Osiris17 is indispensable for morphogenesis of intestinal tract in Locusta migratoria.

The Osiris gene family is believed to play important roles in insect biology. Previous studies mainly focused on the roles of Osiris in Drorophila, how Osiris operates during the development of other species remains largely unknown. Here, we investigated the role of LmOsi17 in development of the hemimetabolous insect Locusta migratoria. LmOsi17 was highly expressed in the intestinal tract of nymphs. Knockdown of LmOsi17 by RNA interference (RNAi) in nymphs resulted in growth defects. The dsLmOsi17-injected nymphs did not increase in body weight or size and eventually died. Immunohistochemical analysis showed that LmOsi17 was localized to the epithelial cells of the foregut and the gastric caecum. Histological observation and hematoxylin-eosin staining indicate that the foregut and gastric caecum are deformed in dsLmOsi17 treated nymphs, suggesting that LmOsi17 is involved in morphogenesis of foregut and gastric caecum. In addition, we observed a significant reduction in the thickness of the new cuticle in dsLmOsi17-injected nymphs compared to control nymphs. Taken together, these results suggest that LmOsi17 contributes to morphogenesis of intestinal tract that affects growth and development of nymphs in locusts.

[Changes in Soil Nitrogen Components and Their Relationship with Environmental Factors with Different Tea Plantation Ages].

Changes in soil nitrogen components in tea gardens affect the soil nitrogen supply capacity and nitrogen cycle. In this study, soil samples were collected from forest land, cultivated land, and tea gardens with different plantation ages (30, 50, and 70 years) to explore the changes in soil nitrogen components and their relationship with physicochemical properties and enzyme activities. The results showed that:① with the increase in tea plantation age, the silt, total phosphorus, and urease and catalase activities gradually increased, whereas the sand, clay, pH, electrical conductivity, soil organic carbon, and the activities of invertase gradually decreased. The alkaline phosphatase activity increased first and then decreased with the increase in tea plantation age, and no significant differences were observed in soil water content and acid phosphatase activity. ② With the increase in tea plantation age, the contents of acid ammonia nitrogen, amino acid nitrogen, and nitrate nitrogen (NO3--N) increased significantly, and the contents of total nitrogen, acid ammonia nitrogen, hydrolyzable unknown nitrogen, and non-hydrolyzable nitrogen in tea gardens were significantly higher than those in forest land. ③ The total phosphorus, alkaline phosphatase, and urease were the main factors affecting soil nitrogen components. Among them, organic nitrogen components were significantly correlated with total phosphorus and alkaline phosphatase, and inorganic nitrogen components were significantly correlated with alkaline phosphatase, whereas total nitrogen had significant correlations with sand, silt, total phosphorus, urease, and alkaline phosphatase.

Osiris17 is essential for stable integrin localization and function during insect wing epithelia remodeling.

The dynamic adhesion between cells and their extracellular matrix is essential for the development and function of organs. During insect wing development, two epithelial sheets contact each other at their basal sites through the interaction of ßPS integrins with the extracellular matrix. We report that Osiris17 contributes to the maintenance of ßPS integrins localization and function in developing wing of Drosophila and locust. In flies with reduced Osiris17 expression the epithelia sheets fail to maintain the integrity of basal cytoplasmic junctional bridges and basal adhesion. In contrast to the continuous basal integrin localization in control wings, this localization is disrupted during late stages of wing development in Osiris17 depleted flies. In addition, the subcellular localization revealed that Osiris17 co-localizes with the endosomal markers Rab5 and Rab11. This observation suggests an involvement of Osiris17 in endosomal recycling of integrins. Indeed, Osiris17 depletion reduced the numbers of Rab5 and Rab11 positive endosomes. Moreover, overexpression of Osiris17 increased co-localization of Rab5 and ßPS integrins and partially rescued the detachment phenotype in flies with reduced ßPS integrins. Taken together, our data suggest that Osiris17 is an endosome related protein that contributes to epithelial remodeling and morphogenesis by assisting basal integrins localization in insects.

Knickkopf (LmKnk) is required for chitin organization in the foregut of Locusta migratoria.

