA), basiconica sensilla II (BAS II), and sensory pits (SP) only occurred on the antennae of adult C. lividipennis of both sexes. Moreover, there was chaetica sensilla III (CHA III) only observed in males. Sixteen types of antennal sensilla were recorded altogether. They were microtrichia sensilla (MIC), three types of trichoidea sensilla (TRI I-III), three types of chaetica sensilla (CHA I-III), three types of basiconica sensilla (BAS I-III), two types of coeloconica sensilla (COE I and COE II), placodea sensilla (PLA), campaniform sensilla (CAM), MPLA, and SP. In the five different nymphal stages of C. lividipennis, the length of their antennae was significantly increased with the increase of the instar, as well as the number of the TRI II and TRI III. Moreover, sexual dimorphism usually occurred not only in the distribution (CHA III and SP) and the number of antennal sensilla (MIC, BAS II, TRI II, TRI III and MPLA), but also in the length of flagellum (F1 and F2). The possible functions of antennal sensilla are discussed. Those observations could contribute to a better understanding of the development of the olfactory system, and facilitate future studies on the antennal functions in C. lividipennis.

Myosin 1b promotes cell proliferation, migration, and invasion in cervical cancer.

OBJECTIVE: Recent evidence suggests an important role of Myosin 1b (Myo1b) in the progression of several cancers, including prostate cancer and head and neck squamous cell carcinoma (HNSCC). However, the contribution of Myo1b to cervical cancer (CC) remains elusive. METHODS: Quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry and western blotting assays were used to confirm the expression of Myo1b in CC tissues compared with matched non-tumor tissues and CC cells, and analyze its clinical significance. In vitro, RNA interference (siRNA or shRNA) was used to investigate the biological function and underlying mechanism of Myo1b in cervical carcinogenesis. Furthermore, tumor growth was evaluated in vivo using a xenogenous subcutaneously implant model. RESULTS: Here, for the first time we reported that Myo1b expression was significantly increased in human CC, compared to cervical intraepithelial neoplasia (CIN) and normal cervical tissues and that the upregulation of Myo1b was significantly correlated with FIGO Stage, HPV infection, lymph node metastasis and pathological grade. In vitro, knockdown of Myo1b significantly suppressed proliferation, migration, and invasion of CaSki and SiHa cells, and markedly decreased the MMP1/MMP9 activities. Also, silencing the expression of Myo1b dramatically repressed tumor growth in a mouse xenograft model. Further investigations showed that HPV16 E6 or E7 could enhance the expression of Myo1b via upregulating c-MYC. CONCLUSION: Taken together, our data suggested a potential role of Myo1b in cervical carcinogenesis and tumor progression and provided novel insights into the mechanism of how this factor promotes cell proliferation, migration, and invasion in CC cells.

High expression of TMEM40 is associated with the malignant behavior and tumorigenesis in bladder cancer.

BACKGROUND: Bladder cancer (BCa) is one of the most common cancers in the urinary system among the world. Previous studies suggested that TMEM40 expression level was significantly associated with clinicopathological parameters including histological grade, clinical stage and pT status of bladder cancer. However, the molecular mechanism of TMEM40 in BCa remains poorly understood. METHODS: Real-time quantitative RT-PCR (qRT-PCR) and western blot (WB) were used to examine the expression levels of TMEM40 in BCa tissues, paired non-cancer tissues and cell lines. A series of experiments, including CCK-8, wound healing, flow cytometry, transwell and EdU assays were performed to assess the effects of TMEM40 on cell proliferation, cell cycle and apoptosis, migration and invasion. In addition, tumor growth was evaluated in vivo using a xenogenous subcutaneously implant model. All statistical analyses were executed by using the SPSS 20.0 software. All experimental data from three independent experiments were analyzed by Student's t test and results were expressed as mean ± standard deviation. RESULTS: In this study, we identified the role of TMEM40 in the tumorigenesis of bladder cancer and found that it was upregulated in bladder cancer tissues and cell lines, compared with their normal counterparts. The results demonstrated that effective silence of TMEM40 expression suppressed cell proliferation, blocked G1-to-S cell cycle transition, and inhibited cell migration and invasion in human bladder 5637 and EJ cell lines. Consistently, in vivo data showed that TMEM40 silencing could dramatically decreased tumor growth. Further study revealed that TMEM40 knockdown resulted in accumulation of p53 and p21 protein and decrease of c-MYC and cyclin D1 protein. CONCLUSION: These data suggest that TMEM40 represents a potential oncogene, which exert a crucial role in the proliferation and apoptosis via the p53 signaling pathway in BCa, thus probably serve as a novel candidate biomarker and a potential therapeutic target for patients with BCa.

