The Role of Glutamine Homeostasis in Emotional and Cognitive Functions.

Glutamine (Gln), a non-essential amino acid, is synthesized de novo by glutamine synthetase (GS) in various organs. In the brain, GS is exclusively expressed in astrocytes under normal physiological conditions, producing Gln that takes part in glutamatergic neurotransmission through the glutamate (Glu)-Gln cycle. Because the Glu-Gln cycle and glutamatergic neurotransmission play a pivotal role in normal brain activity, maintaining Gln homeostasis in the brain is crucial. Recent findings indicated that a neuronal Gln deficiency in the medial prefrontal cortex in rodents led to depressive behaviors and mild cognitive impairment along with lower glutamatergic neurotransmission. In addition, exogenous Gln supplementation has been tested for its ability to overcome neuronal Gln deficiency and reverse abnormal behaviors induced by chronic immobilization stress (CIS). Although evidence is accumulating as to how Gln supplementation contributes to normalizing glutamatergic neurotransmission and the Glu-Gln cycle, there are few reviews on this. In this review, we summarize recent evidence demonstrating that Gln supplementation ameliorates CIS-induced deleterious changes, including an imbalance of the Glu-Gln cycle, suggesting that Gln homeostasis is important for emotional and cognitive functions. This is the first review of detailed mechanistic studies on the effects of Gln supplementation on emotional and cognitive functions.

Glutamine Supplementation Preserves Glutamatergic Neuronal Activity in the Infralimbic Cortex, Which Delays the Onset of Mild Cognitive Impairment in 3×Tg-AD Female Mice.

It was recently found that glutamine (Gln) supplementation activates glutamatergic neurotransmission and prevents chronic-stress-induced mild cognitive impairment (MCI). In this study, we evaluated the effects of Gln on glutamatergic activity in the medial prefrontal cortex and the onset of cognitive impairment in a triple-transgenic Alzheimer's disease mouse model (3×Tg-AD). Female 3×Tg-AD mice were fed a normal diet (3×Tg) or a Gln-supplemented diet (3×Tg+Gln) from 2 to 6 months of age. Glutamatergic neuronal activity was analyzed at 6 months, and cognitive function was examined at 2, 4, and 6 months. 3×Tg mice exhibited a decrease in glutamatergic neurotransmission in the infralimbic cortex, but 3×Tg+Gln mice did not. The 3×Tg group showed MCI at 6 months of age, but the 3×Tg+Gln group did not. The expressions of amyloid peptide, inducible nitric oxide synthase, and IBA-1 were not elevated in the infralimbic cortex in the 3×Tg+Gln group. Therefore, a Gln-supplemented diet could delay the onset of MCI even in a mouse model predisposed to cognitive impairment and dementia through genetic modification.

Long-term exposure changes the environmentally relevant bis(2-ethylhexyl) phthalate to be a neuro-hazardous substance disrupting neural homeostasis in emotional and cognitive functions.

Bis(2-ethylhexyl) phthalate (DEHP) is the most used member of the phthalate class of compounds. Extensive use of this plasticizer allows daily exposure to humans via various routes. A positive relationship between DEHP exposure and neurobehavioral disorders is suspected. But, there are insufficient data on the harmfulness of neurobehavioral disorders caused by DEHP exposure, particularly at daily exposure levels. In this study, we assessed the consequences of daily DEHP ingestion (2 and 20 mg/kg diets) in male mice for at least 100 days and examined its effects on neuronal functions associated with neurobehavioral disorders, such as depression and cognitive decline. We found the marked depressive behaviors and reduced learning and memory function in the DEHP-ingestion groups, and that biomarkers related to chronic stress were increased in plasma and brain tissues. Long-term DEHP ingestion induced collapse of glutamate (Glu) and glutamine (Gln) homeostasis as a result of disruption of the Glu-Gln cycle in the medial prefrontal cortex and hippocampus. The reduced glutamatergic neurotransmission activity caused by DEHP ingestion was demonstrated using an electrophysiological method. This study revealed that long-term exposure to DEHP is hazardous and can cause neurobehavioral disorders, even at daily exposure levels.

Long-Term Hyperglycemia Causes Depressive Behaviors in Mice with Hypoactive Glutamatergic Activity in the Medial Prefrontal Cortex, Which Is Not Reversed by Insulin Treatment.

