Midgut Extracts Research Articles

Functional characterization of novel RbTI gene from ricebean and validation of its insecticidal properties in transgenic tobacco.

Plant protease inhibitors (PI's) inhibit the activity of gut proteases and thus provide resistance against insect attack. Previously we have published first report on cloning and characterization of a novel Bowman-Birk protease inhibitor gene (RbTI) from ricebean (Vigna umbellata). In this study, the RbTI gene was further characterized and validated as a potential candidate for transferring insect resistance in economically important crops. We have successfully generated transgenic tobacco plants expressing RbTI gene constitutively under CaMV35S promoter using Agrobacterium transformation. Genomic PCR and GUS analysis confirmed the successful integration of RbTI gene into tobacco plant genome. qRT-PCR analysis revealed highest RbTI gene expression in transformed tobacco leaves nearing maturity. Feeding of transformed tobacco leaf tissue showed prominent effect on larval mortality throughout the larval growth stages mainly during first three days of feeding. For functional analysis of RbTI gene, we estimated the inhibitory activity of protein extracts from normal and transformed tobacco plants against gut proteases of Spodoptera litura and H. armigera larval instars. Maximum inhibition of trypsin (82.42% and 73.25%) and chymotrypsin (69.50% and 60.64%) enzymes was recorded at early larval stages of both insects. The results of this study validated the future use of RbTI gene from ricebean legume as a potential candidate for transferring insect resistance in economically important crops. Insight, innovation, integration: Present study was conducted with the aim to utilize the state of art biotechnological techniques for transferring key pest resistant genes from underutilized promising crop ricebean. The tobacco plant has been utilized as modern plant for proof of concept where a protease inhibitor gene from Ricebean has been transferred to tobacco plant which induced larval mortality within first three days of feeding at all larval developmental stages. The biochemical assays on mid-gut total protein extract showed that the transgenic tobacco leaves have inhibiting effect on trypsin and chymotrypsin enzymes of insect which is otherwise required for digestion of food by them. Hence, we provide a novel gene that could be utilized for pest resistance in other crops different developmental stages.

Processing Properties and Potency of Bacillus thuringiensis Cry Toxins in the Rice Leaffolder Cnaphalocrocis medinalis (Guenée).

Different Cry toxins derived from Bacillus thuringiensis (Bt) possess different insecticidal spectra, whereas insects show variations in their susceptibilities to different Cry toxins. Degradation of Cry toxins by insect midgut extracts was involved in the action of toxins. In this study, we explored the processing patterns of different Cry toxins in Cnaphalocrocis medinalis (Lepidoptera: Crambidae) midgut extracts and evaluated the impact of Cry toxins degradation on their potency against C. medinalis to better understand the function of midgut extracts in the action of different Cry toxins. The results indicated that Cry1Ac, Cry1Aa, and Cry1C toxins could be degraded by C. medinalis midgut extracts, and degradation of Cry toxins by midgut extracts differed among time or concentration effects. Bioassays demonstrated that the toxicity of Cry1Ac, Cry1Aa, and Cry1C toxins decreased after digestion by midgut extracts of C. medinalis. Our findings in this study suggested that midgut extracts play an important role in the action of Cry toxins against C. medinalis, and the degradation of Cry toxins by C. medinalis midgut extracts could reduce their toxicities to C. medinalis. They will provide insights into the action of Cry toxins and the application of Cry toxins in C. medinalis management in paddy fields.

Effects of saline extract and lectin from Punica granatum seed sarcotesta on Aedes aegypti larvae

