Alkaline Midgut Research Articles

A nanoformulation that enhance the efficiency of camptothecin against lepidopteran pests by controlled release and ‘poison bait’ strategies

Pesticides are critical to securing crop yields, but the extremely low utilization of conventional pesticides puts a serious strain on human development and the environment. Natural product pesticides meet the needs of sustainable development, but challenges like high costs and slow efficacy hinder their widespread use. Camptothecin (CPT) is a natural product with potent bioactivity for various insect pests, but its bioactivity for lepidopteran pests is limited due to the ring-opening in their alkaline midgut. To enhance CPT’s efficacy, a nanopesticide CPT@Zein-FA, which consists of the plant-derived zein and ferulic acid (FA), was designed and verified to be able to improve the bioactivity of CPT against lepidopteran pests including Ostrinia furnacalis, Spodoptera frugiperda and Mythimna separata. The nanopesticide CPT@Zein-FA approximately 100 nm in size, exhibited uniform dispersion, strong adhesion, and stability. CPT@Zein-FA utilizes pH and redox dual response mechanisms to achieve protection in the midgut environment and release in the hemocoel/intracellular environment. With the help of zein bait, CPT@Zein-FA can also promote the feeding of insects and accelerate their death. Metabolomic analysis revealed CPT’s lethal impact on insect metabolism by inhibiting nucleotide synthesis, disrupting body size and development through lipid metabolism and insect hormone synthesis inhibition. This work provides new ideas for the large-scale application of natural product pesticides and contributes to the sustainable development of the world.

PH/redox/α-amylase triple responsive metal-organic framework composites for pest management and plant growth promotion

The development of stimuli-responsive controlled release formulations is a promising strategy to enhance the utilization efficiency and reduce the environmental pollution of pesticides. In this work, a smart pH/redox/α-amylase triple stimuli-responsive pesticide delivery system was constructed by bonding carboxymethyl starch (CMS) with metal-organic frameworks (MIL-101(Fe)–NH2 nanoparticles) which loaded with CAR (CAR@MIL-101(Fe)-CMS nanoparticles). The results indicated that the loading capacity of CAR@MIL-101(Fe)-CMS nanoparticles for CAR was approximately 18.3% and the CMS attached on the surface of CAR@MIL-101(Fe)–NH2 nanoparticles could protect CAR from photolysis effectively. Meanwhile, the coated CMS and metal-organic framework structure of the nanoparticles could be decomposed and consequently release pesticide active ingredients on demand when CAR@MIL-101(Fe)-CMS nanoparticles were metabolized by glutathione and α-amylase in the insect alkaline midgut. The bioactivity survey confirmed that CAR@MIL-101(Fe)-CMS nanoparticles had a longer duration in controlling Spodoptera frugiperda larvae compared to CAR suspension concentrate. In addition, the MIL-101(Fe)-CMS nanocarriers were safe for the growth of maize and could be used as fertilizer to promote the growth and development of crops. Thus, this smart stimuli-responsive pesticide-fertilizer all-in-one combination strategy has great potential in pest management.

Lignocellulose degradation in Protaetia brevitarsis larvae digestive tract: refining on a tightly designed microbial fermentation production line

BackgroundThe Scarabaeidae insect Protaetia brevitarsis (PB) has recently gained increasing research interest as a resource insect because its larvae can effectively convert decaying organic matter to plant growth-promoting frass with a high humic acid content and produce healthy, nutritional insect protein sources. Lignocellulose is the main component of PB larvae (PBL) feed, but PB genome annotation shows that PBL carbohydrate-active enzymes are not able to complete the lignocellulose degradation process. Thus, the mechanism by which PBL efficiently degrade lignocellulose is worthy of further study.ResultsHerein, we used combined host genomic and gut metagenomic datasets to investigate the lignocellulose degradation activity of PBL, and a comprehensive reference catalog of gut microbial genes and host gut transcriptomic genes was first established. We characterized a gene repertoire comprising highly abundant and diversified lignocellulose-degrading enzymes and demonstrated that there was unique teamwork between PBL and their gut bacterial microbiota for efficient lignocellulose degradation. PBL selectively enriched lignocellulose-degrading microbial species, mainly from Firmicutes and Bacteroidetes, which are capable of producing a broad array of cellulases and hemicellulases, thus playing a major role in lignocellulosic biomass degradation. In addition, most of the lignocellulose degradation-related module sequences in the PBL microbiome were novel. PBL provide organic functional complementarity for lignocellulose degradation via their evolved strong mouthparts, alkaline midgut, and mild stable hindgut microenvironment to facilitate lignocellulosic biomass grinding, dissolving, and symbiotic microbial fermentation, respectively.ConclusionsThis work shows that PBL are a promising model to study lignocellulose degradation, which can provide highly abundant novel enzymes and relevant lignocellulose-degrading bacterial strains for biotechnological biomass conversion industries. The unique teamwork between PBL and their gut symbiotic bacterial microbiota for efficient lignocellulose degradation will expand the knowledge of holobionts and open a new beginning in the theory of holobionts.BPGV8LV-Txp2PAmT3d1J6VVideo

