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.

Construction of Midgut Tissue-Specific cDNA Library of Bombyx mandarina M. and Isolation and Sequence Analysis of Serine Protease Gene Fragment

[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.

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.

Intrauterine midgut volvulus without malrotation:Diagnosis from the 'coffee bean sign'

Fetal midgut volvulus is quite rare, and most cases are associated with abnormalities of intestinal rotation or fixation. We report a case of midgut volvulus without malrotation, associated with a meconium pellet, during the gestation period. This 2.79 kg, 33-wk infant was born via a spontaneous vaginal delivery caused by preterm labor. Prenatal ultrasound showed dilated bowel loops with the appearance of a 'coffee bean sign'. This patient had an unusual presentation with a distended abdomen showing skin discoloration. An emergency laparotomy revealed a midgut volvulus and a twisted small bowel, caused by complicated meconium ileus. Such nonspecific prenatal radiological signs and a low index of suspicion of a volvulus during gestation might delay appropriate surgical management and result in ischemic necrosis of the bowel. Preterm labor, specific prenatal sonographic findings (for example, the coffee bean sign) and bluish discoloration of the abdominal wall could suggest intrauterine midgut volvulus requiring prompt surgical intervention.

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.

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.

Midgut volvulus due to jejunal diverticula:A case report

Jejunal diverticulosis is uncommon and often asymptomatic.It can produce significant complications,and some complications are potentially life threatening and require early surgical treatment,such as obstruction,hemorrhage and perforation.There is no consensus on the management of this disease.Only a few cases of jejunal diverticulosis with midgut volvulus have been reported.We herein report a case of 57-year-old woman with jejunal diverticulosis causing small bowel volvulus who complained of intermittent upper abdominal pin-prick for 5 years that eventually progressed to a complete obstruction.The computed tomography scans revealed a mesenteric vessel "whirlpool" and laparotomy showed midgut volvulus secondary to jejunal diverticula.This case highlights jejunal diverticulosis causing small bowel volvulus as an uncommon mechanism of small bowel obstruction,which should be included in the differential diagnosis of small bowel obstruction.

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.

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.

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.

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.

Effect of Zinc Sulfate against the Red Palm Weevil Rhynchophorus ferrugineus with Reference to Their Histological Changes on the Larval Midgut and Adult Reproductive System

The red palm weevil Rhynchophorusferrugineus is the most important insect pest for the date palm trees in the Middle East and Gulf states. Zinc sulfate （ZnSOa＇H20） was toxicologically, biologically and physiologically evaluated as insect development disruptors against the RPW. LCs0 of Zinc sulfate against 10 days old larvae was 0.566%, 3% concentration inhibited the larvae from feeding. LCs0 concentration of zinc sulfate solution was capable to disrupting growth, development and reproduction of R. ferrugineus. Growth and development disruptions are resulted from larval feeding on the treated diet that causing percentage of mortality rate, retardation in larval and pupal duration, percentage of pupation, adults emergence, reduction in deposited eggs that laid by the resulting adults and in the egg fertility. The study further reveals that Zinc sulfate effects on histological structure of epithelial cells of the larval midgut including separation and elongation of their cells, rupture in the peritrophic membrane and in some microvilli and some degeneration of the surrounding muscles. Histological changes also was observed on the adults ovary, in this way, disrupt female gamete production through their action on the accumulation of yolk granules （vitellogenesis） and follicular epithelial cells. Disruption of male gamete production was detected by disorganized of testicular cysts and depopulation of these cysts in addition to degeneration in germ cells. Zinc sulfate may be used among other control methods by injection into the tree trunk or as fertilizer around the tree roots which in turn may have an indirect effect for controlling the RPW.

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.

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.

Integrated analysis of miRNAs and transcriptomes in Aedes albopictus midgut reveals the differential expression profiles of immune-related genes during dengue virus serotype-2 infection

Mosquito microRNAs （miRNAs） are involved in host-virus interaction, and have been reported to be altered by dengue virus （DENV） infection in Aedes albopictus （Diptera： Culicidae）. However, little is known about the molecular mechanisms of Aedes albopictus midgut--the first organ to interact with DENV--involved in its resistance to DENV. Here we used high-throughput sequencing to characterize miRNA and messenger RNA （mRNA） expression patterns in Aedes albopictus midgut in response to dengue virus serotype 2. A total of three miRNAs and 777 mRNAs were identified to be differentially expressed upon DENV infection. For the mRNAs, we identified 198 immune-related genes and 31 of them were differentially expressed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses also showed that the differentially expressed immune-related genes were involved in immune response. Then the differential expression patterns of six immune-related genes and three miRNAs were confirmed by real-time reverse transcription polymerase chain reaction. Furthermore, seven known miRNA-mRNA interaction pairs were identified by aligning our two datasets. These analyses of miRNA and mRNA transcriptomes provide valuable information for uncovering the DENV response genes and provide a basis for future study of the resistance mechanisms in Aedes albopictus midgut.

Midgut immune responses induced by bacterial infection in the silkworm, Bombyx mori

Insect gut epithelial cells produce reactive oxygen species （ROS） and antimicrobial peptides （AMPs） to protect hosts from pathogenic microorganisms. In this study, we evaluate the pathogenicity of Pseudomonas aeruginosa and Bacillus bombysepticus in the silkworm, Bombyx mori. Survival curves show that B. bombysepticus is deadly when larval silkworms are infected orally. Bacterial infection caused intestinal hydrogen peroxide （H202） and nitric oxide （NO） levels to increase significantly by 8 and 16 h post-infection （hpi）, respectively. Real-time quantitative polymerase chain reaction （qPCR） analysis shows that the transcription levels of dual oxidase （Duox） and catalase （CAT） are highly up-regulated by P. aeruginosa infection at 8 hpi. P. aeruginosa infection induced nitric oxide synthase 2 （NOS2） expression at 16 hpi, which contributes to the generation of NO. mRNA levels of AMP genes, specifically Glovorin 2 and Glovorin 3, which obviously increase during the eady infection stage. These results indicate that invading bacteria ele.vate intestinal ROS and NO levels and induce AMP gene transcription, which contributes to intestinal immune defense.