• Salin extract contained lectin, ellagic acid, α and β punicalagins, ellagic acid derivatives, and cinnamic acid derivatives. • The extract was able to induce mortality in A. aegypti larvae. • The lectin PgTeL did not show larvicidal activity. • The extract altered midgut morphology and inhibited amylase and trypsin activity in the larval gut. Currently, one of the main strategies to reduce the incidence of the arboviruses such as Zika, dengue, and chikungunya is through the population control of their vector, Aedes aegypti . Lectins are carbohydrate-binding proteins with several biological properties, including insecticidal activity against A. aegypti. Punica granatum sarcotesta contains a chitin-binding lectin (PgTeL), which prompted our evaluation of its toxicity against A. aegypti . Here, we analyzed the effects of saline extract from P. granatum sarcotesta and PgTeL on the survival of A. aegypti larvae. Saline extract of P. granatum was evaluated for the presence of secondary metabolites; PgTeL was further isolated and purified from this extract. Larvicidal assay was performed using P. granatum saline extract (0.17–1.20 mg/mL of protein) and isolated PgTeL (0.1–1.0 mg/mL). Histological analysis was performed to evaluate the effects of the extract on the larvae midgut morphophysiology. Additionally, the activity of digestive enzymes in midgut extracts of the larvae was determined. P. granatum extract, containing protein (17.1 mg/mL), ellagic acid (2.1 mg/mL), α-punicalagin (2.2 mg/mL), β-punicalagin (2.3 mg/mL), ellagic acid derivatives (2.2 mg/mL), and cinnamic acid derivatives (3.9 mg/mL), caused larvae mortality (LC 50/48 h : 0.67 mg/mL of protein) and altered the midgut morphology; midgut cells were affected, showing protrusions in the apical region and a thickening of the peritrophic matrix. P. granatum extract inhibited amylase and trypsin activities in the larval gut. However, PgTeL did not display larvicidal activity. In conclusion, the sarcotesta saline extract from pomegranate shows larvicidal activity against A. aegypti, and the mode of action may involve disruption of the digestive processes of the larvae. These findings may represent a first step towards the availability of a new tool to control A. aegypti .

Immunization of guinea pigs with cement extract induces resistance against Ixodes scapularis ticks

As hematophagous parasites, many tick species are important vectors of medical and veterinary disease agents. Proteins found in tick saliva and midgut have been used with some success in immunizations of animal hosts against feeding ticks, and whole saliva has been used effectively in this capacity against Ixodes scapularis, the primary vector of tickborne pathogens in the United States. Tick saliva is a complex substance containing hundreds of proteins, and the identification of specific protective antigens is ongoing. We performed a series of experiments immunizing guinea pigs with extracts prepared from midgut or attachment cement collected from adult female I. scapularis followed by challenge with nymphs of the same species. Midgut extract did not induce protective immunity, while immunization with cement extract resulted in partial protection of hosts as evidenced by premature tick detachment and 34-41% reduction in tick engorgement weights. Proteomic characterization of I. scapularis cement was performed, demonstrating that the cement extract was compositionally different from tick saliva, and vitellogenin-like lipoproteins were the most abundant proteins in cement extract (>40%). Cement was also heavily enriched with lysozymes and defensins, including those originating from both the mammalian host as well as ticks. These results demonstrate that I. scapularis cement contains immunogenic components capable of stimulating host resistance against tick feeding. Because the cement is present at the tick-host interface for an extended period of time during the feeding process, these antigens present auspicious candidates for further evaluation and potential inclusion in an anti-tick vaccine.

Resistance in Spodoptera litura (F.) to Insecticides and Detoxification Enzymes

Standard leaf dip bioassay experiments were conducted to study the insecticide resistance in Spodoptera litura (F.) in soybean. Chlorantraniliprole was the most toxic while, cypermethrin and organophosphates (profenophos and triazophos) recorded least toxicity against S. litura populations. Field population of S. litura showed the highest resistance to cypermethrin with resistance ratios (RRs) ranging from 244 to 376. The field populations collected from three locations of Southern Rajasthan exhibited low resistance to newer insecticides, chlorantraniliprole and spinetoram with Lab-SS strain. All the enzyme activities were significantly higher in the whole larval extracts and midgut extracts of S. litura larvae when compared to the Lab-SS strain. Analysis of the carboxyl esterase enzyme in the native PAGE assay revealed that the electrophoretic profiles showed presence of different band regions (EST 1 to EST 7) with esterase activity in the field populations along with the laboratory susceptible strain. The susceptibility of three field populations to different group of insecticides along with the profile of detoxification enzymes indicate the need to formulate a region-specific insecticide resistance management (IRM).

Insecticidal Serralysin of Serratia marcescens Is Detoxified in M3 Midgut Region of Riptortus pedestris.

