Morphological and Ultrastructural Characterization of Hemocytes in an Insect Model, the Hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae).

Hemocytes, the cells present in the hemolymph of insects and other invertebrates, perform several physiological functions, including innate immunity. The current classification of hemocyte types is based mostly on morphological features; however, divergences have emerged among specialists in triatomines, the insect vectors of Chagas' disease (Hemiptera: Reduviidae). Here, we have combined technical approaches in order to characterize the hemocytes from fifth instar nymphs of the triatomine Dipetalogaster maxima. Moreover, in this work we describe, for the first time, the ultrastructural features of D. maxima hemocytes. Using phase contrast microscopy of fresh preparations, five hemocyte populations were identified and further characterized by immunofluorescence, flow cytometry and transmission electron microscopy. The plasmatocytes and the granulocytes were the most abundant cell types, although prohemocytes, adipohemocytes and oenocytes were also found. This work sheds light on a controversial aspect of triatomine cell biology and physiology setting the basis for future in-depth studies directed to address hemocyte classification using non-microscopy-based markers.

The entomotoxin Jack Bean Urease changes cathepsin D activity in nymphs of the hematophagous insect Dipetalogaster maxima (Hemiptera: Reduviidae).

In insects, cathepsin D is a lysosomal aspartic endopeptidase involved in several functions such as digestion, defense and reproduction. Jack Bean Urease (JBU) is the most abundant urease isoform obtained from the seeds of the plant Canavalia ensiformis. JBU is a multifunctional protein with entomotoxic effects unrelated to its catalytic activity, by mechanisms not yet fully understood. In this work, we employed nymphs of the hematophagous insect Dipetalogaster maxima as an experimental model in order to study the effects of JBU on D. maxima CatD (DmCatD). In insects without treatment, immunofluorescence assays revealed a conspicuous distribution pattern of DmCatD in the anterior and posterior midgut as well as in the fat body and hemocytes. Western blot assays showed that the active form of DmCatD was present in the fat body, the anterior and posterior midgut; whereas the proenzyme was visualized in hemocytes and hemolymph. The transcript of DmCatD and its enzymatic activity was detected in the anterior and posterior midgut as well as in fat body and hemocytes. JBU injections induced a significant increase of DmCatD activity in the posterior midgut (at 3 h post-injection) whereas in the hemolymph, such an effect was observed after 18 h. These changes were not correlated with modifications in DmCatD mRNA and protein levels or changes in the immunofluorescence pattern. In vitro experiments might suggest a direct effect of the toxin in DmCatD activity. Our findings indicated that the tissue-specific increment of cathepsin D activity is a novel effect of JBU in insects.

Jaburetox, a urease-derived peptide: Effects on enzymatic pathways of the cockroach Nauphoeta cinerea.

Jaburetox is a recombinant peptide derived from one of the Canavalia ensiformis urease isoforms. This peptide induces several toxic effects on insects of different orders, including interference on muscle contractility in cockroaches, modulation of UDP-N-acetylglucosamine pyrophosphorylase (UAP) and nitric oxide synthase (NOS) activities in the central nervous system of triatomines, as well as activation of the immune system in Rhodnius prolixus. When injected, the peptide is lethal for R. prolixus and Triatoma infestans. Here, we evaluated Jaburetox toxicity to Nauphoeta cinerea cockroaches, exploring the effects on the central nervous system through the activities of UAP, NOS, acid phosphatases (ACP), and acetylcholinesterase (AChE). The results indicated that N. cinerea is not susceptible to the lethal effect of the peptide. Moreover, both in vivo and in vitro treatments with Jaburetox inhibited NOS activity, without modifying the protein levels. No alterations on ACP activity were observed. In addition, the enzyme activity of UAP only had its activity affected at 18 hr after injection. The peptide increased the AChE activity, suggesting a mechanism involved in overcoming the toxic effects. In conclusion, our findings indicate that Jaburetox affects the nitrinergic signaling as well as the AChE and UAP activities and establishes N. cinerea as a Jaburetox-resistant model for future comparative studies.

Soyuretox, an Intrinsically Disordered Polypeptide Derived from Soybean (Glycine Max) Ubiquitous Urease with Potential Use as a Biopesticide.

