A Study on the Activity of Carboxylesterase and the Differential Expression of Its Gene in the Midguts of Bombyx mori Resistant to BmDNV-Z

This study was to discuss the relationship among the change in the activity of Bombyx mori carboxylesterase （BmCarE） in the midguts, the differential expression of BmCarE gene （bmcare） in the midguts, and the ability of Bombyx mori resistant to densonucleosis virus （BmDNV）, and to elucidate the molecular mechanism of resistance to BmDNV-Z. With two silkworm strains, HUABA, which is susceptible to BmDNV-Z, and BC8 （a near isogenic line of HUABA）, which is completely resistant to the same virus, as materials, the activity of BmCarE in the midgut was determined by Bio-Tek Synergy, and the differential expression of bmcare between the two strains was investigated by real-time fluorescence quantitative PCR, both at 12, 36, and 72 h post oral inoculation of the two strains with virus （hereafter referred as inoculation）. While the activity of BmCarE in the midguts of BC8 inoculation group at 12 h post inoculation was higher than that in the BC8 control group, the HUABA inoculated group, and the HUABA control group by 3.28, 2.26, and 3.02 times, respectively, with the difference being highly significant （P 〈 0.01）, there was no statistical difference among the other groups. The relative expression level of bmcare in the midguts of BC8 inoculation group at 12 h post inoculation was higher than that in the BC8 control group, the HUABA inoculation group, and the HUABA control group by 17.714, 21.76, and 15.09 times, respectively, with the difference being highly significant （P 〈 0.01）, and there was no statistical difference among other groups. The elevation of BmCarE activity and expression level of bmcare in the resistant strain at 12 h post inoculation may relate to the resistant gene （nsd/nsd） and the stimulation of BmDNV-Z. The molecular basis for the elevation of BmCarE activity in the resistant strain BC8 may be the change in the expression level of bmcare.

Low-density lipoprotein receptor-related protein 2(LRP2)is required for lipid export in the midgut of the migratory locust,Locusta migratoria

Low-density lipoprotein receptor-related protein 2(LRP2)is a multifunctional endocytic receptor expressed in epithelial cells.In mammals,it acts as an endocytic receptor that mediates the cellular uptake of cholesterol-containing apolipoproteins to maintain lipid homeostasis.However,little is known about the role of LRP2 in lipid homeostasis in insects.In the present study,we investigated the function of LRP2 in the migratory locust Locusta migratoria(LmLRP2).The mRNA of LmLRP2 is widely distributed in various tissues,including integument,wing pads,foregut,midgut,hindgut,Malpighian tubules and fat body,and the amounts of LmLRP2 transcripts decreased gradually in the early stages and then increased in the late stages before ecdysis during the nymphal developmental stage.Fluorescence immunohistochemistry revealed that the LmLRP2 protein is mainly located in cellular membranes of the midgut and hindgut.Using RNAi to silence LmLRP2 caused molting defects in nymphs(more than 60%),and the neutral lipid was found to accumulate in the midgut and surface of the integument,but not in the fat body,of dsLmLRP2-treated nymphs.The results of a lipidomics analysis showed that the main components of lipids(diglyceride and triglyceride)were significantly increased in the midgut,but decreased in the fat body and hemolymph.Furthermore,the content of total triglyceride was significantly increased in the midgut,but markedly decreased in the fat body and hemolymph in dsLmLRP2-injected nymphs.Our results indicate that LmLRP2 is located in the cellular membranes of midgut cells,and is required for lipid export from the midgut to the hemolymphand fat body in locusts.