Health Implications of Bt Cotton Seeds in Animals and Man

Cotton (Gossypium spp.) is an arborous plant from the Malvaceae family. Cottonseed meal is a by-product of cotton that is used for animal feeding because it is rich in oil and proteins. However, gossypol toxicity limits cottonseed use in animal feed. Gossypol is a phenolic compound produced by pigment glands in cotton stems, leaves, seeds and flower buds. Cotton is a long duration crop and is attacked by a number of insect pests. Earlier, insecticide quantity applied on cotton was highest but with the advent of Bt cotton, there is a great reduction in insecticide and pesticide usage as majority of the bollworms are controlled by it. Bt cotton refers to transgenic cotton which contains endotoxin protein inducing gene from soil bacterium Bacillus thuringiensis. It was discovered by a Japanese scientist Ishiwata in 1901. B.thuringiensis strains produces 3 types of insecticidal toxins that are Crystal toxins (Cry), Cytolytic toxins (Cyt) and vegetatively expressed insecticidal proteins (vip). Bt-delta-endotoxins which functions as oral toxins are ingested by insect and protoxins are proteolytically activated to trypsin-resistant active core δ- endotoxin in alkaline mid-gut. The active toxin binds to cadherin receptors present on the brush border membrane of the insect midgut. Cadherins process the toxins to form homo-oligomers, bind to specific receptors like alkaline phosphatases and aminopeptidases before causing pores in the epithelial membrane, resulting in osmotic lysis of the cells which results in cessation of feeding and finally mortality of the insect. Cry toxins have not been reported to be toxic to higher animals such as goats, sheep and cattle in any part of the world as scientific evidences indicate that the Cry toxins do not get activated under the acidic conditions of non-target animals such as goat, sheep and cattle. In one study of acute oral toxicity study in rats, Bt cotton seed material did not induce any treatment related observable toxic effects when compared with Non-Bt cottonseeds. Histological studies in another study revealed that there were no difference in cellular architecture in liver, heart, kidney and intestine of Bt and non-Bt diet fed rats. The Cry toxins Cry1Ac, Cry1Ab, Cry2Ab, Cry1F and Cry1C are considered to be safe to human beings. The stomach of humans, being the first organ of digestion which the Bt protein encounters, is acidic and contains proteases like pepsin which degrade the Bt protein. Thus, the alkaline conditions needed for pro-toxin solubilization and protease action required for toxin activation are absent in the stomach. More importantly, the human intestine lacks the specific receptors to which the activated Bt protein binds and initiates the physiological effect. However, there are several reports that Bt genes cause some serious problems to human health. Cry1Ac toxin at higher concentration have lethal cytotoxic and genotoxic effects on the human lymphocytes. The present review is an attempt to provide details from the available literature regarding uses of Bt cotton and its implications on animal and human health.

Preparation of sodium alginate-poly (vinyl alcohol) blend beads for base-triggered release of dinotefuran in Spodoptera litera midgut

This study investigated the ability of dual crosslinked interpenetrating polymer network (IPN) blend beads (DIN:SA/PVA-beads), composed of sodium alginate (SA) and poly (vinyl alcohol) (PVA), as a base-triggered carrier for the controlled release of dinotefuran (DIN) in Spodoptera litera midgut. The blend beads were characterized for morphology, encapsulation efficiency, swelling degree, and in vitro release of the blend beads were characterized. The results revealed that the double-crosslinked gel beads had a tightly interpenetrating network structure and exhibited a satisfactory embedding effect for DIN. The maximum of the DIN loading capacity was approximately 1.01%, with a high encapsulation efficiency of 83.19%. The triggered release of DIN from the blend beads was studied in deionized water (pH 3.0–11.0) via high-performance liquid chromatography (HPLC); it was found that the release rate was higher in alkaline pH conditions than in acidic and neutral conditions. An in vivo dynamics and degradation study also demonstrated that the excellent release characteristics of DIN:SA/PVA-beads in the midgut of S. litera. This study provides a promising controlled-release form of dinotefuran that is more effective and can be used for the targeted control of pests with alkaline midgut.

