Digestive physiological characteristics of black soldier fly larvae reared on five organic wastes

Black soldier fly larvae (BSFL) are able to utilise a broad range of organic wastes to fulfil their growth needs. To acquire this basic knowledge of its digestive adaptation to various food, five organic wastes (soybean meal [SBM], wheat bran [WB], beer yeast [BY], kitchen waste [KW] and chicken manure [CM]) were fed to 3-day-old BSFL for 16 days. The growth performance, luminal pH of the gut, midgut histology, digestive enzyme activity and intestinal bacterial microbiota of the larvae were assessed. Growth performance was greatest in the KW group followed by the SBM group and was worst in the CM group. Nutrient compositions of larvae were not significantly affected by those of the food sources, with the exception of crude ash. The ultrastructural observations of midgut showed the number of mitochondria in CM and BY groups was less than other three groups. Twenty-four hours after feeding, weakly acidic, acidic, strongly acidic, and alkaline luminal pH values were detected in the anterior, middle and posterior segments of the gut in all groups, but the luminal pH values of the hindgut varied with food source. Significant lipase and esterase activity, cellulase activity, and amylase activity were detected in the intestines of larvae reared on KW, WB and BY, respectively, revealing that digestive enzyme activity was closely associated with the nutrient composition of the food source. Bacterial composition and diversity differed significantly between groups and were characterised by specific indicator bacteria, which may play important roles in food digestibility. The results suggested that different food sources induced adaptive physical, chemical and biological changes in the digestive tracts of BSFL and may indicate that BSFL developed specific strategies for nutrient utilisation and accumulation. The knowledge acquired here will be beneficial for developing rearing protocols to optimise bioconversion in this insect for their various applications.

Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

The acidic luminal pH of lysosomes, maintained within a narrow range, is essential for proper degrative function of the organelle and is generated by the action of a V-type H+ ATPase, but other pathways for ion movement are required to dissipate the voltage generated by this process. ClC-7, a Cl-/H+ antiporter responsible for lysosomal Cl- permeability, is a candidate to contribute to the acidification process as part of this “counterion pathway”. The signaling lipid PI(3,5)P2 modulates lysosomal dynamics, including by regulating lysosomal ion channels, raising the possibility that it could contribute to lysosomal pH regulation. Here we demonstrate that depleting PI(3,5)P2 by inhibiting the PIKfyve kinase causes lysosomal hyperacidification, primarily via an effect on ClC-7. We further show that PI(3,5)P2 directly inhibits ClC-7 transport and that this inhibition is eliminated in a disease-causing gain-of-function ClC-7 mutation. Together these observations suggest an intimate role for ClC-7 in lysosomal pH regulation.

Tubular lysosomes harbor active ion gradients and poise macrophages for phagocytosis

Lysosomes adopt dynamic, tubular states that regulate antigen presentation, phagosome resolution, and autophagy. Tubular lysosomes are studied either by inducing autophagy or by activating immune cells, both of which lead to cell states where lysosomal gene expression differs from the resting state. Therefore, it has been challenging to pinpoint the biochemical properties lysosomes acquire upon tubulation that could drive their functionality. Here we describe a DNA-based assembly that tubulates lysosomes in macrophages without activating them. Proteolytic activity maps at single-lysosome resolution revealed that tubular lysosomes were less degradative and showed proximal to distal luminal pH and Ca2+ gradients. Such gradients had been predicted but never previously observed. We identify a role for tubular lysosomes in promoting phagocytosis and activating MMP9. The ability to tubulate lysosomes without starving or activating immune cells may help reveal new roles for tubular lysosomes.

Enteric nervous system modulation of luminal pH modifies the microbial environment to promote intestinal health

The enteric nervous system (ENS) controls many aspects of intestinal homeostasis, including parameters that shape the habitat of microbial residents. Previously we showed that zebrafish lacking an ENS, due to deficiency of the sox10 gene, develop intestinal inflammation and bacterial dysbiosis, with an expansion of proinflammatory Vibrio strains. To understand the primary defects resulting in dysbiosis in sox10 mutants, we investigated how the ENS shapes the intestinal environment in the absence of microbiota and associated inflammatory responses. We found that intestinal transit, intestinal permeability, and luminal pH regulation are all aberrant in sox10 mutants, independent of microbially induced inflammation. Treatment with the proton pump inhibitor, omeprazole, corrected the more acidic luminal pH of sox10 mutants to wild type levels. Omeprazole treatment also prevented overabundance of Vibrio and ameliorated inflammation in sox10 mutant intestines. Treatment with the carbonic anhydrase inhibitor, acetazolamide, caused wild type luminal pH to become more acidic, and increased both Vibrio abundance and intestinal inflammation. We conclude that a primary function of the ENS is to regulate luminal pH, which plays a critical role in shaping the resident microbial community and regulating intestinal inflammation.Author SummaryThe intestinal microbiota is an important determinant of health and disease and is shaped by the environment of the gut lumen. The nervous system of the intestine, the enteric nervous system (ENS), helps maintain many aspects of intestinal health including a healthy microbiota. We used zebrafish with a genetic mutation that impedes ENS formation to investigate how the ENS prevents pathogenic shifts in the microbiota. We found that mutants lacking an ENS have a lower luminal pH, higher load of pathogenic bacteria, and intestinal inflammation. We showed that correcting the low pH, using the commonly prescribed pharmacological agent omeprazole, restored the microbiota and prevented intestinal inflammation. Conversely, we found that lowering the luminal pH of wild type animals, using the drug acetazolamide, caused expansion of pathogenic bacteria and increased intestinal inflammation. From these experiments, we conclude that a primary function of the ENS is to maintain normal luminal pH, thereby constraining intestinal microbiota community composition and promoting intestinal health.