The etiology of hyperglycemic-induced depressive behaviors is unclear. We hypothesized that long-term hyperglycemia may induce long-lasting disturbances in glutamatergic signaling and neural damages, causing depressive behaviors. To prove our hypothesis, a C57BL/6N mouse model of hyperglycemia was maintained for 4 weeks (equivalent to approximately 3 years in humans), after which insulin treatment was administered for an additional 4 weeks to normalize hyperglycemia-induced changes. Hyperglycemic mice showed depressive-like behaviors. Glutamatergic neurons and glial cells in the medial prefrontal cortex (mPFC) were affected by hyperglycemia. Insulin treatment improved blood glucose, water intake, and food intake to normoglycemic levels, but did not improve depressive-like behaviors. Glutamatergic signaling decreased with long-term hyperglycemia and did not normalize with insulin-induced normoglycemia. Importantly, hyperglycemia-induced changes in the mPFC were almost not reversed by the 4-week insulin treatment. In particular, levels of insulin receptor beta subunit (IRß), IRS-1, vesicular glutamate transporter 1, glutamine transporter SNAT2, phosphate-activated glutaminase, and GLUT-3 were not changed by insulin. Nitration and the dephosphorylation of IRß in the PFC also did not improve with insulin treatment. Therefore, our results suggest that hypoactive glutamatergic activity in the mPFC is involved in diabetic-associated depressive behaviors, and it is difficult to cure with glycemic regulation alone.

Isoflavone-Enriched Soybean Leaves (Glycine Max) Alleviate Cognitive Impairment Induced by Ovariectomy and Modulate PI3K/Akt Signaling in the Hippocampus of C57BL6 Mice.

(1) Background: The estrogen decline during perimenopause can induce various disorders, including cognitive impairment. Phytoestrogens, such as isoflavones, lignans, and coumestans, have been tried as a popular alternative to avoid the side effects of conventional hormone replacement therapy, but their exact mechanisms and risk are not fully elucidated. In this study, we investigated the effects of isoflavone-enriched soybean leaves (IESLs) on the cognitive impairment induced by ovariectomy in female mice. (2) Methods: Ovariectomy was performed at 9 weeks of age to mimic menopausal women, and the behavior tests for cognition were conducted 15 weeks after the first administration. IESLs were administered for 18 weeks. (3) Results: The present study showed the effects of IESLs on the cognitive function in the OVX (ovariectomized) mice. Ovariectomy markedly increased the body weight and fat accumulation in the liver and perirenal fat, but IESL treatment significantly inhibited them. In the behavioral tests, ovariectomy impaired cognitive functions, but administration of IESLs restored it. In addition, in the OVX mice, administration of IESLs restored decreased estrogen receptor (ER) ß and PI3K/Akt expression in the hippocampus. (4) Conclusions: The positive effects of IESLs on cognitive functions may be closely related to the ER-mediated PI3/Akt signaling pathway in the hippocampus.

Combined Water Extracts from Oxidation-Treated Leaves and Branches of Hovenia dulcis Has Anti-Hangover and Liver Protective Effects in Binge Alcohol Intake of Male Mice.

Hovenia dulcis, known as the oriental raisin tree, is used for food supplements and traditional medicine for the liver after alcohol-related symptoms. However, little information exists about the use of its leaves and branches. In this study, we established a method to use the leaves and branches to develop anti-hangover treatment and elucidated the underlying mechanisms. Oxidation-treated leaves (OL) exhibited high antioxidant content comparable to that of the peduncles and showed an anti-hangover effect in male mice. The branch extract (BE) was enriched in the flavonoid catechin, approximately five times more than OL extract. The mixture of OL and BE (OLB) was formulated in a 2:1 ratio with frozen-dried extract weight and was tested for anti-hangover effects and protective properties against binge alcohol-induced liver injury. OLB showed better anti-hangover effect than OL. In addition to this anti-hangover effect, OLB protected the liver from oxidative/nitrosative damage induced by binge alcohol intake.

Ingestion of Bis(2-ethylhexyl) phthalate (DEHP) during adolescence causes depressive-like behaviors through hypoactive glutamatergic signaling in the medial prefrontal cortex.