Alkaline guts contribute to immunity during exposure to acidified seawater in the sea urchin larva.

Larval stages of members of the Abulacraria superphylum including echinoderms and hemichordates have highly alkaline midguts. To date, the reason for the evolution of such extreme pH conditions in the gut of these organisms remains unknown. Here, we test the hypothesis that, analogous to the acidic stomachs of vertebrates, these alkaline conditions may represent a first defensive barrier to protect from environmental pathogens. pH-optimum curves for five different species of marine bacteria demonstrated a rapid decrease in proliferation rates by 50-60% between pH8.5 and 9.5. Using the marine bacterium Vibrio diazotrophicus, which elicits a coordinated immune response in the larvae of the sea urchin Strongylocentrotus purpuratus, we studied the physiological responses of the midgut pH regulatory machinery to this pathogen. Gastroscopic microelectrode measurements demonstrate a stimulation of midgut alkalization upon infection with V. diazotrophicus accompanied by an upregulation of acid-base transporter transcripts of the midgut. Pharmacological inhibition of midgut alkalization resulted in an increased mortality rate of larvae during Vibrio infection. Reductions in seawater pH resembling ocean acidification conditions lead to moderate reductions in midgut alkalization. However, these reductions in midgut pH do not affect the immune response or resilience of sea urchin larvae to a Vibrio infection under ocean acidification conditions. Our study addressed the evolutionary benefits of the alkaline midgut of Ambulacraria larval stages. The data indicate that alkaline conditions in the gut may serve as a first defensive barrier against environmental pathogens and that this mechanism can compensate for changes in seawater pH.

Elementary budget of stag beetle larvae associated with selective utilization of nitrogen in decaying wood.

Wood degradation by insects plays important roles for the forest matter cycling. Since wood is deficient in nitrogen compared to the insect body, wood-feeding insects need to assimilate the nitrogen selectively and discard an excess carbon. Such a stoichiometric imbalance between food and body will cause high metabolic cost; therefore, wood-feeding insects may somehow alleviate the stoichiometric imbalance. Here, we investigated the carbon and nitrogen budgets of the larvae of stag beetle, Dorcus rectus, which feed on decaying wood. Assimilation efficiency of ingested wood was 22%, and those values based on the carbon and nitrogen were 27 and 45%, respectively, suggesting the selective digestion of nitrogen in wood. Element-based gross growth efficiency was much higher for nitrogen (45%) than for carbon (3%). As a result, the larvae released 24% of the ingested carbon as volatile, whereas almost no gaseous exchange was observed for nitrogen. Moreover, solubility-based elementary analysis revealed that the larvae mainly utilized alkaline-soluble-water-insoluble fraction of wood, which is rich in nitrogen. Actually, the midgut of the larvae was highly alkaline (pH10.3). Stag beetle larvae are known to exhibit coprophagy, and here we also confirmed that alkaline-soluble-water-insoluble nitrogen increased again from fresh feces to old feces in the field. Stable isotope analysis suggested the utilization of aerial nitrogen by larvae; however, its actual contribution is still disputable. Those results suggest that D. rectus larvae selectively utilize alkaline-soluble nitrogenous substrates by using their highly alkaline midgut, and perhaps associate with microbes that enhance the nitrogen recycling in feces.

Biopolymer microencapsulations of Bacillus thuringiensis crystal preparations for increased stability and resistance to environmental stress.