Riptortus pedestris insect indiscriminately acquires not only the symbiotic bacterium Burkholderia insecticola, but also entomopathogens that are abundant in the soil via feeding. However, it is unclear how the host insect survives oral infections of entomopathogens. A previous study suggested that serralysin, a potent virulence factor produced by Serratia marcescens, suppresses cellular immunity by degrading adhesion molecules, thereby contributing to bacterial pathogenesis. Here, we observed that S. marcescens orally administered to R. pedestris stably colonized the insect midgut, while not exhibiting insecticidal activity. Additionally, oral infection with S. marcescens did not affect the host growth or fitness. When co-incubated with the midgut lysates of R. pedestris, serralysin was remarkably degraded. The detoxification activity against serralysin was enhanced in the midgut extract of gut symbiont-colonizing insects. The mRNA expression levels of serralysin genes were negligible in M3-colonizing S. marcescens. M3-colonizing S. marcescens did not produce serralysin toxin. Immunoblot analyses revealed that serralysin was not detected in the M3 midgut region. The findings of our study suggest that orally infected S. marcescens lose entomopathogenicity through host-derived degrading factors and suppression of serralysin.

A versatile inhibitor of digestive enzymes in Aedes aegypti larvae selected from a pacifastin (TiPI) phage display library

In Brazil, the major vector of arboviruses is Aedes aegypti, which can transmit several alpha and flaviviruses. In this work, a pacifastin protease inhibitor library was constructed and used to select mutants for Ae. aegypti larvae digestive enzymes. The library contained a total of 3.25 × 105 cfu with random mutations in the reactive site (P2–P2’). The most successfully selected mutant, TiPI6, a versatile inhibitor, was able to inhibit all three Ae. aegypti larvae proteolytic activities, trypsin-like, chymotrypsin-like and elastase-like activities, with IC50 values of 0.212 nM, 0.107 nM and 0.109 nM, respectively. In conclusion, the TiPI mutated phage display library was shown to be a useful tool for the selection of an inhibitor of proteolytic activities combined in a mix. TiPI6 is capable of controlling all three digestive enzyme activities present in the larval midgut extract. To our knowledge, this is the first time that one inhibitor containing a Gln at the P1 position showed inhibitory activity against trypsin, chymotrypsin, and elastase-like activities. TiPI6 can be a candidate for further larvicidal studies.

Opuntia ficus-indica cladode extract is an embryotoxic, larvicidal, and oviposition-deterrent agent for the diamondback moth, Plutella xylostella

The search for new compounds to control Plutella xylostella, the main pest of Brassica crops, has been stimulated by the environmental impact of the insecticides currently used and reports of resistant populations. This work evaluated the effects of a cladode extract from Opuntia ficus-indica (CE) on the survival and development of P. xylostella, as well as on its oviposition behavior. Additionally, the effects on the histological organization of the larval midgut, as well as digestive enzyme activities, were evaluated. CE contained flavonoids, cinnamic derivatives, terpenes, steroids, and reducing sugars. High-performance liquid chromatography analysis confirmed the presence of rutin and compounds containing aliphatic chains and aromatic rings in their structures. CE reduced the viability of eggs with an EC50 (concentration that reduces egg viability by 50%) at 5.37% (w/v) and killed larvae with an LC50 (concentration that kills 50% of larvae) at 7.75% (w/v). Histological analysis revealed several alterations in the midgut of larvae exposed to the extract, including the presence of spherites, cell nuclei expulsion, the disappearance of the peritrophic membrane, and an increase in the number of goblet cells. CE inhibited the in vitro activity of proteases from larvae midgut extracts. The extract also presented oviposition-deterrent activity. In conclusion, cladodes of O. ficus-indica are a source of compounds toxic to eggs and larvae of P. xylostella and are able to interfere with oviposition site choice. The results suggest potential of CE as a botanical insecticide, though field trials are needed to confirm activity.

Bacillus thuringiensis Cry1C resistance development and its processing pattern in Egyptian cotton leaf worm: Spodoptera littoralis (Boisd.) (Lepidoptera:Noctuidae)

Biopesticides based on the entomopathogenic bacterium, Bacillus thuringiensis toxins, have shown high ability to control several pests belonging to order Lepidoptera. Egyptian cotton leaf worm, Spodoptera littoralis (Boisd.) (Lepidoptera:Noctuidae), is a major pest that attacks various crops in Egypt, and Cry1C toxin-based formulations are heavily used to its control. A laboratory investigation was conducted to study the resistance development in S. littoralis against Cry1C toxin. Thus, a field strain of S. littoralis was selected and maintained under laboratory conditions for 12 generations. The selection pressure resulted in 32.12-fold of resistance ratio after 12 generations. The hydrolysis of Cry1C toxin by the midgut extracts of the susceptible line, and the resistant line was compared. Results revealed that Cry1C toxin was hydrolyzed more rapidly in the resistant line than the susceptible one. The Cry1C toxin was processed till 1 hour after incubation in the susceptible line; but in case of resistant line, the Cry1C toxin was degraded in 1 hour after incubation.