The foregut, located at the front of the digestive tract, serves a vital role in insects by storing and grinding food into small particles. The innermost layer of the foregut known as the chitinous intima, comes into direct contact with the food and acts as a protective barrier against abrasive particles. Knickkopf (Knk) is required for chitin organization in the chitinous exoskeleton, tracheae and wings. Despite its significance, little is known about the biological function of Knk in the foregut. In this study, we found that LmKnk was stably expressed in the foregut, and highly expressed before molting in Locusta migratoria. To ascertain the biological function of LmKnk in the foregut, we synthesized specific double-stranded LmKnk (dsLmKnk) and injected it into locusts. Our findings showed a significant decrease in the foregut size, along with reduced food intake and accumulation of residues in the foregut after dsLmKnk injection. Morphological observations revealed that newly formed intima became thinner and lacked chitin lamella. Furthermore, fluorescence immunohistochemistry revealed that LmKnk was located in the apical region of new intima and epithelial cells. Taken together, this study provides insights into the biological function of LmKnk in the foregut, and identifies the potential target gene for exploring biological pest management strategies.

[Prediction of Spatial Distribution of Heavy Metals in Cultivated Soil Based on Multi-source Auxiliary Variables and Random Forest Model].

Spatial prediction of the concentrations of soil heavy metals (HMs) in cultivated land is critical for monitoring cultivated land contamination and ensuring sustainable eco-agriculture. In this study, 32 environmental variables from terrain, climate, soil attributes, remote-sensing information, vegetation indices, and anthropogenic activities were used as auxiliary variables, and random forest (RF), regression Kriging (RK), ordinary Kriging (OK), and multiple linear regression (MLR) models were proposed to predict the concentrations of As, Cd, Cr, Cu, Hg, Ni, Pb, and Zn in cultivated soils. In comparison to those of RK, OK, and MLR, the RF model had the best prediction performance for As, Cd, Cr, Hg, Pb, and Zn, whereas the OK and RK models had highest prediction performance for Cu and Ni, respectively, showing that R2 was the highest, and mean absolute error (MAE) and root mean square error (RMSE) were the lowest. The prediction performance of the spatial distribution of soil HMs under different prediction methods was basically consistent. The high value areas of eight HMs concentrations were all distributed in the southern plain area. However, the RF model depicted the details of spatial prediction more prominently. Moreover, the importance ranking of influencing factors derived from the RF model indicated that the spatial variation in concentrations of the eight HMs in Lanxi City were mainly affected by the combined effects of Se, TN, pH, elevation, annual average temperature, annual average rainfall, distance from rivers, and distance from factories. Given the above, random forest models could be used as an effective method for the spatial prediction of soil heavy metals, providing scientific reference for regional soil pollution investigation, assessment, and management.

Transcription factors, cap 'n' collar isoform C regulates the expression of CYP450 genes involving in insecticides susceptibility in Locusta migratoria.

BACKGROUND: The cap 'n' collar (Cnc) belongs to the Basic Leucine Zipper (bZIP) transcription factor super family. Cap 'n' collar isoform C (CncC) is highly conserved in the animal kingdom. CncC contributes to the regulation of growth, development, and aging and takes part in the maintenance of homeostasis and the defense against endogenous and environmental stress. Insect CncC participates in the regulation of various kinds of stress-responsive genes and is involved in the development of insecticide resistance. RESULTS: In this study, one full-length CncC sequence of Locusta migratoria was identified and characterized. Upon RNAi silencing of LmCncC, insecticide bioassays showed that LmCncC played an essential role in deltamethrin and imidacloprid susceptibility. To fully investigate the downstream genes regulated by LmCncC and further identify the LmCncC-regulated genes involved in deltamethrin and imidacloprid susceptibility, a comparative transcriptome was constructed. Thirty-five up-regulated genes and 73 down-regulated genes were screened from dsLmCncC-knockdown individuals. We selected 22 LmCncC-regulated genes and verified their gene expression levels using RT-qPCR. Finally, six LmCYP450 genes belonging to the CYP6 family were selected as candidate detoxification genes, and LmCYP6FD1 and LmCYP6FE1 were further validated as detoxification genes of insecticides via RNAi, insecticide bioassays, and metabolite identification. CONCLUSIONS: Our data suggest that the locust CncC gene is associated with deltamethrin and imidacloprid susceptibility via the regulation of LmCYP6FD1 and LmCYP6FE1, respectively.