Parasporal crystals synthesized by Bacillus thuringiensis (Bt) have been widely used as microbial pesticides because of their toxicity to the larval stages of specific insects. However, parasporal crystals can be damaged by environmental stresses, such as high temperature, ultraviolet radiation, and desiccation. To reduce environmental susceptibility of parasporal crystals and extend the duration of their activity, we developed a new type of protection by making microcapsules of crystals (MCs). The microcapsules were self-assembled by alternate deposition (layer by layer) of low-cost chitosan and sodium alginate (or sodium carboxymethyl cellulose) on the crystal surface. Crystal toxins (Cry1Ac) were released from microcapsules at pH values above 9.0. Bioassay results demonstrated that microencapsulated preparations had larvicidal toxicity equivalent to the non-encapsulated form. Microencapsuled crystals were protected from environmental stresses such as high temperature and desiccation. The results indicate that microcapsule protection can enhance the efficacy of Bt in pest control, especially to Lepidoptera larvae that have a alkaline midgut.

Microprofiles of oxygen, redox potential, and pH, and microbial fermentation products in the highly alkaline gut of the saprophagous larva of Penthetria holosericea (Diptera: Bibionidae)

The saprophagous larvae of bibionid flies harbor bacteria in their alkaline intestinal tracts, but little is known about the contribution of the gut microbiota to the digestion of their recalcitrant diet. In this study, we measured oxygen and hydrogen partial pressure, redox potential and pH in the midgut, gastric caeca and hindgut of larvae of the bibionid fly Penthetria holosericea with Clark-type O2 and H2 microsensors, platinum redox microelectrodes, and LIX-type pH microelectrodes. The center of the midgut lumen was anoxic, whereas gastric caeca and hindgut were hypoxic. However, redox potential profiles indicated oxidizing conditions throughout the gut, with lowest values in the midgut (+20 to +60mV). Hydrogen production was not detected. The midgut was extremely alkaline (pH around 11), whereas hindgut and gastric caeca were neutral to slightly alkaline. While HPLC analysis showed high concentrations of glucose in the midgut (15mM) and gastric caeca (27mM), the concentrations of microbial fermentation products such as lactate (2–4mM), acetate (<1mM) and succinate (<0.5mM) were low in all gut regions, suggesting that the contribution of microorganisms to the digestive process, particularly in the alkaline midgut, is only of minor importance. We conclude that the digestive strategy of the saprophytic larva of P. holosericea, which feeds selectively on decomposed leaves and its own microbe-rich faeces, differs fundamentally from those of detritivorous and humivorous insects, which host a highly active, fermentative microbiota in their alkaline midgut or hindgut compartments.

The multiple strategies of an insect herbivore to overcome plant cyanogenic glucoside defence.

Cyanogenic glucosides (CNglcs) are widespread plant defence compounds that release toxic hydrogen cyanide by plant β-glucosidase activity after tissue damage. Specialised insect herbivores have evolved counter strategies and some sequester CNglcs, but the underlying mechanisms to keep CNglcs intact during feeding and digestion are unknown. We show that CNglc-sequestering Zygaena filipendulae larvae combine behavioural, morphological, physiological and biochemical strategies at different time points during feeding and digestion to avoid toxic hydrolysis of the CNglcs present in their Lotus food plant, i.e. cyanogenesis. We found that a high feeding rate limits the time for plant β-glucosidases to hydrolyse CNglcs. Larvae performed leaf-snipping, a minimal disruptive feeding mode that prevents mixing of plant β-glucosidases and CNglcs. Saliva extracts did not inhibit plant cyanogenesis. However, a highly alkaline midgut lumen inhibited the activity of ingested plant β-glucosidases significantly. Moreover, insect β-glucosidases from the saliva and gut tissue did not hydrolyse the CNglcs present in Lotus. The strategies disclosed may also be used by other insect species to overcome CNglc-based plant defence and to sequester these compounds intact.

Effect of midgut proteolytic activity on susceptibility of lepidopteran larvae to Bacillus thuringiensis subsp. Kurstaki