Blood Digestion by Trypsin-Like Serine Proteases in the Replete Lyme Disease Vector Tick, Ixodes scapularis.

Ixodes scapularis is the major vector of Lyme disease in the Eastern United States. Each active life stage (larva, nymph, and adult) takes a blood meal either for developing and molting to the next stage (larvae and nymphs) or for oviposition (adult females). This protein-rich blood meal is the only food taken by Ixodes ticks and therefore efficient blood digestion is critical for survival. Studies in partially engorged ticks have shown that the initial stages of digestion are carried out by cathepsin proteases within acidic digestive cells. In this study, we investigated the potential role of serine proteases in blood digestion in replete ticks. RNA interference was used for functional analysis and a trypsin-benzoyl-D, L-arginine 4-nitoanilide assay was used to measure active trypsin levels. Hemoglobinolytic activity was determined in vitro, with or without a serine protease inhibitor. Our data suggest that trypsin levels increase significantly after repletion. Knockdown of serine proteases negatively impacted blood feeding, survival, fecundity, levels of active trypsin in the midgut, and resulted in lower hemoglobin degradation. Incubation of midgut extract with a trypsin inhibitor resulted in 65% lower hemoglobin degradation. We provide evidence of the serine proteases as digestive enzymes in fully engorged, replete females. Understanding the digestive profile of trypsin during blood meal digestion in I. scapularis improves our understanding of the basic biology of ticks and may lead to new methods for tick control.

PH influences the profiles of midgut extracts in Cnaphalocrocis medinalis (Guenée) and its degradation of activated Cry toxins

Midgut extracts play crucial roles in food digestion and detoxification. We evaluated the effect of pH on the profiles of the midgut extracts in rice leaffolder, Cnaphalocrocis medinalis and the degradation of activated Bt-toxins by the midgut extracts under different pH conditions. Total protease activity increased slightly with the increase with the simulated pH in the midgut extracts and the maximal protease activity was observed at pH 10.5, while an upward trend was observed as the pH of reaction buffer increased. Activity of chymotrypsin-like enzymes increased with pH, both in the buffer and midgut extracts, while the activity of trypsin-like enzyme was unaffected. Degradation of the activated Cry2A by the midgut extracts enhanced as the pH increased. Cry2A was fully degraded into smaller segments at pH 9.0–10.5. Activated Cry1C protein at pH 9.0–10.5 was partially degraded by the midgut extracts. Activated Cry1Aa and Cry1Ac were partially degraded into fragments by the midgut extracts at high pH. These results will facilitate our further understanding of the interactions between C. medinalis and the Cry toxin.

Enzymatic hydrolysis of cellulose using extracts from insects

The use of Zophobas morio extracts in the aspect of cellulose hydrolysis is presented for the first time. The aim of this study was to investigate the action of enzymes obtained from Z. morio on cellulose hydrolysis and to determine their influence on the structural properties of cellulose with use the Fourier transform infrared spectroscopy (FTIR) and gel permeation chromatography (GPC). Cellulose hydrolysis products were analyzed by high performance liquid chromatography (HPLC). This analysis indicated that microcrystalline cellulose with smaller particle size was more susceptible to enzymatically treatment.Moreover, our investigation of cellulase activity showed a different profile of the used enzyme during particular developmental stages of Z. morio. Midgut extracts obtained from adult insects are more effective in degrading cellulose than extracts from larvae. The analysis of cellulose hydrolysis confirms that the efficiency of this reaction also depends on the structure of cellulosic materials and internal conditions of enzymatic reaction. In this study the cellulolytic activity of Z. morio midgut extracts showed that these insects could be valuable sources of cellulases.

Preliminary Evaluation of Tick Protein Extracts and Recombinant Ferritin 2 as Anti-tick Vaccines Targeting Ixodes ricinus in Cattle.