Azole selenourea disrupted the midgut and caused malformed development of Plutella xylostella

Chemical insecticides targeting the digestive system of diamondback moth(DBM),Plutella xylostella,have not been developed.The discovery of an insecticide with novel mode of action is a challenge for the control of DBM.In this study,a class of selenium-and difluoromethyl-modified azoles(fluoroazole selenoureas,FASU)were designed and synthesized for the control of DBM.Of these azoles,compound B4 showed the highest insecticidal activity against DBM.The LC50of third-and second-instar larvae reached 32.3 and 4.6μg mL^(–1),respectively.The midgut tissue of larvae was severely disrupted,and the larval intestinal tissue was dotted with unique red spots after treatment with compound B4.Compound B4 led to disintegration of the peritrophic matrix,swelling of the midgut epithelium,fracture of the microvilli,and extensive leakage of cellular debris in the midgut lumen.Surviving larvae grew very slowly,and the larval duration was significantly prolonged after exposure to compound B4 at sublethal doses(LC10,LC25and LC50).Furthermore,the pupation rate,emergence rate and pupae weight were significantly decreased.Compound B4 also induced abnormal pupae,causing adults to be trapped in the cocoon or failure to fly due to twisted wings.These results demonstrated that FASU could reduce the population of DBM in sublethal doses.FASU is the first synthetic insecticidal lead compound that has been shown to disrupt the midgut tissue of the larvae of DBM,and its mode of action totally differs from that of commercial chemical insecticides.

野桑蚕中肠组织cDNA文库的构建及丝氨酸蛋白酶基因片段的克隆与序列分析(英文)

[Objective] The aim of the study is to construct cDNA library of midgut tissue of wild silkworm and isolate the serine protease gene. [Method] The midgut tissue-specific cDNA library of wild silkworm was constructed via cDNA Library Construction Kit (TaKaRa), then the serine protease gene was cloned via sequencing of the yielded cDNA library. [Result] The titer of cDNA library reached 6.2×105 pfu/ml, average insert size was about 1.2 kb. The serine protease gene cDNA fragment was obtained from colony sequencing (Accession No: EU672968). The nucleotide sequence of the cloned 854 bp fragment encodes 284 amino acid residues. Homology analyses showed some homology between putative amino acid sequence of the cloned fragment and amino acid sequences of serine proteases from other ten insects. [Conclusion] The results may avail to reveal the resistance of silkworm and wild silkworm to exotic intrusion.

Left-sided appendicitis:Review of 95 published cases and a case report

AIM:To give an overview of the literature on left-sided acute appendicitis (LSAA) associated with situs inversus totalis (SIT) and midgut malrotation (MM).METHODS:We present a new case of LSAA with SIT and a literature review of studies published in the English language on LSAA,accessed via PubMed and Google Scholar databases.RESULTS:Ninety-five published cases of LSAA were evaluated and a 25-year-old female,who presented to our clinic with left lower abdominal pain caused by LSAA,is reported.In the reviewed literature,fiftyseven patients were male and 38 were female with an age range of 8 to 82 years and a median age of 29.1 ± 15.9 years.Sixty-six patients had SIT,23 had MM,three had cecal malrotation,and two had a previously unnoted congenital abnormality.Fifty-nine patients had presentedto the hospital with left lower,14 with right lower and seven with bilateral lower quadrant pain,and seven subjects complained of left upper quadrant pain.The diagnosis was established preoperatively in 49 patients,intraoperatively in 19,and during the postoperative period in five;14 patients were aware of having this anomaly.The data of eight patients were not unavailable.Eleven patients underwent laparoscopic appendectomy,which was combined with cholecystectomy in two cases.Histopathological examination of the appendix specimens revealed adenocarcinoma in only two of 95 patients.CONCLUSION:The diagnosis of left lower quadrant pain is based on well-established clinical symptoms,physical examination and physician's experience.