Over the past decades, the production and use of hazardous chemicals has increased worldwide, and the incidence of neurological diseases is increasing proportionately. Among these chemicals, Bis(2-ethylhexyl) phthalate (DEHP) is the most common member of the phthalate family used as a plasticizer. The present study assessed the consequences of daily DEHP ingestion and its effects on brain functions related to depressive-like behaviors. Adolescent C57BL/6 male mice ingested different concentrations of DEHP in their diet (2, 20, and 200 mg/kg of diet), and behavioral changes in anxiety, despair, anhedonia, and sociality were investigated. DEHP exposure evoked depressive-like behaviors in a dose-dependent manner for each symptom. The levels of corticosterone and reactive oxygen species/reactive nitrogen species increased in DEHP-exposed groups, suggesting chronic stress-like responses. In the medial prefrontal cortex (mPFC), glutamate and glutamine were decreased, and glutamine synthetase showed lower activity compared to the control group, suggesting imbalanced glutamatergic signaling. Measuring the spontaneous excitatory postsynaptic current of glutamatergic neurons, we found that DEHP ingestion resulted in hypoactive glutamatergic signaling in the mPFC.

Acutely increased ß-hydroxybutyrate plays a role in the prefrontal cortex to escape stressful conditions during the acute stress response.

Ketone bodies can be increased in the blood under certain physiological conditions, but their role under such conditions remains to be clarified. In the present study, we found the increment and usage of ß-hydroxybutyrate (BHB) in the prefrontal cortex (PFC) during acute stress. BHB levels increased in the blood and PFC after 30-min acute immobilization stress, and BHB dehydrogenase 1 increased in the PFC simultaneously, but not in the hippocampus. Moreover, increased levels of acetyl-CoA, pyruvate carboxylase, and glutamate dehydrogenase 1 were found in the PFC, implicating the metabolism of increased BHB in the brain. Thus, we checked the levels of glutamate, glutamine, and GABA and found increased levels of glutamate and glutamine in the stressed group compared with that in the control group in the PFC. Exogenous administration of BHB enhanced struggling behaviors under stressful conditions. Our results suggest that the metabolism of BHB from peripheral blood in the PFC may contribute to acute stress responses to escape stressful conditions.

Glutamine Supplementation Prevents Chronic Stress-Induced Mild Cognitive Impairment.

We recently reported that glutamine (Gln) supplementation protected glutamatergic neurotransmission from the harmful effects of chronic stress. Altered glutamatergic neurotransmission is one of the main causes of cognitive disorders. However, the cognitive enhancer function of Gln has not been clearly demonstrated thus far. Here, we evaluated whether and how Gln supplementation actually affects chronic stress-induced cognitive impairment. Using a chronic immobilization stress (CIS) mouse model, we confirmed that chronic stress induced mild cognitive impairment (MCI) and neuronal damage in the hippocampus. In contrast, Gln-supplemented mice did not show evidence of MCI. To investigate possible underlying mechanisms, we confirmed that CIS increased plasma corticosterone levels as well as brain and plasma levels of reactive oxygen/nitrogen species. CIS also increased levels of inducible nitric oxide synthase and NADPH oxidase subunits (p47phox and p67phox) in both the prefrontal cortex and CA1 region of the hippocampus. CIS decreased the number of synaptic puncta in the prefrontal cortex and hippocampus, but these effects were inhibited by Gln supplementation. Taken together, the present results suggest that Gln is an effective agent against chronic stress-induced MCI.

Glutamine Supplementation Ameliorates Chronic Stress-induced Reductions in Glutamate and Glutamine Transporters in the Mouse Prefrontal Cortex.

Chronic immobilization stress (CIS) induces low levels of glutamate (Glu) and glutamine (Gln) and hypoactive glutamatergic signaling in the mouse prefrontal cortex (PFC), which is closely related to the Glu-Gln cycle. A Gln-supplemented diet ameliorates CIS-induced deleterious changes. Here, we investigated the effects of CIS and Gln supplementation on Glu-Gln cycle-related proteins to characterize the underlying mechanisms. Using the CIS-induced depression mouse model, we examined the expression of 11 proteins involved in the Glu-Gln cycle in the PFC. CIS decreased levels of glutamate transporter 1 (GLT1) and sodium-coupled neutral amino acid transporter (SNAT) 1, SANT2, SNAT3, and SNAT5. Gln supplementation did not affect the non-stressed group but significantly increased GLT1 and SNATs of the stressed group. By immunohistochemical analysis, we confirmed that SNAT1 and SNAT2 were decreased in neurons and GLT1, SNAT3, and SNAT5 were decreased in astrocytes in the medial PFC of the stressed group, but Gln-supplemented diet ameliorated these decrements. Collectively, these results suggest that CIS may cause depressive-like behaviors by decreasing Glu and Gln transportation in the PFC and that a Gln-supplemented diet could prevent the deleterious effects of CIS.