Anti-tick vaccines have the potential to be an environmentally friendly and cost-effective option for tick control. In vaccine development, the identification of efficacious antigens forms the major bottleneck. In this study, the efficacy of immunization with recombinant ferritin 2 and native tick protein extracts (TPEs) against Ixodes ricinus infestations in calves was assessed in two immunization experiments. In the first experiment, each calf (n = 3) was immunized twice with recombinant ferritin 2 from I. ricinus (IrFER2), TPE consisting of soluble proteins from the internal organs of partially fed I. ricinus females, or adjuvant, respectively. In the second experiment, each calf (n = 4) was immunized with protein extracts from the midgut (ME) of partially fed females, the salivary glands (SGE) of partially fed females, a combination of ME and SGE, or adjuvant, respectively. Two weeks after the booster immunization, calves were challenged with 100 females and 200 nymphs. Blood was collected from the calves before the first and after the second immunization and fed to I. ricinus females and nymphs using an in vitro artificial tick feeding system. The two calves vaccinated with whole TPE and midgut extract (ME) showed hyperemia on tick bite sites 2 days post tick infestation and exudative blisters were observed in the ME-vaccinated animal, signs that were suggestive of a delayed type hypersensitivity (DTH) reaction. Significantly fewer ticks successfully fed on the three animals vaccinated with TPE, SGE, or ME. Adults fed on the TPE and ME vaccinated animals weighed significantly less. Tick feeding on the IrFER2 vaccinated calf was not impaired. The in vitro feeding of serum or fresh whole blood collected from the vaccinated animals did not significantly affect tick feeding success. Immunization with native I. ricinus TPEs thus conferred a strong immune response in calves and significantly reduced the feeding success of both nymphs and adults. In vitro feeding of serum or blood collected from vaccinated animals to ticks did not affect tick feeding, indicating that antibodies alone were not responsible for the observed vaccine immunity.

Protease inhibitory, insecticidal and deterrent effects of the trypsin-inhibitor benzamidine on the velvetbean caterpillar in soybean.

The recognition of protease inhibitors with insecticidal activity is important as a basis for the development of mimetic peptides with potential use as biorational insecticides. We sprayed benzamidine on soybean plants and assessed whether this potent synthetic trypsin-inhibitor has protease inhibitory, insecticidal and deterrent effects on the velvetbean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Erebidae). Activity of trypsin inhibition in soybean leaves was increased and total proteolytic activity in the midgut extract from larvae fed on these leaves was reduced by benzamidine. Different concentrations of benzamidine sprayed on the plant caused approximately 50 % of larval mortality, and larval choice and moth preference and oviposition were all negatively affected. Low concentrations of benzamidine increased mortality and hindered insect choice and oviposition as well as higher doses. Since many synthetic protease inhibitors are usually expensive, small doses of benzamidine may be effective to protect soybean against A. gemmatalis attack. Our results highlight the potential of synthetic protease inhibitors for insecticidal and deterrent purposes in insect pest management.

Decreased Cry1Ac activation by midgut proteases associated with Cry1Ac resistance in Helicoverpa zea.

Field-evolved resistance of Helicoverpa zea to Bacillus thuringiensis (Bt) toxin Cry1Ac was first reported more than a decade ago, yet the underlying mechanisms remain elusive. Towards understanding the mechanisms of resistance to Cry1Ac, we analyzed a susceptible (LAB-S) and two resistant (GA and GA-R) strains of H. zea. The GA strain was derived from Georgia and exposed to Bt toxins only in the field. The GA-R strain was derived from the GA strain and selected for increased resistance to Cry1Ac in the laboratory. Resistance to MVPII, a liquid formulation containing a hybrid protoxin similar to Cry1Ac, was 110-fold for GA-R and 7.8-fold for GA relative to LAB-S. In midgut brush border membrane vesicles, activity of alkaline phosphatase and aminopeptidase N did not vary significantly among strains. The activity of total proteases, trypsin-like proteases and chymotrypsin-like proteases was significantly lower for GA-R and GA than LAB-S, but did not differ between GA-R and GA. When H. zea midgut cells were exposed to Cry1Ac protoxin that had been digested with midgut extracts, toxicity was significantly lower for extracts from GA-R and GA relative to extracts from LAB-S, but did not differ between GA-R and GA. Transcriptional analysis showed that none of the five protease genes examined was associated with the decline in Cry1Ac activation in GA-R and GA relative to LAB-S. The results suggest that decreased Cry1Ac activation is a contributing field-selected mechanism of resistance that helps explain the reduced susceptibility of the GA-R and GA strains. Relative to the LAB-S strain, the two Cry1Ac-resistant strains had lower total protease, trypsin and chymotrypsin activities, a lower Cry1Ac activation rate, and Cry1Ac protoxin incubated with their midgut extracts was less toxic to H. zea midgut cells. © 2018 Society of Chemical Industry.