Maturing Autophagosomes are Transported Towards the Cell Periphery

Autophagosome maturation comprises fusion with lysosomes and acidification. It is a critical step in the degradation of cytosolic protein aggregates that characterize many neurodegenerative diseases. In order to better understand this process, we studied intracellular trafficking of autophagosomes and aggregates of α-synuclein, which characterize Parkinson’s disease and other synucleinopathies. The autophagosomal marker LC3 and the aggregation prone A53T mutant of α-synuclein were tagged by fluorescent proteins and expressed in HEK293T cells and primary astrocytes. The subcellular distribution and movement of these vesicle populations were analyzed by (time-lapse) microscopy. Fusion with lysosomes was assayed using the lysosomal marker LAMP1; vesicles with neutral and acidic luminal pH were discriminated using the RFP-GFP “tandem-fluorescence” tag. With respect to vesicle pH, we observed that neutral autophagosomes, marked by LC3 or synuclein, were located more frequently in the cell center, and acidic autophagosomes were observed more frequently in the cell periphery. Acidic autophagosomes were transported towards the cell periphery more often, indicating that acidification occurs in the cell center before transport to the periphery. With respect to autolysosomal fusion, we found that lysosomes preferentially moved towards the cell center, whereas autolysosomes moved towards the cell periphery, suggesting a cycle where lysosomes are generated in the periphery and fuse to autophagosomes in the cell center. Unexpectedly, many acidic autophagosomes were negative for LAMP1, indicating that acidification does not require fusion to lysosomes. Moreover, we found both neutral and acidic vesicles positive for LAMP1, consistent with delayed acidification of the autolysosome lumen. Individual steps of aggregate clearance thus occur in dedicated cellular regions. During aggregate clearance, autophagosomes and autolysosomes form in the center and are transported towards the periphery during maturation. In this process, luminal pH could regulate the direction of vesicle transport. Graphic Abstract (1) Transport and location of autophagosomes depend on luminal pH: Acidic autophagosomes are preferentially transported to the cell periphery, causing more acidic autophagosomes in the cell periphery and more neutral autophagosomes at the microtubule organizing center (MTOC). (2) Autolysosomes are transported to the cell periphery and lysosomes to the MTOC, suggesting spatial segregation of lysosome reformation and autolysosome fusion. (3) Synuclein aggregates are preferentially located at the MTOC and synuclein-containing vesicles in the cell periphery, consistent with transport of aggregates to the MTOC for autophagy.

Maturing autophagosomes are transported towards the cell periphery

Autophagosome maturation comprises fusion with lysosomes and acidification. It is a critical step in the degradation of cytosolic protein aggregates that characterize many neurodegenerative diseases. In order to better understand this process, we studied intracellular trafficking of autophagosomes and aggregates of α-synuclein, which characterize Parkinson’s disease and other synucleinopathies. The autophagosomal marker LC3 and the aggregation prone A53T mutant of α-synuclein were tagged by fluorescent proteins and expressed in HEK293T cells and primary astrocytes. The subcellular distribution and movement of these vesicle populations were analyzed by (time-lapse) microscopy. Fusion with lysosomes was assayed using the lysosomal marker LAMP1; vesicles with neutral and acidic luminal pH were discriminated using the RFP-GFP “tandem fluorescence” tag. With respect to vesicle pH, we observed that neutral autophagosomes, marked by LC3 or synuclein, were located more frequently in the cell center, and acidic autophagosomes were observed more frequently in the cell periphery. Acidic autophagosomes were transported towards the cell periphery more often, indicating that acidification occurs in the cell center before transport to the periphery. With respect to autolysosomal fusion, we found that lysosomes preferentially moved towards the cell center whereas autolysosomes moved towards the cell periphery, suggesting a cycle where lysosomes are generated in the periphery and fuse to autophagosomes in the cell center. Unexpectedly, many acidic autophagosomes were negative for LAMP1, indicating that acidification does not require fusion to lysosomes. Moreover, we found both neutral and acidic vesicles positive for LAMP1, consistent with delayed acidification of the autolysosome lumen. Individual steps of aggregate clearance thus occur in dedicated cellular regions. During aggregate clearance, autophagosomes and autolysosomes form in the center and are transported towards the periphery during maturation. In this process, luminal pH could regulate the direction of vesicle transport.