Chronological Aging Standard Curves of Telomere Length and Mitochondrial DNA Copy Number in Twelve Tissues of C57BL/6 Male Mouse.

The changes in telomere length and mitochondrial DNA copy number (mtDNAcn) are considered to be aging markers. However, many studies have provided contradictory or only fragmentary information about changes of these markers in animal models, due to inaccurate analysis methods and a lack of objective aging standards. To establish chronological aging standards for these two markers, we analyzed telomere length and mtDNAcn in 12 tissues-leukocytes, prefrontal cortex, hippocampus, pituitary gland, adrenal gland, retina, aorta, liver, kidney, spleen, skeletal muscle, and skin-from a commonly used rodent model, C57BL/6 male mice aged 2⁻24 months. It was found that at least one of the markers changed age-dependently in all tissues. In the leukocytes, hippocampus, retina, and skeletal muscle, both markers changed age-dependently. As a practical application, the aging marker changes were analyzed after chronic immobilization stress (CIS) to see whether CIS accelerated aging or not. The degree of tissue-aging was calculated using each standard curve and found that CIS accelerated aging in a tissue-specific manner. Therefore, it is expected that researchers can use our standard curves to objectively estimate tissue-specific aging accelerating effects of experimental conditions for least 12 tissues in C57BL/6 male mice.

Insufficient glutamine synthetase activity during synaptogenesis causes spatial memory impairment in adult mice.

Glutamatergic synapses constitute a major excitatory neurotransmission system and are regulated by glutamate/glutamine (Gln) cycling between neurons and astrocytes. Gln synthetase (GS) produced by astrocytes plays an important role in maintaining the cycle. However, the significance of GS during synaptogenesis has not been clarified. GS activity and expression significantly increase from postnatal day (PD) 7 to 21, and GS is expressed prior to glial fibrillary acidic protein (GFAP) and is more abundant than GFAP throughout synaptogenesis. These observations suggest that GS plays an important role in synaptogenesis. We investigated this by inhibiting GS activity in neonatal mice and assessed the consequences in adult animals. Lower expression levels of GS and GFAP were found in the CA3 region of the hippocampus but not in the CA1 region. Moreover, synaptic puncta and glutamatergic neurotransmission were also decreased in CA3. Behaviorally, mice with inhibited GS during synaptogenesis showed spatial memory-related impairment as adults. These results suggest that postnatal GS activity is important for glutamatergic synapse development in CA3.

Glutamine has antidepressive effects through increments of glutamate and glutamine levels and glutamatergic activity in the medial prefrontal cortex.

Emerging evidence has shown the low levels of glutamate (Glu) and glutamine (Gln) and the hypoactivity in the cortex of patients with depression. The hypoactivity is closely related with low frequency of glutamatergic signaling that is affected by the levels of Glu and Gln. Thus, we hypothesized that there might be a causality among low levels of Glu and Gln, hypoactive glutamatergic neurotransmissions, and depressive behaviors. Here, we found low Glu and Gln levels and low frequency of spontaneous excitatory postsynaptic current (sEPSC) of glutamatergic neurons in the medial prefrontal cortex (mPFC) of chronic immobilization stress (CIS)-induced depressed mice. The depressed mice also showed hypoactive Gln synthetase (GS). Inhibition of GS by methionine sulfoximine (MSO) decreased Glu and Gln levels and increased depressive behaviors with low frequency of sEPSC in the mPFC, indicating that Glu and Gln decrements cause hypoactive glutamatergic neurotransmissions and depressive behaviors. Both Glu and Gln could increase sEPSC of glutamatergic neurons in the mPFC on slice patch, but only Gln overcame MSO to increase sEPSC, suggesting that exogenous Gln would recover CIS-induced low frequency of sEPSC caused by hypoactive GS and act as an antidepressant. Expectedly, Gln supplementation showed antidepressant effects against CIS; it increased glutamatergic neurotransmissions with Glu and Gln increment in the mPFC and attenuated depressive behaviors. Moreover, selective glutamatergic activation in the mPFC by optogenetics decreased depressive behavior. In conclusion, depressive behaviors evoked by chronic stress were due to hypoactive glutamatergic neurons in the mPFC caused by low levels of Glu and Gln, and exogenous Gln can be used as an alternative antidepressant to increase glutamatergic neurotransmission.