Benzothiadiazole and B-Aminobutyricacid Induce Resistance to Ectropis Obliqua in Tea Plants (Camellia Sinensis (L.) O. Kuntz).

In order to investigate the effect of benzothiadiazole (BTH) and β-aminobutyric acid (BABA) on the resistance of tea plants (Camellia sinensis) to tea geometrid (Ectropis obliqua), three levels each of benzothiadiazole (BTH) and β-aminobutyric acid (BABA) were sprayed on 10-year-old tea plants. Generally PPO and PAL activities increased with low concentrations of BTH and BABA treatments. Quantitative RT-PCR revealed a 1.43 and 2.72-fold increase in PPO gene expression, and 3.26 and 3.99-fold increase in PAL gene expression with 75 mg/L BTH and 400 mg/L BABA respectively. Analysis of hydrolysis of synthetic substrates also revealed that chymotrypsin-like enzyme activity present in larval midgut extracts was not significantly inhibited by BTH and BABA. However, proteinase activity was found to be inversely proportional to the age of tea geometrid. Larvae pupation rate decreased by 8.10, 10.81 and 21.62% when tea geometrid were fed with leaves treated with 25, 50 and 75 mg/L BTH solutions, while 100, 200 and 400 mg/L BABA solutions decreased same by 8.10, 16.21 and 13.51% respectively. Also, larvae development period delayed to 23.33 and 26.33 days with 75 mg/L BTH and 400 mg/L BABA treatments respectively. The results in this study; therefore, suggest that benzothiadiazole (BTH) and β-aminobutyric acid (BABA) play a role in inducing resistance in tea plants to tea geometrid, with the optimal effect achieved at BTH-3 (75 mg/L) and BABA-3 (400 mg/L), respectively.

Dietary Protein and Carbohydrate Levels Affect Performance and Digestive Physiology of Plodia interpunctella (Lepidoptera: Pyralidae).

In this study, life history and nutritional indices of Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) was evaluated on six food commodities: dried fig, dried wheat germ, dried white mulberry, groundnut, pistachio, and raisin, compared with artificial diet. The influence of dietary macronutrient content on digestive α-amylase was also assessed. A delay in the developmental time of P. interpunctella immature stages was detected when larvae were fed on raisin. The highest survival rate of immature stages was on the artificial diet, and the lowest was on raisin. The highest realized fecundity and fertility were recorded for the females reared on artificial diet. Overall, fourth instar P. interpunctella reared on artificial diet had the highest relative consumed and growth rate, and lowest rates were observed in larvae fed raisin. Amylolytic activity and isoform patterns varied depending on larval instar and diets, but were higher for larvae fed artificial diet with moderate carbohydrate and protein. Zymograms showed the presence of three isoforms of α-amylase in midgut extracts of P. interpunctella fed different diets. Larvae fed dried white mulberry, fig, and raisin had one (A2) α-amylase isoform. The data suggest that dietary carbohydrate and protein content induce changes in nutritional efficiency, development, and α-amylase activity. A survey of the differences in digestive enzyme activity in response to macronutrient balance and imbalance highlight their importance in the nutrition of insects.