Histopathological Effects of the Protein Toxin from Xenorhabdus nematophila on the Midgut of Helicoverpa armigera

Xenorhabdus nematophila HB310, which is highly virulent for many insects, is symbiotic with Steinernema carpocapsae HB310. Toxin II was obtained using methods such as salting out and native-PAGE from the cells of X. nematophila HB310. The histopathology of toxin II on H. armigera larvae was studied by dissecting an olefin slice of the midgut. The symptoms showed that the histopathology of the H. armigera midgut was similar to that of other novel midgut-active toxins such as the δ-endotoxins from Bacillus thuringiensis, as well as Tca from Photorhabdus luminescens W14. The midgut tissues of H. armigera fourth-instar larvae began to transform after the oral intake of the toxin Ⅱ over 6 h. First, the anterior region of the peritrophic membrane （PM） began to degrade followed by the elongation of the columnar cells. The epithelium decomposed gradually, and the midgut tissues were either loose or disordered. The PM disappeared after 12 h but reappeared after 72 h following transient or sublethal exposure to the toxin Ⅱ. Toxin Ⅱ also directly destroyed in vitro PMs of H. armigera.

Malrotation: Current strategies navigating the radiologic diagnosis of a surgical emergency

The most accurate and practical imaging algorithm for the diagnosis of intestinal malrotation can be a complex and sometimes controversial topic.Since 1900,sig-nificant advances have been made in the radiographic assessment of infants and children suspected to have anomalies of intestinal rotation.We describe the cur-rent methods of abdominal imaging of malrotation along with their pros and cons.When associated with volvulus,malrotation is a true surgical emergency re-quiring rapid diagnosis and treatment.We emphasize the importance of close cooperation and communica-tion between radiology and surgery to perform an effective and efficient diagnostic evaluation allowing prompt surgical decision making.

Effects of inhibitors on the protease profiles and degradation of activated Cry toxins in larval midgut juices of Cnaphalocrocis medinalis (Lepidoptera:Pyralidae)

Midgut juice plays an important role in food digestion and detoxification in insects.In order to understand the potential of midgut juice of Cnaphalocrocis medinalis(Guenée)to degrade Bt proteins,the enzymatic activity of midgut juice and its degradation of Bt proteins(Cry2A,Cry1C,Cry1Aa,and Cry1Ac)were evaluated in this study through protease inhibitor treatments.The activities of total protease in midgut juices were significantly inhibited by phenylmethylsulfonyl fluoride(PMSF),tosyl-L-lysine chloromethyl ketone(TLCK),pepstatin A and leupeptin.The enzymatic activity of chymotrypsin was significantly inhibited by PMSF,and enzymatic activity of trypsin was significantly inhibited by ethylenediaminetetraacetic acid(EDTA),PMSF,tosyl phenylalanine chloromethyl ketone(TPCK),TLCK and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane(E-64).EDTA could significantly inhibit the degradation of Cry2A by C.medinalis.EDTA,PMSF,TPCK,and TLCK could inhibit the degradation of Cry1C and Cry1Aa.EDTA,PMSF,TPCK,TLCK,and E-64 could inhibit the degradation of Cry1Ac.Our results indicated that some protease inhibitors hindered various enzymatic activities in the larval midgut of C.medinalis,which may reduce the insect’s ability to degrade Bt toxins.These findings may aid the application of protease inhibitors in the management of this insect pest in the future.

Effect of Exogenous MJA Treatment of Tea Plants on the Growth of Geometrid Larvae

The effect of tea plant Camellia sinensis induced by exogenous methyl jasmonate （MJA） on lipoxygenase （LOX）, polyphenol oxidase （PPO） and proteinase inhibitor （PI） activity in the leaves of tea plants, as well as the growth and midgut proteinase activity of the geometrid Ectropis obliqua larvae were studied. MJA significantly induced LOX, PPO and PI activity in leaves of tea plants. When geometrid larvae have fed on leaves of tea plants treated with MJA, the activities of the high alkaline trypsin-like enzyme and chymotrypsin-like enzyme in their midgut were significantly inhibited, but the activities of the low alkaline trypsin-like enzyme in their midgut were unaffected, leading to imbalance between different types of proteinase activity in the midgut of the larvae and in turn, the growth were inhibited. These chains of response may be an important mechanism of the direct resistance induced by MJA-treatment of tea plant on geometrid larvae.