Jinggangmycin stimulates reproduction and increases CHCs-dependent desiccation tolerance in Drosophila melanogaster.

Jinggangmycin (JGM), an agricultural antibiotic compound, is mainly used against the rice sheath blight (RSB) Rhizoctonia solani. However, its application may lead to unexpected consequences in insects. In this study, the effects of JGM on the physiological parameters of Drosophila melanogaster were investigated. The results showed that 0.005 g/ml JGM exposure increased female daily egg production and extended the oviposition period, while there was no significant effect on reproduction at 0.016 g/ml. At the same time, desiccation tolerance increased in flies fed 0.005 g/ml JGM. The RT-qPCR results revealed that FAS1 and FAS3 expression were upregulated in 0.005 g/ml JGM treated flies. Consistently, the amount of CHCs accumulated on the cuticle surface increased upon JGM treatment at 0.005 g/ml. Moreover, RNAi for FAS3 decreased desiccation tolerance of JGM-treated flies. These results suggest that JGM affects fatty acid biosynthesis, which in turn enhances reproduction and desiccation tolerance in Drosophila.

[Effect of Spartina alterniflora Invasion on Soil C:N:P Stoichiometry in Coastal Wetland of Hangzhou Bay].

The invasion of Spartina alterniflora poses a great threat to coastal wetland ecosystems. In this study, the stoichiometric characteristics of soil carbon, nitrogen, and phosphorus under a Spartina alterniflora invasion were explored using ANOVA in a coastal wetland in Hangzhou Bay, and the driving coupling relationship between soil environmental factors and soil C:N:P stoichiometric characteristics of the coastal wetland were further explored based on the redundancy analysis (RDA), boosted regression tree (BRT), and partial least squares-structural equation (PLS-SEM) model. The results showed that:① after the invasion of Spartina alterniflora, soil N:P and total nitrogen (TN) in the wetland increased significantly, and with the increase in invasion time, TN and N:P decreased significantly, whereas soil organic carbon (SOC), C:N, and C:P increased significantly. ② The RDA model revealed that the main factors affecting the stoichiometric characteristics of topsoil C:N:P were SOC>electrical conductivity (EC)>TN in winter and SOC>bulk density (BD)>TN in summer. ③ The BRT model showed that under the invasion of Spartina alterniflora, TN was the key factor affecting soil C:N and N:P, and SOC was the key factor affecting C:P. ④ The PLS-SEM model showed that clay and water content directly affected SOC, thus affecting C:N and C:P; the clay and EC directly affected total phosphorus (TP), thus affecting N:P and C:P; and the EC directly affected TN, thus affecting C:N and N:P. In conclusion, the invasion of Spartina alterniflora had a significant impact on soil C:N:P stoichiometric characteristics in the study area. Soil physical properties and nutrient content directly or indirectly affected soil C:N:P stoichiometric characteristics to varying degrees.

Chitinase 6 is required for procuticle thickening and organ shape in Drosophila wing.

The polysaccharide chitin is a major scaffolding molecule in the insect cuticle. In order to be functional, both chitin amounts and chitin organization have been shown to be important parameters. Despite great advances in the past decade, the molecular mechanisms of chitin synthesis and organization are not fully understood. Here, we have characterized the function of the Chitinase 6 (Cht6) in the formation of the wing, which is a simple flat cuticle organ, in the fruit fly Drosophila melanogaster. Reduction of Cht6 function by RNA interference during wing development does not affect chitin organization, but entails a thinner cuticle suggesting reduced chitin amounts. This phenotype is opposed to the one reported recently to be caused by reduction of Cht10 expression. Probably as a consequence, cuticle permeability to xenobiotics is enhanced in Cht6-less wings. We also observed massive deformation of these wings. In addition, the shape of the abdomen is markedly changed upon abdominal suppression of Cht6. Finally, we found that suppression of Cht6 transcript levels influences the expression of genes coding for enzymes of the chitin biosynthesis pathway. This finding indicates that wing epidermal cells respond to activity changes of Cht6 probably trying to adjust chitin amounts. Together, in a working model, we propose that Cht6-introduced modifications of chitin are needed for chitin synthesis to proceed correctly. Cuticle thickness, according to our hypothesis, is in turn required for correct organ or body part shape. The molecular mechanisms of this processes shall be characterized in the future.