Low expression of PinX1 is associated with malignant behavior in basal-like breast cancer.

Human Pinx1 protein, associated with shelterin proteins, is widely revealed as a haploinsufficient tumor suppressor. Growing evidence has manifested the deregulation of PinX1 in distinct cancers. Nonetheless, the loss status of PinX1 and its diagnostic, prognostic and clinicopathological significance in Basal-like breast cancer are still unclear. In the present study, the PinX1 expression levels of breast cancer tissues were investigated by qRT-PCR and immunoblotting assays. Then immunohistochemistry (IHC) was performed to detect PinX1 expression on a tissue microarray. The optimal threshold for PinX1 positivity was determined by receiver operating characteristic (ROC) curve analysis. To clarify the probable role of PinX1 in BLBC, the PinX1 knockout and stably over-expressed MDA-MB-231 cell lines were constructed by the CRISPR-Cas9 system and gene transfection. The association of PinX1 expression with cell proliferation, migration and apoptosis of MDA-MB-231 cells were observed by CCK-8 assay, wound healing assay, transwell assay, flow cytometric analysis and immunoblotting of the cleaved caspase-3 protein level. Our results showed that both PinX1 mRNA and protein expression were downregulated in breast cancer tissues (P<0.05). In IHC analysis, the optimal cut-off parameter for PinX1 positive expression was 62.5% (the AUC was 0.749, P<0.01). PinX1 positivity was 76.9% (10/14) in luminal subtypes, 50% (5/10) in Her2-enriched breast cancer and 27.3% (9/33) in basal-like subtypes. Besides, in 59 invasive ductal breast carcinomas, PinX1 expression was inversely related to histology grade (P<0.05) while it was positively associated with PR status (P<0.05) and ER status (P<0.05). These results indicated that low expression of PinX1 correlated with aggressive clinicopathological significance of breast cancer, especially in the basal-like subtype. Besides, we identified that overexpression of PinX1 inhibited the proliferation rates and migration ability and increased the apoptosis rates of BLBC. Our findings demonstrated that low expression of PinX1 was associated with malignant behaviors in basal-like subtype of breast cancer. PinX1 is likely a feasible biomarker and molecular target of BLBC.

Expression of TMEM40 in bladder cancer and its correlation with clinicopathological parameters.

Transmembrane protein 40 (TMEM40) is a 23-kDa protein in cell membrane. There is no report that TMEM40 is associated with cancer. However, our study found that TMEM40 was high expressed in bladder cancer tissues. Immunohistochemical analyses of TMEM40 expression were performed on a tissue microarray including 72 transitional cell carcinomas and 43 normal bladder tissues to investigate the expression and clinical significance of TMEM40 in bladder cancer. We adopted receiver operating characteristic (ROC) analysis to select the optimal cut-off score. TMEM40 expression was defined positive if above 62.5% of cells were stained, and below it was negative. Then, the expression of TMEM40 in bladder cancer cells was evaluated by quantitative real-time PCR and western blot analysis. A significantly high level of TMEM40 in bladder cancer cells was proved. On the basis of ROC curve analysis, TMEM40 expression was positive in 68.1% (n=49) and negative in 31.9% (n=23) of bladder cancer cases. TMEM40 staining was positive in 2.3% (n=1) and negative in 97.7% (n=42) of normal bladder tissues. It showed that TMEM40 was up-regulated in bladder cancer tissues compared to normal bladder tissues. Moreover, TMEM40 expression was significantly associated with histological grade (P<0.05), clinical stage (P<0.05), pT status (P<0.05), but not age. Our study demonstrates that high TMEM40 expression is associated with bladder cancer, and it could be a diagnostic biomarker for bladder cancer.

[Geostatistic analysis of spatial pattern of Fruhstorferiola tonkinensis Will].

By the methods of geostatistics, this paper studied the spatial pattern of Fruhstorferiola tonkinensis nymphs in mulberry fields of north Guangdong. The results showed that the semivariogram of F. tonkinensis nymphs at all development stages could be described by spherical model, implying an aggregative distribution, and the range of their spatial correlation was 1. 973, 1. 968, 2. 169, 2. 302 and 4. 307 m from 1st to 5th instars. Based on the information obtained from investigation site, the isoclines maps of the instars were created by the geostatistical software Surfer 8. 0 with Kriging interpolation, and the resulting maps gave a clear indication about the spatial patterns of the instars. Unlike other locusts, the younger instars of F. tonkinensis had an aggregative distribution, while the elder ones were dispersive in distribution.