Subunit E isoform 1 of vacuolar H+-ATPase OsVHA enables post-Golgi trafficking of rice seed storage proteins

Dense vesicles (DVs) are Golgi-derived plant-specific carriers that mediate post-Golgi transport of seed storage proteins in angiosperms. How this process is regulated remains elusive. Here, we report a rice (Oryza sativa) mutant, named glutelin precursor accumulation8 (gpa8) that abnormally accumulates 57-kDa proglutelins in the mature endosperm. Cytological analyses of the gpa8 mutant revealed that proglutelin-containing DVs were mistargeted to the apoplast forming electron-dense aggregates and paramural bodies in developing endosperm cells. Differing from previously reported gpa mutants with post-Golgi trafficking defects, the gpa8 mutant showed bent Golgi bodies, defective trans-Golgi network (TGN), and enlarged DVs, suggesting a specific role of GPA8 in DV biogenesis. We demonstrated that GPA8 encodes a subunit E isoform 1 of vacuolar H+-ATPase (OsVHA-E1) that mainly localizes to TGN and the tonoplast. Further analysis revealed that the luminal pH of the TGN and vacuole is dramatically increased in the gpa8 mutant. Moreover, the colocalization of GPA1 and GPA3 with TGN marker protein in gpa8 protoplasts was obviously decreased. Our data indicated that OsVHA-E1 is involved in endomembrane luminal pH homeostasis, as well as maintenance of Golgi morphology and TGN required for DV biogenesis and subsequent protein trafficking in rice endosperm cells.

Determinants of Phagosomal pH During Host-Pathogen Interactions

The ability of phagosomes to halt microbial growth is intimately linked to their ability to acidify their luminal pH. Establishment and maintenance of an acidic lumen requires precise co-ordination of H+ pumping and counter-ion permeation to offset the countervailing H+ leakage. Despite the best efforts of professional phagocytes, however, a number of specialized pathogens survive and even replicate inside phagosomes. In such instances, pathogens target the pH-regulatory machinery of the host cell in an effort to survive inside or escape from phagosomes. This review aims to describe how phagosomal pH is regulated during phagocytosis, why it varies in different types of professional phagocytes and the strategies developed by prototypical intracellular pathogens to manipulate phagosomal pH to survive, replicate, and eventually escape from the phagocyte.

Histochemical localisation of carbonic anhydrase in the digestive tract and salivary glands of the house cricket, Acheta domesticus

The house cricket (Acheta domesticus) is one of several cricket species with great potential to be farmed as a sustainable protein source. In order to succeed in large-scale cricket farming, knowledge of cricket digestion is essential. The digestive tract morphology of A. domesticus is well documented, but knowledge of the salivary glands is lacking. In the digestive tract of insects, the carbonic anhydrase (CA) enzyme family is believed to contribute to the luminal pH gradient. Presence of CA in the digestive tract of A. domesticus has been reported, but not the cellular localisation. This study examined the digestive tract of A. domesticus, including salivary glands, and the cellular localisation and activity of CA in fed or starved (48 h) males and females. Tissues were collected from third-generation offspring of wild A. domesticus captured in Sweden and the histology of the salivary glands and the cellular localisation of CA in the digestive tract of A. domesticus were determined, to our knowledge for the first time. The salivary glands resembled those of grasshoppers and locusts, and we suggest the two main cell types present to be parietal and zymogenic cells. Histochemical analysis revealed that CA activity was localised in midgut epithelium, both main cell types of salivary gland, and muscle along the entire digestive tract. These findings support the suggestion that CA contributes to digestive tract luminal pH gradient, by driving acidic secretions from the salivary glands and alkaline secretions from the midgut. Starvation resulted in significantly reduced body size and weight, but neither starvation nor sex had any effect on CA activity or localisation.

Cation Chloride Cotransporter 1 (CCC1) regulates pH and ionic conditions in the TGN/EE and is required for endomembrane trafficking

The secretory and endocytic pathways intersect at the Trans-Golgi Network/Early Endosome (TGN/EE). TGN/EE function depends on luminal pH adjustment, which is regulated by the combined activity of a proton pump and several proton/ion antiporters. The identity of the proton pump is known, as well the antiporters that catalyse cation and anion import into the TGN/EE. However, the protein(s) required to complete the transport circuit, and that mediates cation and anion efflux has not been identified. Here, we characterise Arabidopsis Cation Chloride Cotransporter (AtCCC1) and show that it is localised in the TGN/EE. We further demonstrate that regulation of both luminal pH and ion concentrations are dependent on AtCCC1 function, using pharmacological treatments and genetically encoded fluorescent sensors. Loss of AtCCC1 leads to alterations in cellular functions dependent on the TGN/EE including endo- and exocytosis, trafficking to the vacuole and trafficking of the plasma membrane protein PIN2. This discovery provides the cellular role for CCC1s and can explain the multitude of phenotypic defects observed in loss-of-function plants. Collectively, our results demonstrate that non-proton-coupled ion transport contributes to the regulation of TGN/EE luminal ionic and pH conditions; and that CCC1 is an essential missing component of the TGN/EE ion transport circuit.One sentence summaryThe TGN/EE-localised Cation Chloride Cotransporter 1 maintains optimal luminal ionic and pH conditions required for intracellular protein trafficking and cell elongation.