Ureases from different biological sources display non-ureolytic properties that contribute to plant defense, in addition to their classical enzymatic urea hydrolysis. Antifungal and entomotoxic effects were demonstrated for Jaburetox, an intrinsically disordered polypeptide derived from jack bean (Canavalia ensiformis) urease. Here we describe the properties of Soyuretox, a polypeptide derived from soybean (Glycine max) ubiquitous urease. Soyuretox was fungitoxic to Candida albicans, leading to the production of reactive oxygen species. Soyuretox further induced aggregation of Rhodnius prolixus hemocytes, indicating an interference on the insect immune response. No relevant toxicity of Soyuretox to zebrafish larvae was observed. These data suggest the presence of antifungal and entomotoxic portions of the amino acid sequences encompassing both Soyuretox and Jaburetox, despite their small sequence identity. Nuclear Magnetic Resonance (NMR) and circular dichroism (CD) spectroscopic data revealed that Soyuretox, in analogy with Jaburetox, possesses an intrinsic and largely disordered nature. Some folding is observed upon interaction of Soyuretox with sodium dodecyl sulfate (SDS) micelles, taken here as models for membranes. This observation suggests the possibility for this protein to modify its secondary structure upon interaction with the cells of the affected organisms, leading to alterations of membrane integrity. Altogether, Soyuretox can be considered a promising biopesticide for use in plant protection.

The Fat Body of the Hematophagous Insect, Panstrongylus megistus (Hemiptera: Reduviidae): Histological Features and Participation of the ß-Chain of ATP Synthase in the Lipophorin-Mediated Lipid Transfer.

In insects, lipid transfer to the tissues is mediated by lipophorin, the major circulating lipoprotein, mainly through a nonendocytic pathway involving docking receptors. Currently, the role of such receptors in lipid metabolism remains poorly understood. In this work, we performed a histological characterization of the fat body of the Chagas' disease vector, Panstrongylus megistus (Burmeister), subjected to different nutritional conditions. In addition, we addressed the role of the ß-chain of ATP synthase (ß-ATPase) in the process of lipid transfer from lipophorin to the fat body. Fifth-instar nymphs in either fasting or fed condition were employed in the assays. Histological examination revealed that the fat body was composed by diverse trophocyte phenotypes. In the fasting condition, the cells were smaller and presented a homogeneous cytoplasmic content. The fat body of fed insects increased in size mainly due to the enlargement of lipid stores. In this condition, trophocytes contained abundant lipid droplets, and the rough endoplasmic reticulum was highly developed and mitochondria appeared elongated. Immunofluorescence assays showed that the ß-ATPase, a putative lipophorin receptor, was located on the surface of fat body cells colocalizing partially with lipophorin, which suggests their interaction. No changes in ß-ATPase expression were found in fasting and fed insects. Blocking the lipophorin-ß-ATPase interaction impaired the lipophorin-mediated lipid transfer to the fat body. The results showed that the nutritional status of the insect influenced the morphohistological features of the tissue. Besides, these findings suggest that ß-ATPase functions as a lipophorin docking receptor in the fat body.

Jaburetox-induced toxic effects on the hemocytes of Rhodnius prolixus (Hemiptera: Reduviidae).

Jaburetox is a recombinant peptide derived from a Canavalia ensiformis urease that presents toxic effects upon several species of insects, phytopathogenic fungi and yeasts of medical importance. So far, no toxicity of Jaburetox to mammals has been shown. Previous reports have identified biochemical targets of this toxic peptide in insect models, although its mechanism of action is not completely understood. In this work, we aimed to characterize the effects of Jaburetox in hemolymphatic insect cells. For this purpose, the model insect and Chagas' disease vector Rhodnius prolixus was used. In vivo and in vitro experiments indicated that Jaburetox interacts with a subset of hemocytes and it can be found in various subcellular compartments. In insects injected with Jaburetox there was an increase in the gene expression of the enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP), chitin synthase and nitric oxide synthase (NOS). Nevertheless, the expression of NOS protein, the enzyme activities of UAP and acid phosphatase (a possible link between UAP and NOS) as well as the phosphorylation state of proteins remained unchanged upon the in vivo Jaburetox treatment. Nitric oxide (NO) imaging using fluorescent probes showed that Jaburetox augmented NO production in the hemocyte aggregates when compared to controls. Even though Jaburetox activated the hemocytes, as demonstrated by wheat germ agglutinin binding assays, the peptide did not lead to an increase of their phagocytic behavior. Taken together, these findings contribute to our understanding of toxic effects of Jaburetox, a peptide with biotechnological applications and a prospective tool for rational insect control.

Jaburetox affects gene expression and enzyme activities in Rhodnius prolixus, a Chagas' disease vector.