SDE5, a putative RNA export protein, participates in plant innate immunity through a flagellin-dependent signaling pathway in Arabidopsis.

In eukaryotes, RNA silencing, mediated by small interfering RNAs, is an evolutionarily widespread and versatile silencing mechanism that plays an important role in various biological processes. Increasing evidences suggest that various components of RNA silencing pathway are involved in plant defense machinery against microbial pathogens in Arabidopsis thaliana. Here, we show genetic and molecular evidence that Arabidopsis SDE5 is required to generate an effective resistance against the biotrophic bacteria Pseudomonas syringae pv. tomato DC3000 and for susceptibility to the necrotrophic bacteria Erwinia caratovora pv. caratovora. SDE5, encodes a putative mRNA export factor that is indispensable for transgene silencing and the production of trans-acting siRNAs. SDE5 expression is rapidly induced by exogenous application of phytohormone salicylic acid (SA), methyl jasmonate (MeJA), phytopathogenic bacteria, and flagellin. We further report that SDE5 is involved in basal plant defense and mRNA export. Our genetic data suggests that SDE5 and Nonexpressor of PR Gene1 (NPR1) may contribute to the same SA-signaling pathway. However, SDE5 over-expressing transgenic plant exhibits reduced defense responsive phenotype after flagellin treatment. Taken together, these results support the conclusion that SDE5 contributes to plant innate immunity in Arabidopsis.

Development of an antibody-based diagnostic method for the identification of Bemisia tabaci biotype B.

The whitefly Bemisia tabaci is a very destructive pest. B. tabaci is composed of various morphologically undistinguishable biotypes, among which biotypes B and Q, in particular, draw attention because of their wide distribution in Korea and differential potentials for insecticide resistance development. To develop a biotype-specific protein marker that can readily distinguishes biotypes B from other biotypes in the field, we established an ELISA protocol based on carboxylesterase 2 (COE2), which is more abundantly expressed in biotypes B compared with Q. Recombinant COE2 was expressed, purified and used for antibody construction. Polyclonal antibodies specific to B. tabaci COE2 [anti-COE2 pAb and deglycosylated anti-COE2 pAb (DG anti-COE2 pAb)] revealed a 3-9-fold higher reactivity to biotype B COE2 than biotype Q COE2 by Western blot and ELISA analyses. DG anti-COE2 pAb exhibited low non-specific activity, demonstrating its compatibility in diagnosing biotypes. Western blot and ELISA analyses determined that one of the 11 field populations examined was biotype B and the others were biotype Q, suggesting the saturation of biotype Q in Korea. DG anti-COE2 pAb discriminates B. tabaci biotypes B and Q with high specificity and accuracy and could be useful for the development of a B. tabaci biotype diagnosis kit for on-site field applications.

An insulin-binding protein from the venom of a solitary wasp Eumenes pomiformis binds to apolipophorin III in lepidopteran hemolymph.

EpIBP, an insulin-like peptide-binding protein, is a major protein component of the venom of a solitary hunting wasp, Eumenes pomiformis. To evaluate the bioactivity, bacteria-expressed EpIBP was injected into Spodoptera exigua larvae, resulting in a higher survival rate and reduced loss of body weight under starvation conditions than control larvae. EpIBP was found to interact with apolipophorin III (apoLp III), implying that EpIBP might function by altering the apoLp III-mediated metabolism of prey.

Differential Properties of Venom Peptides and Proteins in Solitary vs. Social Hunting Wasps.

The primary functions of venoms from solitary and social wasps are different. Whereas most solitary wasps sting their prey to paralyze and preserve it, without killing, as the provisions for their progeny, social wasps usually sting to defend their colonies from vertebrate predators. Such distinctive venom properties of solitary and social wasps suggest that the main venom components are likely to be different depending on the wasps' sociality. The present paper reviews venom components and properties of the Aculeata hunting wasps, with a particular emphasis on the comparative aspects of venom compositions and properties between solitary and social wasps. Common components in both solitary and social wasp venoms include hyaluronidase, phospholipase A2, metalloendopeptidase, etc. Although it has been expected that more diverse bioactive components with the functions of prey inactivation and physiology manipulation are present in solitary wasps, available studies on venom compositions of solitary wasps are simply too scarce to generalize this notion. Nevertheless, some neurotoxic peptides (e.g., pompilidotoxin and dendrotoxin-like peptide) and proteins (e.g., insulin-like peptide binding protein) appear to be specific to solitary wasp venom. In contrast, several proteins, such as venom allergen 5 protein, venom acid phosphatase, and various phospholipases, appear to be relatively more specific to social wasp venom. Finally, putative functions of main venom components and their application are also discussed.