[Spatial Heterogeneity of PM2.5 Concentration in Response to Land Use/Cover Conversion in the Yangtze River Delta Region].

The sustainable management direction of PM2.5 concentrations in the Yangtze River Delta region remains unclear due to regional spatial effects. This study combined the random forest model, spatial econometric model, and multi-scale geographically weighted regression model (MGWR) to explore the multi-scale spatial response of PM2.5 concentration to land use/cover conversion. The results show that:① PM2.5 concentrations in the Yangtze River Delta region from 2000 to 2018 showed four types of spatial-temporal patterns of spatially continuous aggregation, with strong regional synchronous changes. ② The relative influence of land conversion on PM2.5 concentrations showed a complex performance, and the source-sink effect of cultivated land and forest land was obvious. Neighborhood analysis indicated that the effect of surrounding aggregated land use conversion was generally more significant than that of single cells on PM2.5 concentration change, and the spatial effect was obvious. ③ PM2.5 concentration changes were mostly significantly negatively correlated with forest land and grassland conversion types and significantly positively correlated with conversion types between cropland, construction land, and water bodies. The importance ranking of the random forest model and correlation coefficient intensity indicated that the conversion between cropland-cropland (29.65%; 0.650), forest land-forest land (26.98%; 0.726), construction land-cropland (22.57%; 0.519), cropland-forestland (17.84%; 0.602), and cropland-construction land (16.34%; 0.424) contributed more to the variation in PM2.5 concentration. The spatial Durbin model revealed a significant spatial dependence of the change in PM2.5 concentration and a strong spatial spillover effect. ④ The MGWR model revealed the scale effects and non-stationary characteristics of the spatial relationships between different land use conversions acting on PM2.5 concentration change, and its spatial relationship showed strong differences in transfer types. However, the multi-models revealed that different land conversions drove the PM2.5 concentration change in different ways, so it is necessary to formulate targeted joint management strategies in a categorical and hierarchical manner.

DEAD-Box RNA Helicase DDX47 Maintains Midgut Homeostasis in Locusta migratoria.

Tissue homeostasis is critical for maintaining organ shape, size, and function. The condition is regulated by the balance between the generation of new cells and the loss of senescent cells, and it involves many factors and mechanisms. The midgut, an important part of the intestinal tract, is responsible for digestion and nutrient absorption in insects. LmDDX47, the ortholog of DEAD-box helicase 47 from Locusta migratoria, is indispensable for sustaining a normal midgut in the nymphs. However, the underlying cellular and molecular mechanisms remain to be elucidated. In this study, LmDDX47 knockdown resulted in atrophy of the midgut and gastric cecum in both nymph and adult locusts. After LmDDX47 knockdown, the number of regenerative and columnar cells in the midgut was significantly reduced, and cell death was induced in columnar tissue. LmDDX47 was localized to the nucleolus; this was consistent with the reduction in 18S rRNA synthesis in the LmDDX47 knockdown group. In addition, the acetylation and crotonylation levels of midgut proteins were significantly increased. Therefore, LmDDX47 could be a key regulator of midgut homeostasis, regulating 18S rRNA synthesis as well as protein acetylation and crotonylation in the migratory locust.

Silencing of transcription factor E93 inhibits adult morphogenesis and disrupts cuticle, wing and ovary development in Locusta migratoria.

