Inhibition of trehalase affects the trehalose and chitin metabolism pathways in Diaphorina citri(Hemiptera:Psyllidae)

The Asian citrus psyllid,Diaphorina citri is the principal vector of huanglongbing,which transmits Candidatus Liberibacter asiaticus.Trehalase is a key enzyme involved in trehalose hydrolysis and plays an important role in insect growth and development.The specific functions of this enzyme in D.citri have not been determined.In this study,three trehalase genes(DcTreJ-1,DcTrel-2,and DcTre2)were identified based on the D.citri genome database.Bioinformatic analysis showed that DcTrel-1 and DcTrel-2 are related to soluble trehalase,whereas DcTre2 is associated with membrane-bound trehalase.Spatiotemporal expression analysis indicated that DcTrel-1 and DcTrel-2 had the highest expression levels in the head and wing,respectively,and DcTre2 had high expression levels in the fat body.Furthermore,DcTrel-1 and DcTrel-2 expression levels were induced by 20-hydroxyecdysone and juvenile hormone III,but DcTre2 was unaffected.The expression levels of DcTrel-1,DcTrel-2,and DcTre2 were significantly up-regulated,which resulted in high mortality after treatment with validamycin.Trehalase activities and glucose contents were downregulated,but the trehalose content increased after treatment with validamycin.In addition,the expression levels of chitin metabolism-related genes significantly decreased at 24 and 48 h after treatment with validamycin.Furthermore,silencing of DcTrel-1,DcTrel-2,and DcTre2 reduced the expression levels of chitin metabolism-related genes and led to a malformed phenotype of D.citri.These results indicate that D.citri trehalase plays an essential role in regulating chitin metabolism and provides a new target for control of D.citri.

Phospholipase C gamma(PLCγ)regulates soluble trehalase in the 20E-induced fecundity of Apolygus lucorum

Apolygus lucorum is the dominant pathogenic insect attacking Bacillus thuringiensis(Bt)cotton in China.Additionally,20-hydroxyecdysone(20E)has important functions in many biological processes,including insect reproduction.Phospholipase C(PLC),which is an essential enzyme for phosphoinositide metabolism,is involved in 20E signal transduction,but its function in 20E-mediated reproduction in A.lucorum remains unclear.In this study,20E increased A/PLCγ transcription as well as the abundance and activity of the encoded protein during molting and metamorphosis.The 20E treatment also induced the considerable accumulation of two second messengers,inositol triphosphate and diacylglycerol.The expression levels of genes encoding vitellogenin(AlVg)and soluble trehalase(AlTre-1)were similar to those of AlPLCy,and were upregulated in response to 20C.The silencing of AlPLCγ resulted in downregulated expression o f AITre-γ smdAlVg.However,the silencing of AlTre-1 and AlVg did not affect AlPLCγ expression.Moreover,the silencing of AlVg did not alter AlTre-1 expression.Furthermore,an examination of the insect specimens indicated that AlPLCy is required for female adult reproduction,and that downregulated expression of this gene is associated with decreases in fecundity,adult longevity,and egg hatching rate as well as delayed oocyte maturation.We propose that 20E regulates AlTre-1 expression via AlPLCy and affects Vg expression as well as ovary development to facilitate the reproductive activities of A.lucorum females.

Characterization,heterologous expression and engineering of trehalase for biotechnological applications

Trehalose is a non-reducing disaccharide connected byα-1,1-glycosidic bonds;it is widely distributed in bacteria,fungi,yeast,insects,and plant tissues and plays various roles.It can be hydrolyzed by trehalase into two glucose molecules.Trehalases from different sources have been expressed in Escherichia coli,Pichia pastoris,Saccharomyces cerevisiae,baculovirus-silkworm,and other expression systems;however,it is most common in E.coli.The structural characteristics of different glycoside hydrolase(GH)family trehalases and the sources of trehalase have been analyzed.The catalytic mechanism of GH37 trehalase has also been elucidated in detail.Moreover,the molecular modification of trehalase has mainly focused on directed evolution for improving enzyme activity.We comprehensively reviewed the current application status and adaptable transformations was comprehensively overviewed in the context of industrial performance.We suggest that the level of recombinant production is far from meeting industrial requirements,and the catalytic performance of trehalase needs to be improved urgently.Finally,we discuss developmental prospects and future trends.

Determining the IgG concentrations in bovine colostrum and calf sera with a novel enzymatic assay

Background: Immune protection in newborn calves relies on a combination of the timing,volume and quality of colostrum consumed by the calf after birth.Poor quality colostrum with inadequate immunoglobulin concentration contributes to failed transfer of passive immunity in calves,leading to higher calf morbidity and mortality.Therefore,estimating colostrum quality and ensuring the transfer of passive immunity on farm is of critical importance.Currently,there are no on-farm tools that directly measure immunoglobulin content in colostrum or serum.The aim of this study was to apply a novel molecular assay,split trehalase immunoglobulin G assay(STIGA),to directly estimate immunoglobulin content in dairy and beef colostrum and calf sera,and to examine its potential to be developed as on-farm test.The STIGA is based on a split version of trehalase TreA,an enzyme that converts trehalose into glucose,enabling the use of a common glucometer for signal detection.In a first study,60 dairy and64 beef colostrum and 83 dairy and 84 beef calf sera samples were tested with STIGA,and the resulting glucose production was measured and compared with radial immunodiffusion,the standard method for measuring immunoglobulin concentrations.Results: Pearson correlation coefficients between the methods were determined and the sensitivity,specificity,and accuracy of the test were calculated for different colostrum quality and failed transfer of passive immunity cut-off points.The correlations of the STIGA measured by colorimetric enzymatic reaction compared to radial immunodiffusion for dairy and beef colostrum were 0.72 and 0.73,respectively,whereas the correlations for dairy and beef sera were 0.9 and 0.85,respectively.Next,STIGA was tested in a blinded study with fresh colostrum and serum samples where the correlation coefficient was 0.93 and 0.94,respectively.Furthermore,the performance of STIGA followed by glucometer readings resulted in correlations with radial immunodiffusion of 0.7 and 0.85 for dairy and beef colostrum and 0.94 and 0.83 for dairy and beef calf serum.Conclusions: A split TreA assay was validated for measurement of the immunoglobulin content of colostrum and calf sera using both a lab-based format and in a more user-friendly format compatible with on-farm testing.