Bacillus thuringiensis (Bt) is the most effective microbial control agent for controlling numerous species from different insect orders. All subspecies and strains of B. thuringiensis can produce a spore and a crystalline parasporal body. This crystal which contains proteinaceous protoxins is dissolved in the alkaline midgut, the resulting molecule is then cleaved and activated by proteolytic enzymes and acts as a toxin. An interesting aspect of this activation process is that variations in midgut pH and protease activity have been shown to account for the spectrum of some Bt proteins activity. Thus, an important factor that could be a determinant of toxin activity is the presence of proteases in the midgut microenvironment of susceptible insects. Reciprocally, any alteration in the midgut protease composition of the host can result in resistance to Bt. Here in this paper, we reviewed this processes in general and presented our assays to reveal whether resistance mechanism to Bt in Diamondback Moth (DbM) larvae could be due to the function of the midgut proteases? We estimated LC50 for both probable susceptible and resistant populations in laboratory and greenhouse tests. Then, the midgut protease activities of the B. thuringiensis induced-resistant and susceptible populations of the DbM were assayed on Hemoglubin and on N-alpha-benzoyl-DL-arginine-p-nitroanilide (BapNA) for total and tryptic activities, respectively. Six hours after feeding on Bt treated and untreated canola leaves, the midguts of instar larvae of both populations were isolated. Following related protocols, peptides released through the activity of proteinases on Hemoglubin and BApNA were recorded using microplate reader. Control (Blank) was also considered with adding TCA to reaction mix before adding enzymatic extract. Data analysis indicated that there are significant differences for tryptic activity on BApNA and also for total proteolytic activity on Hemoglubin between susceptible and resistant populations fed on Bt treated leaves. But these differences were not significant for larvae fed on healthy canola leaves between these two populations. These results which supported the role of DbM's proteolytic system in development of resistance to Bt, will be discussed in details.

Insect virus polyhedra, infectious protein crystals that contain virus particles

High-resolution atomic structures have been reported recently for two types of viral polyhedra, intracellular protein crystals produced by ubiquitous insect viruses. Polyhedra contain embedded virus particles and function as the main infectious form for baculoviruses and cypoviruses, two distinct classes of viruses that infect mainly Lepitoptera species (butterflies and moths). Polyhedra are extremely stable and protect the virus particles once released in the environment. The extensive crystal contacts observed in the structures explain the remarkable stability of viral polyhedra and provide hints about how these crystals dissolve in the alkaline midgut, releasing embedded virus particles to infect feeding larvae. The stage is now set to answer intriguing questions about the in vivo crystallization of polyhedra, how virus particles are incorporated into polyhedra, and what determines the size and shape of the crystals. Large quantities of polyhedra can be obtained from infected larvae and polyhedra can also be produced using insect cell expression systems. Modified polyhedra encapsulating other entities in place of virus particles have potential applications as a means to stabilize proteins such as enzymes or growth factors, and the extremely stable polyhedrin lattice may provide a framework for future engineered micro-crystal devices.

A Novel Biorational Pesticide: Efficacy of Methionine Against Heraclides (Papilio) cresphontes, a Surrogate of the Invasive Princeps (Papilio) demoleus (Lepidoptera: Papilionidae)

ABSTRACT The Southeast Asian citrus-feeding butterfly Princeps (Papilio) demoleus (L.) was recently introduced into the Americas, causing an imminent threat to citrus production and ornamental flora. The human nutrient amino acid methionine has been shown by us to disrupt aminoacid-modulated ion transport systems in caterpillars and other insect larvae that possess an alkaline midgut. Heraclides (Papilio) cresphontes was bioassayed as a United States Department of Agriculture permitted surrogate of the Florida quarantined P. demoleus to test the potential efficacy of methionine. Larvae were allowed to feed ad libitum on wild lime plants with leaves treated with methionine or proline. Methionine caused 100% mortality in first through fourth instars in a time- and dose-dependent manner, as determined by probit analysis whereas proline was not toxic. Wild lime host plants did not exhibit phytotoxicity with methionine treatments during a 14 d test period. It is concluded that methionine is an effective larvicide against H. cresphontes, and therefore may be a candidate environmentally safe biorational pesticide for use against invasive P. demoleus in the Americas.

Characterization of a β-1,3-glucanase active in the alkaline midgut of Spodoptera frugiperda larvae and its relation to β-glucan-binding proteins

Spodoptera frugiperda β-1,3-glucanase (SLam) was purified from larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against β-1,3-glucan (laminarin), but cannot hydrolyze yeast β-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive β-1,3-glucanases from insects, SLam is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLam was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. Multiple sequence alignment of β-1,3-glucanases and β-glucan-binding protein supports the assumption that the β-1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the derived β-1,3-glucanases by the loss of an extended N-terminal region and the β-glucan-binding proteins by the loss of the catalytic residues. SLam homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLam antiserum reacts with a single protein in the insect midgut. Immunocytolocalization shows that the enzyme is present in secretory vesicles and glycocalyx from columnar cells.