Ecdysone-induced protein 93F (E93) plays important roles during the metamorphosis process in insects. In this study, a cDNA of the LmE93 gene was identified from the transcriptome of Locusta migratoria, which consists of the 3378-nucleotide open-reading frame (ORF) and encodes 1125 amino acids with helix-turn-helix (HTH) motifs. Reverse transcription quantitative polymerase chain reaction analysis revealed that LmE93 was highest expressed in ovary. The LmE93 expression level was markedly low from the 3rd to 4th instar nymphs, and greatly increased in 1-day-old 5th instar nymphs with a peak on middle nymphal days, then declined in the late nymphal days. Moreover, injected dsLmE93 into 4th and 5th instar nymphs greatly reduced LmE93 transcripts, respectively, and prevented the process of metamorphosis, causing supernumerary nymphal stages. Hematoxylin-eosin staining of the integument showed that the apolysis occurred in advance in 4th instar nymphs, and old cuticle degradation was decreased in dsLmE93-injected locusts of 5th instar nymphs. Smaller and no fully developed wings with reduced columns between the anterior and posterior regions were found in N6 and N7 supernumerary nymphs. In addition, the development of the ovary in dsLmE93-injected locusts was severely blocked, the yolk was almost not formed and there was no development of ovarioles. The results indicated that LmE93 play key roles in the metamorphosis, cuticle, wing and ovarian development of locusts.

Characteristics of Halloween genes and RNA interference-mediated functional analysis of LmCYP307a2 in Locusta migratoria.

Halloween genes are involved in the biosynthesis of the molting hormone, which plays a key role in insect ecdysis, development, metamorphosis, and reproduction. Our previous work identified five Halloween genes from Locusta migratoria, but their functions are currently unknown. In this study, the sequences of these five Halloween genes were analyzed and characterized. LmCYP307a2, LmCYP306a1, LmCYP302a1, and LmCYP315a1 were primarily expressed in the prothoracic glands, while LmCYP314a1 was universally expressed in peripheral tissues, especially in the ovaries and Malpighian tubules. All five Halloween genes were mainly expressed from the 5th to the 7th d in 5th-instar nymphs. RNA interference (RNAi) silencing of LmCYP307a2 resulted in severe molting delays and molting failure, which could be rescued by supplementary 20-hydroxyecdysone. A hematoxylin and eosin staining analysis suggested that the RNAi of LmCYP307a2 inhibited the ecdysis process by inhibiting the apolysis and degradation of the old cuticle, and by promoting the synthesis of a new cuticle. Quantitative reverse transcription polymerase chain reaction results showed that the expressions of LmE74, LmCht5, and LmCht10 were dramatically down-regulated, while that of LmChsI was substantially up-regulated, after knockdown of LmCYP307a2. The results suggest that LmCYP307a2 is related to the molt process via regulation of chitin synthesis and degradation.

Identification of Rab family genes and functional analyses of LmRab5 and LmRab11A in the development and RNA interference of Locusta migratoria.

Rab proteins constitute the largest family of small GTPases, which play pivotal roles in intracellular membrane trafficking in all eukaryotes. A number of Rab genes have been identified in eukaryotes; however, very little information about these genes has been reported in insects. In the current study, for the first time we identified and characterized 27 Rab family genes from Locusta migratoria. Phylogenetic analysis and comparison of domain architecture indicated that Rab family genes are highly conserved among insect species. Tissue-dependent expression profiles indicated that expression of Rab genes was highest in the ovary, except for LmRab3, which was most highly expressed in hemolymph. The biological function of each Rab gene was investigated using RNA interference (RNAi). Double-stranded RNA targeting each Rab gene was injected into the hemocoel of nymphs and revealed that suppression of two Rab genes (LmRab5 and LmRab11A) caused 100% mortality. In addition, nymphs injected with dsLmRab5 exhibited severe phenotypic defects in the gastric caeca and midgut, while dsLmRab11A arrested the molting process. We then applied the RNAi of RNAi technique to test if silencing either of these two genes would affect the suppression of the lethal giant larvae (LmLgl) reporter gene and found that suppression of LmRab5 diminished the RNAi efficiency of LmLgl, whereas suppression of LmRab11A enhanced RNAi efficiency of LmLgl. These results indicate that Rab genes contribute differently to RNAi efficiency in different tissues. Our study provides a foundation for further functional investigations of Rab genes and their contributions to RNAi efficiency in L. migratoria.

[Therapeutic effect of auriculotherapy with miniature bian needle on anxiety in the patients after percutaneous coronary intervention].