Intestinal obstruction in pregnancy with reverse rotation of the midgut:A case report

BACKGROUND Reverse rotation of the midgut is a rare type of intestinal malrotation.Volvulus of the right colon or entire midgut,stenosis of the transverse colon and obstruction of the duodenojejunal junction are common complications of reverse rotation.In this study,we report the first case of intestinal obstruction associated with reverse rotation in pregnancy.CASE SUMMARY A 31-year-old woman at 362+wk gestation presented to the emergency department with progressive abdominal cramping,nausea and bilious vomiting.Abdominal ultrasound scanning showed dilatation of the bowel.Computed tomography scanning revealed features of reverse rotation of the midgut with intestinal volvulus.After consultation with the obstetrician,the pregnancy was terminated and exploratory abdominal surgery was performed.Intra-operatively,it was found that the mesentery of the colon and small intestine was insufficiently attached.The right colon and the small intestinal mesentery was twisted,and intestinal necrosis was observed.The duodenum and duodenojejunal junction were curved in front of the transverse colon,and the transverse colon passed through the tunnel behind the mesenteric root.Intestinal reverse rotation with volvulus was confirmed.The necrotic intestine was resected and small intestine mesenteric reconstruction was performed.The patient recovered after surgery.After leaving the hospital,the patient and her daughter remained well during an 8-month follow-up period.CONCLUSION We report the diagnosis,treatment and etiology of a pregnant patient with intestinal obstruction due to reverse rotation of the midgut.For similar cases,appropriate diagnosis and treatment should be carried out according to the condition of the fetus and pregnant woman.

Environmental stress upon hepatopancreatic cells of freshwater prawns (Decapoda: Caridea) from the floodplain of ParanáRiver

In order to evaluate the influence of stressed environments on hepatopancreatic cells of fre- shwater prawns, Macrobachium borellii Nobili, 1896 and Palaemonetes argentinus Nobili 1901, (Crustacea, Decapoda, Palaemonidae) were collected at three different aquatic environments with different relationship to urban development in Argentina. Furthermore the effects of several cypermethryn concentration on hepa- topancreatic cell of M. borellii and P. argentinus were evaluated in a laboratory assays. The “N°1” lake (Santa Fe Argentina) which is more affected by the anthropogenic pressure and “Don Felipe” lake which is still not strongly by human activities were the studied sites from the floodplain of Paranáriver. While Alejandra lake was the intermedia effects sites. Different damaged ultra-structures were found in F- and R-cells of prawns in the stressed lake. The predominant features were: disrupted the microvillous border, swelled mitochondria, reduction of endoplasmic reticulum, dyctiosomes, glycoproteins, desna-turalization of vacuole membrane and premature autolysis. Moreover the F-cell number was higher in the environment near to city than in the others sites. Similar effects were observed in the cypermethryn assays. The observations clearly indicate that the ultrastructure of midgut gland in the both palaemonids varies depending on the site from which animals are collected and the biocid presence. So, in this case it can be stated that the hepatopancreas histology of fre- shwater prawns is a good tool to monitor the impact of a stressed environment upon freshwater prawns.

Analysis of Protein Expression Patterns of Silkworm Jinqiu and Its Cross Parents

The differences of protein expression between the improved cross breeding race Jinqiu and its parents were analyzed to discuss the gene construction, and to form a base for illuminating the molecular mechanisms of successful cross breeding in silkworm. Protein samples from silk gland, hemolymph, and midgut were separated by 2-dimensional gel electrophoresis （2-DE）. In the three tissues the matched protein spots between Jinqiu and its cross parents were approximately 70% with approximately 30% specific protein spots. In the matched protein spots, 9-24% was differentially expressed representing up- and down-regulated expression. These specific protein spots might be either the newly appeared, which were produced from the genic interaction of cross parents＇ genes in cross breeding, or posttranscriptionally modified, which were produced from the different modifications on the same original proteins. These results indicate that it is important for a new successful breed, by cross breeding, relying on the actions of some newly produced functional proteins from genic interaction, in addition to marshaling excellent genes of cross parents.