The scarab gut: A potential bioreactor for bio‐fuel production

Abstract Cellulose and hemicelluloses are the most prevalent sources of carbon in nature. Currently many approaches employ micro‐organisms and their enzyme products to degrade plant feedstocks for production of bioenergy. Scarab larvae are one such model. They consume celluloses from a variety of sources including plant roots, soil organic matter and decaying wood, and are able to extract nutrients and energy from these sources. In this paper, we review the physicochemical properties of the scarab larval gut, the diversity and digestive role that microflora play in the scarab gut and discuss the potential for applying these digestive processes in bioreactors for improving bio‐fuel production. Scarab larvae are characterised by their highly alkaline midgut which is dominated by serine proteinase enzymes, and a modified hindgut which harbors the majority of the intestinal microbiota under anaerobic conditions. Evidence suggests that digestion of recalcitrant organic matter in scarab larvae likely results from a combination of endogenous gut proteinases and cellulolytic enzymes produced by symbiotic micro‐organisms. Most of the easily digestible proteins are mobilized and absorbed in the midgut by endogenous proteinases. The hindgut contents of scarab larvae are characterized by high concentrations of volatile fatty acids, the presence of fermenting bacteria, and typical anaerobic activities, such as methanogenesis. The hindgut typically contains a wide diversity of micro‐organisms, some of which appear to be obligate symbionts with cellulolytic potential. As a result, the scarab larval gut can be regarded as a small bioreactor resembling the rumen of sheep or cattle, where solid food particles composed of cellulose, hemicellulose, pectin and polysaccharides are degraded through enzymatic and fermentation processes. Together these observations suggest scarab larvae have potential to assist the bio‐fuel industry by providing new sources of (hemi)cellulolytic bacteria and bacterial (hemi)cellulolytic enzymes.

Transformation and mineralization of soil organic nitrogen by the humivorous larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

We studied transformation and mineralization of nitrogenous soil polymers by the humivorous larva of the scarabaeid beetle Pachnoda ephippiata, using 14C-labeled peptides, peptidoglycan, and chitin, both native and stabilized by co-polymerization with synthetic humic acids. The results indicated that these materials were solubilized mainly in the alkaline midgut. The solubilization rate of peptides to TCA-soluble products was higher than those of peptidoglycan and chitin. Solubilization rates were lower when the polymers were stabilized in humic acids. Incubation of soil with midgut fluid released amino acids, which explains the high in vivo concentrations of amino acids observed in the midgut fluid. The large amounts of \( {\text{NH}}^{ + }_{4} \) in the fresh fecal pellets indicated that at least 1.7% of the total nitrogen (7% of the acid-hydrolyzable peptides) in the ingested soil was mineralized during gut passage; also the amount of soluble protein and the solubility of humic substances increased. The combined evidence supports the hypothesis that not only microbial biomass but also the nitrogen-rich components of humus are important dietary components for humivorous insects, and that feeding activities of soil macroinvertebrates strongly impact transformation and mineralization of soil organic matter.

Peptidic soil components are a major dietary resource for the humivorous larvae of Pachnoda spp. (Coleoptera: Scarabaeidae)

Humivorous scarab beetle larvae can thrive exclusively on soil organic matter. Feeding experiments have revealed that the larva of Pachnoda ephippiata mineralizes all major humus components except the polyphenolic fraction. High proteolytic activity in the alkaline midgut fluid and an enormous ammonia production during gut passage suggested that peptidic soil components are an important dietary resource for the larva. By comparing acid-hydrolyzable amino acids in food soil and feces, we showed that a significant fraction of the peptides in soil are removed during gut passage. This agrees well with the high concentrations of free amino acids found the midgut section. Incubation experiments revealed the presence of substantial particle-associated proteolytic activity also in the hindgut, most probably due to microbial activity. High rates of ammonia formation in hindgut homogenates and the conversion of radiolabeled amino acids to acetate and propionate indicated that microbial fermentations of soil peptides play an important role in the hindgut. This was corroborated by viable counts of amino-acid-fermenting bacteria, which formed a substantial fraction of the hindgut microbiota. A complete inventory of organic and inorganic nitrogen species before, during, and after gut passage revealed the formation of nitrite and nitrate in midgut and hindgut, and a substantial nitrogen deficit in the feces, suggesting that part of the ammonia formed by mineralization is subjected to oxidation and subsequent denitrification to N 2. Together, the results strongly support the hypothesis that peptidic soil components form a major energy and nutrient source for humivorous insects, supplying the animal with microbial fermentation products and essential amino acids.