Expression analysis of chlorophyllid a binding protein a secretory, red fluorescence protein in the midgut of silkworm, Bombyx mori

Chlorophyllid a binding protein （ chbp） was recently characterized by its ability to bind the prosthetic group of chlorophylls and little information is known regarding its expression. In the present study, we found that chpb was expressed highly and exclusively in the midgut of silkworm, Bombyx mori. The expression level of chbp was very high in the newly molted fifth instar larvae followed by gradual decline in the same instar. Our results demonstrated that CHBP was a secretory protein and located mainly in the apical of midgut epithelial cells. Real-time polymerase chain reaction analysis results showed that chpb highly expressed in the anterior midgut, threefold and sixfold higher compared with that of the middle midgut and posterior midgut, respectively, and chpb expression declined in darkness. In addition, the expression of chbp was affected by high-dose virus or bacterium infection.

Multiple microRNAs control ecdysone signaling in the midgut of Spodoptera litura

Metamorphosis is one of the most important physiological processes in insects.It is regulated by a serial of ecdysone cascade genes.Recently,lots o f microRNAs(miRNAs)were investigated in insects;however,their function in metamorphosis is largely unknown.In the present study,the dynamics of a small RNA population was investigated by RNA sequencing from the midgut of a lepidopteran pest Spodoptera litura during larval-pupal metamorphosis.A total of 101 miRNAs were identified,and 75 miRNAs were differentially expressed during the metamorphic process.The relationship between these differentially expressed miRNAs and 12 ecdysone cascade genes was analyzed by four classical software programs,and a multiple-to-multiple regulatory network was found to exist between these miRNAs and their targets.Among them,miR-14-3p and its two targets(EcR and E75)were chosen for further validation.MiR-14-3p had higher expression level in the 6 th instar larvae as compared with either the prepupae or pupae,which was opposite to that of both EcR and E75,two ecdysone cascade genes.Luciferase reporter assay confirmed that both EcR and E75 were regulated by miR-14-3p.Interestingly,the 3'untranslated regions are nearly identical to each other among different transcript variants of the ecdysone cascade genes,including EcR,USP,E75,E74,E78,E93,Hr3,Hr4,Hr39,K rhl and F tzfl.Thus,different transcript variants of one ecdysone cascade gene could be regulated by the same mi RNA.The above data suggest that the ecdysone signaling pathway is under the tight control of miRNA.These findings expand our understanding of the mechanism of insect metamorphosis and may also provide a novel possibility for the control of pest insects in the future.

A midgut-specific lytic polysaccharide monooxygenase of Locusta migratoria is indispensable for the deconstruction of the peritrophic matrix

Lytic polysaccharide monooxygenases(LPMOs)are important enzymes that boost the hydrolysis of recalcitrant polysaccharides,such as chitin.They are found extensively in different insect species and are classified as auxiliary activities family 15(AA15)LPMOs(LPMO15).Some of them were identified from the insect midgut and proven to act on chitin.However,knowledge about their physiological roles during insect growth and development remains limited.Here,we found that midgut-specific LPMO15s are widely distributed in different insect orders,such as the orthopteran Locusta migratoria and the lepidopteran Bombyx mori.Using L.migratoria as a model insect,the function of midgut-specific LmLPMO15-3 during development was investigated.Double-stranded RNA-mediated downregulation of LmLPMO15-3 expression at the 4th or 5th instar nymph stage severely decreased the survival rate and resulted in lethal phenotypes.Hematoxylin and eosin staining results indicated that the deficient individuals exhibited incompletely digested peritrophic matrix(PM),which suggested that LmLPMO15-3 is essential for the deconstruction of the PM during molting.This study provides direct evidence of the physiological importance of a midgut-specific LPMO15 during insect development.As L.migratoria is one of the most destructive agricultural pests,LmLPMO15-3 is a potential target for pest management.

Ferritin acts as a target site for the snowdrop lectin （GNA） in the midgut of the cotton leafworm Spodoptera littoralis

The snowdrop lectin GNA （Galanthus nivalis agglutinin） has been shown to possess insecticidal activity to a range of economically important insect pests. However, the precise mechanism of insecticidal action of GNA against insects remains unknown. In this investigation, we attempted to purify and identify receptor（s） responsible for binding of GNA in the larval midgut of a major lepidopteran pest （the cotton leafworm, Spodoptera littoralis） to better understand its mode of action. Therefore, cytoplasmic as well as membrane proteins from 800 larval midguts were chromatographed on a column with immobilized GNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the proteins eluted from the GNA column followed by sequencing of the GNA-binding proteins and BLAST analyses revealed that the N-terminal sequences of a 24 kDa polypeptide purified from the cytoplasmic and membrane protein fraction revealed sequence similarity to sequences encoding heavy chain homologs of ferritin from Manduca sexta （76% sequence identity）, Calpodes ethlius （80% sequence identity） and Bombyx mori （61% sequence identity）. Furthermore, the N-terminal sequence of a 31 kDa polypeptide from the membrane protein fraction showed sequence similarity to a light chain homolog of ferritin from Manduca sexta （88% sequence identity）.