OBJECTIVE: To observe the effect of auriculotherapy with miniature bian needle on anxious emotion, the condition of angina pectoris attack and sleep quality in the patients with anxiety after percutaneous coronary intervention (post-PCI). METHODS: A total of 74 eligible patients of post-PCI combined with anxious depression were randomized into an auriculotherapy group (37 cases, 2 cases dropped out) and a control group (37 cases, 3 cases dropped out). In the auriculotherapy group, on the base of the conventional secondary prevention medication for coronary heart disease (CHD), auriculotherapy with miniature bian needle was supplemented. In the control group, a proper physical exercise was combined on the base of the secondary prevention medication for CHD. The duration of treatment was 4 weeks in two groups. Separately, the score of Hamilton anxiety scale (HAMA), the score Seattle angina questionnaire (SAQ) and the score of Pittsburgh sleep quality index (PSQI) were assessed in the patients of the two groups before and after treatment. RESULTS: After treatment, the score of HAMA, the score of each item of SAQ and PSQI score were all improved significantly as compared with those before treatment respectively in both the auriculotherapy group and the control group (P<0.001, P<0.05). After treatment, HAMA score, PSQI score and the scores of physical limitation (PL), anginal stability (AS), anginal frequency (AF) and treatment satisfaction (TS) in SAQ in the auriculotherapy group were all better than those in the control group (P<0.001, P<0.05). The total effective rate was 91.43% (32/35) in the auriculotherapy group, obviously higher than 58.82% (20/34) in the control group (P<0.001). CONCLUSION: Auriculotherapy with miniature bian needle effectively relieves anxious emotions and the condition of angina pectrois attack and improves sleep quality in the post-PCI patients with anxiety.

The microRNA miR-184 regulates the CYP303A1 transcript level to control molting of Locusta migratoria.

Cytochrome P450 monooxygenases (CYPs) play essential physiological functions in insects. CYP303A1 is highly conserved in insect species studied to date, and shows an indispensable role for adult eclosion in both Locusta migratoria and Drosophila melanogaster. However, how CYP303A1 is regulated to control insect developmental processes remains uninvestigated. In this study, we discovered functional binding sites for miR-184 in the coding sequence of LmCYP303A1. The luciferase reporter assay showed that miR-184 could target LmCYP303A1 and regulate its expression in vitro. Furthermore, overexpression of miR-184 through microinjection of agomir to locusts reduced the transcripts of LmCYP303A1 and led to abnormal molting, which is similar to the phenotype of silencing LmCYP303A1 by direct injection of dsLmCYP303A1 to locusts. Meanwhile, down-regulation of miR-184 by injection of antagomir increased the LmCYP303A1 transcript and caused molting defects. These findings suggested that miR-184 could target LmCYP303A1 to regulate the molting process in L. migratoria, which might be considered as a novel target for pest control.

LmIntegrinß-PS is required for wing morphogenesis and development in Locusta migratoria.

Wings are an important flight organ of insects and their morphogenesis depends on a series of cell-to-cell and cell-to-extracellular matrix interactions. Integrin as a transmembrane protein receptor mediates cell-to-cell adhesion, cell-to-extracellular matrix interactions and signal transduction. In the present study, we characterized an integrin gene that encodes integrinß-PS protein in Locusta migratoria. LmIntegrinß-PS is highly expressed in the wing pads and the middle stages of 5th instar nymphs. Immunohistochemical analysis revealed that the LmIntegrinß-PS protein was localized at the cell base of the two layers of wings. After suppression of LmIntegrinß-PS by RNA interference, the wing pads or wings were unable to form normally, with a blister wing appearance during nymph to nymph transition and nymph to adult transition. We further found that the dorsal and ventral epidermis of the wings after dsLmIntegrinß-PS injection were improperly connected and formed huge cavities revealed by hematoxylin and eosin staining. Furthermore, the morphology and structure of the wing cuticle was significantly disturbed which affected the stable arrangement and attachments of the wing epidermis. Moreover, the expression of related cell adhesion genes was significantly decreased in LmIntegrinß-PS-suppressed L. migratoria, suggesting that LmIntegrinß-PS is required for the morphogenesis and development of wings during molting by stabilizing cell adhesion and maintaining the cytoskeleton of these cells.

Roles of LmCDA1 and LmCDA2 in cuticle formation in the foregut and hindgut of Locusta migratoria.