Mobilization of soil phosphorus during passage through the gut of larvae of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

Soil macrofauna play important roles in the mobilization of soil nutrients. The influence of gut passage on soil phosphorus availability was investigated using the larvae of the scarabaeid beetle Pachnoda ephippiata. Gut passage significantly increased the levels of available P in the feces over that of the soil consumed. 31P-NMR recorded the changes in the nature of organic P in alkaline extracts from gut content. Alkaline phosphatase activity was high in the alkaline midgut, but low in the hindgut and soil. Ferric iron (Fe3+) in the soil was reduced to ferrous iron (Fe2+) in the gut. The amount of Ca2+ was lower in the gut than in the soil. Iron reduction and the decrease of Ca2+ concentration in the gut would reduce P sorption. We think that the following effects are responsible for increased availability of P during the gut passage: alkaline extraction of soil organic matter; hydrolysis of organic phosphate esters with alkaline phosphatase; digestion by the resident microorganisms; and changes in the concentration of metal ions during gut passage. This study suggests that the feeding activities of humivorous larvae would affect the amount of soil P available to plant growth.

Selective digestion of the peptide and polysaccharide components of synthetic humic acids by the humivorous larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

In order to identify the potential nutrient and energy sources of humivorous beetle larvae, we carried out feeding trials with soil supplemented with specifically 14C-labeled model humic acids synthesized by peroxidase-initiated radical polymerization, using the cetoniid beetle Pachnoda ephippiata (Coleoptera: Scarabaeidae) as a model organism. Ingestion of soil by the larvae significantly increased the mineralization of humic acids labeled in their peptide (HA-*peptide) or polysaccharide components (HA-*peptidoglycan and HA-*chitin), whereas the mineralization of humic acids labeled in the aromatic components (HA-*catechol) did not increase significantly. Mineralization was accompanied by a reduction of residual radiolabel in the acid-soluble fraction and an increase in the humic acid and humin fractions of the fecal pellets. During the gut passage, the residual label in peptide or polysaccharide components was transformed into acid-soluble products, especially in the alkaline midgut. High-performance gel-permeation chromatography demonstrated that the changes in solubility were accompanied by large changes in the molecular weight of the residual material. The amount of radiolabel derived from the peptide and polysaccharide components recovered from the larval body and hemolymph was significantly higher than that derived from the aromatic component, which supports the hypothesis that humivorous beetle larvae selectively digest the peptide and polysaccharide components of humic substances, whereas the aromatic components of humic substances are not an important source of nutrients and energy. This is also the first experimental evidence that also chitin and peptidoglycan, the major structural polymers in fungal and bacterial biomass, can be protected from microbial degradation in soil by a copolymerization with phenols and might contribute substantially to the refractory nitrogen pool in soil organic matter.

Digestion of microbial biomass, structural polysaccharides, and protein by the humivorous larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

In order to investigate whether microbial biomass and its residues are nutrient and energy sources for humivorous beetle larvae, we carried out feeding trials using soil supplemented with 14C-labeled fungal biomass ( Penicillium chrysogenum), bacterial biomass ( Bacillus megaterium), fungal or bacterial structural polysaccharide (chitin, peptidoglycan), bacterial protein, or cellulose, taking the larva of the cetoniid beetle Pachnoda ephippiata (Coleoptera: Scarabaeidae) as a model of a humus-feeding beetle larva with a highly alkaline gut. The results showed that gut passage strongly stimulated the mineralization of the structural polymers. The stimulatory effect correlated positively with the recalcitrance of the preparation in the control soil, and was accompanied by a transformation of the residual radiolabel to alkali-soluble and acid-soluble products. The solubility increase was highest in the extremely alkaline midgut. High-performance gel-permeation chromatography demonstrated that the changes in solubility were accompanied by reciprocal changes in the molecular weight of the residual material and that the residual material in the fecal pellets was more humified than in the control soil. The amount of radiolabel recovered from the body and hemolymph of the larvae indicated that microbial biomass and its structural components were assimilated more efficiently than cellulose, which supports the hypothesis that microorganisms and the nitrogenous components of humus are an important dietary resource for humivorous soil macroinvertebrates.