Jaburetox, a recombinant peptide of ∼11kDa derived from one of the Canavalia ensiformis (Jack Bean) urease isoforms, is toxic and lethal to insects belonging to different orders when administered orally or via injection. Previous findings indicated that Jaburetox acts on insects in a complex fashion, inhibiting diuresis and the transmembrane potential of Malpighian tubules, interfering with muscle contractility and affecting the immune system. In vitro, Jaburetox forms ionic channels and alters permeability of artificial lipid membranes. Moreover, recent data suggested that the central nervous system (CNS) is a target organ for ureases and Jaburetox. In this work, we employed biochemical, molecular and cellular approaches to explore the mode of action of Jaburetox using Rhodnius prolixus, one of the main Chagas' disease vectors, as experimental model. In vitro incubations with fluorescently labeled Jaburetox indicated a high affinity of the peptide for the CNS but not for salivary glands (SG). The in vitro treatment of CNS or SG homogenates with Jaburetox partially inhibited the activity of nitric oxide synthase (NOS), thus disrupting nitrinergic signaling. This inhibitory effect was also observed in vivo (by feeding) for CNS but not for SG, implying differential modulation of NOS in these organs. The inhibition of NOS activity did not correlate to a decrease in expression of its mRNA, as assessed by qPCR. UDP-N-acetylglucosamine pyrophosphorylase (UAP), a key enzyme in chitin synthesis and glycosylation pathways and a known target of Jaburetox in insect CNS, was also affected in SG, with activation of the enzyme seen after both in vivo or in vitro treatments with the peptide. Unexpectedly, incubation of Jaburetox with a recombinant R. prolixus UAP had no effect on its activity, implying that the enzyme's modulation by the peptide requires the participation of other factor(s) present in CNS or SG homogenates. Feeding Jaburetox to R. prolixus decreased the mRNA levels of UAP and chitin synthase, indicating a complex regulation exerted by the peptide on these enzymes. No changes were observed upon Jaburetox treatment in vivo and in vitro on the activity of the enzyme acid phosphatase, a possible link between UAP and NOS. Here we have demonstrated for the first time that the Jaburetox induces changes in gene expression and that SG are another target for the toxic action of the peptide. Taken together, these findings contribute to a better understanding of the mechanism of action of Jaburetox as well as to the knowledge on basic aspects of the biochemistry and neurophysiology of insects, and might help in the development of optimized strategies for insect control.

Humoral and cellular immune responses induced by the urease-derived peptide Jaburetox in the model organism Rhodnius prolixus.

BACKGROUND: Although the entomotoxicity of plant ureases has been reported almost 20 years ago, their insecticidal mechanism of action is still not well understood. Jaburetox is a recombinant peptide derived from one of the isoforms of Canavalia ensiformis (Jack Bean) urease that presents biotechnological interest since it is toxic to insects of different orders. Previous studies of our group using the Chagas disease vector and model insect Rhodnius prolixus showed that the treatment with Jack Bean Urease (JBU) led to hemocyte aggregation and hemolymph darkening, among other effects. In this work, we employed cell biology and biochemical approaches to investigate whether Jaburetox would induce not only cellular but also humoral immune responses in this species. RESULTS: The findings indicated that nanomolar doses of Jaburetox triggered cation-dependent, in vitro aggregation of hemocytes of fifth-instar nymphs and adults. The use of specific eicosanoid synthesis inhibitors revealed that the cellular immune response required cyclooxygenase products since indomethacin prevented the Jaburetox-dependent aggregation whereas baicalein and esculetin (inhibitors of the lipoxygenases pathway) did not. Cultured hemocytes incubated with Jaburetox for 24 h showed cytoskeleton disorganization, chromatin condensation and were positive for activated caspase 3, an apoptosis marker, although their phagocytic activity remained unchanged. Finally, in vivo treatments by injection of Jaburetox induced both a cellular response, as observed by hemocyte aggregation, and a humoral response, as seen by the increase of spontaneous phenoloxidase activity, a key enzyme involved in melanization and defense. On the other hand, the humoral response elicited by Jaburetox injections did not lead to an increment of antibacterial or lysozyme activities. Jaburetox injections also impaired the clearance of the pathogenic bacteria Staphylococcus aureus from the hemolymph leading to increased mortality, indicating a possible immunosuppression induced by treatment with the peptide. CONCLUSIONS: In our experimental conditions and as part of its toxic action, Jaburetox activates some responses of the immune system of R. prolixus both in vivo and in vitro, although this induction does not protect the insects against posterior bacterial infections. Taken together, these findings contribute to the general knowledge of insect immunity and shed light on Jaburetox's mechanism of action.