Chitin deacetylases (CDAs, including CDA1 and CDA2) are considered key enzymes for body cuticle formation and tracheal morphogenesis in various insect species. However, their functions in the formation of the cuticular intima of the foregut and hindgut are unclear. Here, we investigated the roles of their respective genes LmCDA1 and LmCDA2 in this process, in the hemimetabolous insect Locusta migratoria. Transcripts of LmCDA1 and LmCDA2 were highly expressed both before and after molting in the foregut. In the hindgut, their expression was high only before molting. In both the foregut and hindgut, LmCDA1 protein was localized in the basal half of the chitin matrix (procuticle), whereas LmCDA2 was detected in the upper half of the procuticle. Knockdown of LmCDA1 by RNA interference (RNAi) in 5th-instar nymphs caused no visible defects of the hindgut cuticle. By contrast, the chitinous lamellae of the cuticular intima in the foregut of knockdown animals were less compact than in control animals. RNAi against LmCDA2 led to thickening of both the foregut and hindgut cuticles, with a greater number of thinner laminae than in the respective control cuticles. Taken together, our results show that LmCDA1 and LmCDA2 have distinct, but overlapping, functions in chitin organization in the foregut cuticle. However, in the hindgut, this process seems independent of LmCDA1 activity but requires LmCDA2 function. Thus, the CDAs reflect tissue-specific differences in cuticular organization and function, which need further detailed molecular and histological analyses for full comprehension.

A dsRNA-degrading nuclease (dsRNase2) limits RNAi efficiency in the Asian corn borer (Ostrinia furnacalis).

The efficiency of RNA interference (RNAi) varies substantially among different insect species. Rapid degradation of double-stranded RNA (dsRNA) by dsRNA-degrading nucleases (dsRNases) has been implicated to cause low RNAi efficiency in several insect species. In this study, we identified four dsRNase genes (OfdsRNase1, OfdsRNase2, OfdsRNase3 and OfdsRNase4) from the Asian corn borer (Ostrinia furnacalis) transcriptome database. Bioinformatic analyses showed that each deduced protein sequence contained endonuclease NS domains and signal peptides. Gene expression analysis revealed that OfdsRNase2 was exclusively expressed in the midgut of larvae. RNAi efficiency was investigated in 2-d-old fifth-instar larvae (high expression of dsRNase2) and 2-d-old pupae (low expression of dsRNase2) by feeding or injecting dsRNA targeting a marker gene that encodes the lethal giant larvae protein (OfLgl). Our results showed that OfLgl only partially silenced the expression of OfLgl in pupae, but not in larvae, suggesting that OfdsRNase2 could contribute to lower RNAi efficiency in larval stages. This hypothesis was supported by our RNAi-of-RNAi experiment using a tissue culture technique where the silencing efficiency against the reporter gene, OfHex1, was significantly improved after knockdown of OfdsRNase2. When double luciferase assays were performed to evaluate the role of the four dsRNases in vitro, only OfdsRNase2 expressed in S2 cells significantly affected RNAi efficiency by degrading dsRNA. Taken together, our results suggested that the degradation of dsRNA by OfdsRNase2 in the midgut contributed to low RNAi efficiency in O. furnacalis larvae.

Chitinase 10 controls chitin amounts and organization in the wing cuticle of Drosophila.

Wings are essential for insect fitness. A number of proteins and enzymes have been identified to be involved in wing terminal differentiation, which is characterized by the formation of the wing cuticle. Here, we addressed the question whether chitinase 10 (Cht10) may play an important role in chitin organization in the wings of the fruit fly Drosophila melanogaster. Initially, we first found that Cht10 expression coincides with the expression of the chitin synthase coding gene kkv. This suggests that the respective proteins may cooperate during wing differentiation. In tissue-specific RNA interference experiments, we demonstrate that suppression of Cht10 causes an excess in chitin amounts in the wing cuticle. Chitin organization is severely disrupted in these wings. Based on these data, we hypothesize that Cht10 restricts chitin amounts produced by Kkv in order to ensure normal chitin organization and wing cuticle formation. In addition, we found by scanning electron microscopy that Cht10 suppression also affects the cuticle surface. In turn, cuticle inward permeability is enhanced in Cht10-less wings. Moreover, flies with reduced Cht10 function are unable to fly. In conclusion, Cht10 is essential for wing terminal differentiation and function.