English

Midgut symbiotic bacteria are known to play fundamental roles in the biology of mosquitoes, however knowledge of midgut bacterial communities associated with mosquitoes is scanty due to limitation of the isolation techniques based on culturing. In this study, the composition and diversity of midgut bacteria in field collected and lab reared adult female Anopheles gambiae and Culex quinquefasciatus mosquitoes was explored using the Illumina sequencing. Deoxyribonucleic acid was isolated from the pooled midgut extracts and their 16S rRNA gene sequenced using Illumina sequencing platform. Operational taxonomic units (OTUs) were analyzed using QIIME 1.8.0; taxonomy was assigned using BLASTn against SILVA 119 and hierarchical clustering was done using R program software. Out of the total number of sequence reads obtained, 145 OTUs were realized at 3% genetic distance. The 145 OTUs spanned 12 phyla; Proteobacteria, Firmicutes, Cyanobacteria, Euryarchaeota, Gemmatimonadetes, Spirochaetae, Archeabacteria Verrucomicrobia, Chloroflexi, Bacteriodetes, Acidobacteria and Actinobacteria. Microbial community composition based on OTUs showed significant difference between field collected and lab reared mosquitoes (aµa2 = 45.0799, p = 3.2 × 10-5). Similarly, there was a significant difference in community composition at OTU level between Anopheles gambiae and Culex quinquefasciatus (aµa2 = 31.2257, p = 7.7 × 10-4). The bacterial composition and diversity appeared to be influenced by the environment and the species of the mosquitoes. Key words: Anopheles gambiae, Culex quinquefasciatus, midgut, DNA, diversity.

Population-associated heterogeneity of the digestive Cys protease complement in Colorado potato beetle, Leptinotarsa decemlineata

Herbivorous insects use complex protease complements to process plant proteins, useful to adjust their digestive functions to the plant diet and to elude the antidigestive effects of dietary protease inhibitors. We here assessed whether basic profiles and diet-related adjustments of the midgut protease complement may vary among populations of the insect herbivore Colorado potato beetle (Leptinotarsa decemlineata). Two laboratory colonies of this insect were used as models, derived from insect samples collected in potato fields ∼1200km distant from each other in North America. Synchronized 4th-instar larvae reared on potato were kept on this plant, or switched to tomato or eggplant, to compare their midgut cathepsin activities and content of intestain Cys proteases under different diet regimes. Cathepsin D activity, cathepsin L activity, cathepsin B activity and total intestain content shortly after larval molting on potato leaves were about two times lower in one population compared to the other. By comparison, cathepsin D activity, cathepsin B activity, total intestain content and relative abundance of the most prominent intestain families were similar in the two populations after three days regardless of the plant diet, unlike cathepsin L activity and less prominent intestain families showing population-associated variability. Variation in Cys protease profiles translated into the differential efficiency of a Cys protease inhibitor, tomato cystatin SlCYS8, to inhibit cathepsin L activity in midgut extracts of the two insect groups. Despite quantitative differences, SlCYS8 single variants engineered to strongly inhibit Cys proteases showed improved potency against cathepsin L activity of either population. These data suggest the feasibility of designing cystatins to control L. decemlineata that are effective against different populations of this insect. They underline, on the other hand, the practical relevance of considering natural variability of the protease complement among L. decemlineata target populations, eventually determinant in the success or failure of cystatin-based control strategies on a large-scale basis.

Metabolic changes in the midgut of Eri silkworm after Oral administration of 1-deoxynojirimycin: A 1H-NMR-based metabonomic study.

1-deoxynojirimycin (DNJ) is a natural D-glucose analogue and has a strong physiological activity in inhibiting α-glucosidase in vivo. The antidiabetic effects of DNJ in mice or other mammals were extensively explored, but the physiological and toxic roles of DNJ in insects was seldom reported. In this study, the biological effects of DNJ were examined in midgut extracts of fourth-instar larvae of Eri silkworm (Samia cynthia ricini, Saturniidae). Based on nuclear magnetic resonance (NMR) metabonomics technology, we analyzed the alterations of glycometabolism, lipids, and energy metabolism pathways in the midgut of S. cynthia ricini caused by DNJ. Pattern recognition analysis (partial least square-discriminant analysis, PLS-DA) showed that four groups of latex, 0.25% DNJ, 0.5% DNJ and the mixture of 0.5% DNJ and latex (1:1) were distinctly different from the control group. Moreover, several metabolic pathways of DNJ-mediated modulation in the midgut were identified. Compared with the control group, alanine, succinate, glutamate, and fumarate concentrations decreased in three groups of 0.5% DNJ, latex, and the mixture, choline levels increased in two DNJ groups, and trehalose levels increased in all experimental groups. Therefore, these results suggest that DNJ modulated lipid metabolism by limiting the hydrolysis pathways of phospholipids metabolism. Additionally, DNJ has a potent negative effect on energy metabolism by inhibiting the hydrolysis of trehalose, glycolysis and the tricarboxylic acid (TCA) cycle. Overall, DNJ, as a single-ingredient, is an efficient substance for modulating lipid metabolism and inhibiting energy metabolism.