Use of Ultrastructuke and DNA Hybridization to Detect Replication of the Nuclear Polyhedrosis Virus in Midgut Cells of Homologous Host

It was controversial issue if nuclear polyhedrosis virus(NPV) could replicate in midgut cells of host larvae from Lepidoptera by now.The replication of Mythimna separata NPV(MsNPV) in M.separata larvae midgut cells was studied by ultrastructural and DNA hybridized techniques.The paper demonstrated that the MsNPV could neither infect midgut cell nor replicate in midgut cell of homologous host.Therefore MsNPV virions released from the virial occlusion bodies were considered as direct penetration though the intercellular spaces of midgut cells to hemocoel of the host larvae.

Situs Inversus Totalis with Left-Sided Appendicitis: A Case Report

Introduction: Left-sided acute appendicitis (LSAA) develops in association with two types of congenital anomalies: situs inversus totalis (SIT) and midgut malrotation (MM). A Left sided appendicitis is an ambiguous and difficult diagnosis to make. Aim: To present a proven case of left-sided acute appendicitis (LSAA) associated with situs inversus totalis (SIT). Case Report: A case of Left appendicitis was evaluated in a 28-year-old Asian male, who presented to our hospital in Feb. 2016, with lower abdominal pain more on left side and suspected diverticulitis or acute appendicitis with unusual appendix location. The patient doesn’t recall any history of abdominal surgery or about situs inversus totalis, abdominal and pelvic ultrasound was done, left iliac fossa appendicitis was diagnosed, Erect chest X-ray including upper abdomen revealed dextrocardia and stomach air on right side (situs inversus totalis), the patient underwent diagnostic Laproscop and Endoscopic resection of the appendix, with no incidents, and then discharged without complications, follow visits went unremarkable. Conclusion: The diagnosis of left lower quadrant pain is based on well-established clinical symptoms, physical examination and physician’s experience.

The Midgut Bacterial Flora of Laboratory-Reared Hard Ticks, Haemaphysalis longicornis, Hyalomma asiaticum, and Rhipicephalus haemaphysaloides

Ixodid ticks play an important role in the transmission of a variety of zoonoses of viral, bacterial and protozoan origin, and they also harbor a wealth of microorganisms. To gain more detailed insights into the potential interactions between bacterial flora and tick-borne pathogens, we investigated the midgut bacterial flora of laboratory-reared Haemaphysalis longicornis, Hyalomma asiaticum and Rhipicephalus haemaphysaloides. Based on morphological, biochemical, and 16 S rDNA sequencing results, we identified 15 species belonging to 12 genera in the midgut of the three ticks. The bacterial communities were similar to those found in other studies of hematophagous arthropods. Kocuria sp. was the most frequently isolated species and its 16 S rDNA gene sequence was very similar to Kocuria koreensis P31 T. To our knowledge, this is the first report of the bacterial flora of tick midguts and the results show that there were many different bacterial species in each tick species. Among the most common genera, there may have been a novel species in the genus Kocuria. The results might be the first step for looking for different aspects of the pathogen and tick interaction.

Left Sided Acute Appendicitis: Radiological Aspects

Left sided appendicitis is a rare pathology. Two situations may explain the occurrence of the disease: situs inversus or midgut malrotation. Its diagnosis is based on clinical presentation but confirmed by radiological examination. Our observation is based on a left-sided acute appendicitis with a midgut malrotation in a 31-year-old patient with no clinical history.

A Case Report of Adult Patent Vitellointestinal Duct Sac and Midgut Volvulus

Midgut volvulus is a clinical rare condition which appears as recurrent intermittent abdominal pain after consumption of food with intermittent colicky pain and sometimes with completely asymptomatic period. This gut twist may result in complications such as ischemia, obstruction, hemorrhage, or perforation. In the yolk sac during the third week of intrauterine life, there is normal communication with intraembryonic gut. During development proceeds, this communication gets thinner into a tube known as the vitellointestinal duct. Vitellointestinal duct usually becomes obliterated before eighth week of intrauterine life. In about 2% of people this duct persists and gives rise to a group of anomalies such as Meckel’s diverticulum is the commonest and complete patency of the duct is the rarest. Here we present the case of a 34-year-old male who presented with both conditions.