Venom peptides from solitary hunting wasps induce feeding disorder in lepidopteran larvae.

The cell lytic activity and toxicity against lepidopteran larvae of 13 venom peptides (4 OdVPs and 9 EpVPs) from two solitary hunting wasps, Orancistrocerus drewseni and Eumenes pomiformis, were examined with mastoparan as a reference peptide. Of the 13 peptides, 7 were predicted to have α-helical structures that exhibit the typical character of amphipathic α-helical antimicrobial peptides. The remaining peptides exhibited coil structures; among these, EpVP5 possesses two Cys residues that form an internal disulfide bridge. All the helical peptides including mastoparan showed antimicrobial and insect cell lytic activities, whereas only two of them were hemolytic against human erythrocytes. The helical peptides induced a feeding disorder when injected into the vicinity of the head and thorax of Spodoptera exigua larvae, perhaps because their non-specific neurotoxic or myotoxic action induced cell lysis. At low concentrations, however, these helical peptides increased cell permeability without inducing cell lysis. These findings suggest that the helical venom peptides may function as non-specific neurotoxins or myotoxins and venom-spreading factors at low concentrations, as well as preservatives for long-term storage of the prey via antimicrobial, particularly antifungal, activities.

Identification and characterization of venom proteins of two solitary wasps, Eumenes pomiformis and Orancistrocerus drewseni.

Secretory proteins were identified in the venoms of two solitary hunting wasps, Eumenes pomiformis and Orancistrocerus drewseni, by SDS-PAGE in conjunction with mass analysis. More than 30 protein bands (2-300 kDa) were detected from the crude venom of each wasp. With the aid of the previously constructed venom gland/sac-specific EST libraries, a total of 31 and 20 proteins were identified from 18 to 20 distinctive protein bands of E. pomiformis and O. drewseni venoms, respectively. Arginine kinase was the most predominant protein in both wasp venoms. Along with the full-length arginine kinase, a truncated form, which was known to have paralytic activity on a spider, was a common predominant protein in the two wasp venoms. Insulin/insulin-like peptide-binding protein was abundantly found only in E. pomiformis venom, which might be due to its unique behaviors of oviposition and provision. The presence of various immune response-related proteins and antioxidants suggested that wasps might use their venom to maintain prey fresh while feeding wasp larvae by protecting the prey from microbial invasion and physiological stresses. It seemed that some venom proteins are secreted into venom fluid from venom gland cells via exosomes, not by signal sequence-mediated transport processes.

Differential gene expression profiles in the venom gland/sac of Eumenes pomiformis (Hymenoptera: Eumenidae).

To search for novel transcripts encoding biologically active venom components, a subtractive cDNA library specific to the venom gland and sac (gland/sac) of a solitary hunting wasp species, Eumenes pomiformis Fabricius (1781), was constructed by suppression subtractive hybridization. A total of 541 expressed sequence tags (ESTs) were clustered and assembled into 102 contigs (31 multiple sequences and 71 singletons). In total, 37 cDNAs were found in the library via BLASTx searching and manual annotation. Eight contigs (337 ESTs) encoding short venom peptides (10 to 16 amino acids) occupied 62% of the library. The deduced amino acid sequence (78 amino acids) of a novel venom peptide transcript shared sequence similarity with trypsin inhibitors and dendrotoxin-like venom peptides known to be K(+) channel blockers, implying that this novel peptide may play a role in the paralysis of prey. In addition to phospholipase A2 and hyaluronidase, which are known to be the main components of wasp venoms, several transcripts encoding enzymes, including three metallopeptidases and a decarboxylase likely involved in the processing and activation of venomous proteins, peptides, amines, and neurotransmitters, were also isolated from the library. The presence of a transcript encoding a putative insulin/insulin-like peptide binding protein suggests that solitary hunting wasps use their venom to control their prey, leading to larval growth cessation. The abundance of these venom components in the venom gland/sac and in the alimentary canal was confirmed by quantitative real-time PCR. Discovery of venom gland/sac-specific transcripts should promote further studies on biologically active components in the venom of solitary hunting wasps.