Subcellular localization of the stripe disease-specific protein encoded by rice stripe virus (RSV) in its vector,the small brown planthopper,Laodel-phax striatellus

The stripe disease-specific protein (SP) encoded by the rice stripe virus (RSV) was successfully used as a localization signal of the virus in its vector, the small brown lanthopper, Laodelphax striatellus Fallen. Immunogold particles in large numbers were detected in various parts of the viruliferous females: the ovum, surface of chorion, the mid-gut lumen, and the columnar cells. Whereas there was noneof these particles in the non-viruliferous females and males, and testis of viruliferous males. Endosymbionts (mycetocytes) were abundant, harboring ovaries of both viruliferous and non-viruliferous females, but none in the testis of males. The results provided us with the direct proof that RSV is a ciruculative and propagative plant virus and it was transovarially transmitted alongside with endosymbionts of its vector. Therefore, we deem it a nice lead for future studies on the mechanism of RSV transmission and functioning of its viral proteins.

Detoxification of insecticides, allechemicals and heavy metals by glutathione S-transferase SIGSTE1 in the gut of Spodoptera litura

Insect glutathione S-transferases （GSTs） play important roles in detoxifying toxic compounds and eliminating oxidative stress caused by these compounds. In this study, detoxification activity of the epsilon GST S1GSTE1 in Spodoptera litura was analyzed for several insecticides and heavy metals. S1GSTE1 was significantly up-regulated by chlor- pyrifos and xanthotoxin in the midgut of S. litura. The recombinant S1GSTE1 had Vmax （reaction rate of the enzyme saturated with the substrate） and Km （michaelis constant and equals to the substrate concentration at half of the maximum reaction rate of the enzyme） values of 27.95 ± 0.88μmol/min/mg and 0.87 ± 0.028 mmol/L for glutathione, respec- tively, and Vmax and Km values of 22.96 ± 0,78μmol/min/mg and 0.83 ± 0.106 mmol/L for 1-chloro-2,4-dinitrobenzene, respectively. In vitro enzyme indirect activity assay showed that the recombinant S1GSTE 1 possessed high binding activities to the insecticides chlor- pyrifos, deltamethrin, malathion, phoxim and dichloro-diphenyl-trichloroethane （DDT）. S1GSTE1 showed higher binding activity to toxic heavy metals cadmium, chromium and lead than copper and zinc that are required for insect normal growth. Western blot analysis showed that S1GSTE1 was induced in the gut of larvae fed with chlorpyrifos or cadmium. S1GSTE1 also showed high peroxidase activity. All the results together indicate that S1G- STE1 may play an important role in the gut orS. litura to protect the insect from the toxic effects of these compounds and heavy metals.

A midgut-specific serine protease, BmSP36, is involved in dietary protein digestion in the silkworm, Bombyx mori

Serine proteases play important roles in digestion and immune responses during insect development. In the present study, the serine protease gene BmSP36, which encodes a 292-residue protein, was cloned from the midgut cells ofBombyx mori. BmSP36 contains an intact catalytic triad （H57, D102 and S195） and a conserved substrate-binding site （G189, H216 and G226）, suggesting that it is a serine protease with chymotrypsin- like specificity. The temporal and spatial expression patterns of BmSP36 indicated that its messenger RNA and protein expression mainly occurred in the midgut at the feeding stages. Western blotting, immunofluorescence and liquid chromatography-tandem mass spectrometry analyses revealed secretion of BmSP36 protein from epithelial cells into the midgut lumen. The transcriptional and translational expression of BmSP36 was down- regulated after starvation but up-regulated after refeeding. Moreover, expression of the BmSP36 gene could be up-regulated by a juvenile hormone analogue. These results enable us to better define the potential role of BmSP36 in dietary protein digestion at the feeding stages during larval development.