On the mechanisms involved in biological heme crystallization.

Blood-feeding organisms digest hemoglobin, releasing large quantities of heme inside their digestive tracts. Free heme is very toxic, and these organisms have evolved several mechanisms to protect against its deleterious effects. One of these adaptations is the crystallization of heme into the dark-brown pigment hemozoin (Hz). Here we review the process of Hz formation, focusing on organisms other than Plasmodium that have contributed to a better understanding of heme crystallization. Hemozoin has been found in several distinct classes of organisms including protozoa, helminths and insects and Hz formation is the predominant form of heme detoxification. The available evidence indicates that amphiphilic structures such as phospholipid membranes and lipid droplets accompanied by specific proteins play a major role in heme crystallization. Because this process is specific to a number of blood-feeding organisms and absent in their hosts, Hz formation is an attractive target for the development of novel drugs to control illnesses associated with these hematophagous organisms.

Alpha-glucosidase promotes hemozoin formation in a blood-sucking bug: an evolutionary history.

BACKGROUND: Hematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: Hz formation activity of an alpha-glucosidase was investigated. Hz formation was inhibited by specific alpha-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect alpha-glucosidase was able to inhibit Hz formation. The alpha-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that alpha-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of alpha-glucosidase gene, was injected into R. prolixus females' hemocoel. Gene silencing was accomplished by reduction of both alpha-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of alpha-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of alpha-glucosidase shows a high similarity to the insect alpha-glucosidases, with critical histidine and aspartic residues conserved among the enzymes. CONCLUSIONS/SIGNIFICANCE: Herein the Hz formation is shown to be associated to an alpha-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that alpha-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.

Parasitic castration, growth, and sex steroids in the freshwater bonefish Cyphocharax gilbert (Curimatidae) infested by Riggia paranensis (Cymothoidea)

Cyphocharax gilbert shows parasitic castration when infested by the crustacean Riggia paranensis, being unable to reproduce. Fish were sampled in the middle rio Itabapoana, Brazil, to study the prevalence of parasitism, growth, and sex steroid concentrations, considering the body size, sex, and reproductive condition of specimens. Most of the fish analyzed were infested (56.0 percent). The presence of two lines on the scales was more frequent among infested fish (22.0 percent) than among fish without parasites (12.0 percent for females and 10.0 percent for males). The occurrence of three lines on the scales was rare (3.5 percent among infested and 2.0 percent among females without parasites). These results suggest that growth of the host is faster than that of non infested fish. The serum concentrations of sex steroids from fish without parasites varied at different gonadal development stages (17 beta-estradiol: 60.0 to 976.7 pg/ml; total testosterone: 220.0 to 3,887.7 pg/ml). All infested fish had lower levels of the two sex steroids and undeveloped gonads. Sex steroids levels in infested females were close to those in females at post-spawning stages. Total testosterone concentrations of infested males were below those of males at early gonadal maturation stage. These results suggest that R. paranensis reduces the reproductive capacity of C. gilbert by affecting the host endocrine system

Cyphocharax gilbert exibe castração parasitária quando está infestado pelo crustáceo Riggia paranensis, estando impossibilitado de reproduzir. Os peixes foram coletados no trecho médio do rio Itabapoana, Brasil, para analisar a prevalência do parasitismo, quantificar crescimento e as concentrações de esteróides sexuais, considerando o tamanho do corpo, o sexo e a condição reprodutiva dos espécimes. A maioria dos peixes analisados estava infestada (56,0 por cento). A presença de duas linhas em escamas foi mais freqüente entre os peixes infestados (22,0 por cento) que entre os peixes não infestados (12,0 por cento para as fêmeas e 10,0 por cento para os machos). A presença de três linhas na escama foi rara (3,5 por cento entre os peixes infestados e 2,0 por cento entre as fêmeas não infestadas). Estes resultados sugerem que o crescimento no hospeideiro pode ser mais rapido que no peixes não parasitados. As concentrações de esteróides sexuais no soro dos peixes não infestados variaram entre os diferentes estágios reprodutivos (17 beta-estradiol: 60,0 a 976,7 pg/ml; total testosterona: 220,0 a 3.887,7 pg/ml). Todos os peixes infestados apresentaram baixos níveis dos dois hormônios esteroidais e ausência de desenvolvimento gonadal. Os níveis de esteróides sexuais nas fêmeas infestadas foram próximos aos níveis encontrados nas fêmeas pós-desovadas. A concentração de testosterona encontrada nos machos infestados foi inferior àquela obtida nos machos que estavam iniciando o desenvolvimento gonadal. Estes resultados sugerem que R. paranensis impede a reprodução de C. gilbert, afetando o sistema endócrino do hospedeiro

Perimicrovillar membranes promote hemozoin formation into Rhodnius prolixus midgut.

Rhodnius prolixus is a hematophagous insect that ingests large quantities of blood in each blood-feeding session. This ingested blood provides important nutrients to sustain the insect's oogenesis and metabolic pathways. During the digestive process, however, huge amounts of heme are generated as a consequence of the hemoglobin breakdown. Heme is an extremely dangerous molecule, since it can generate reactive oxygen species in the presence of oxygen that impair the normal metabolism of the insect. Part of the hemoglobin-derived heme can associate with the perimicrovillar membranes (PMM) in the gut lumen of R. prolixus; in this study we demonstrate the participation of the PMM in a heme detoxification process. These membranes were able to successfully induce heme aggregation into hemozoin (Hz). Heme aggregation was not dependent on the erythrocyte membranes, since the contribution of these membranes to the process was negligible, demonstrating that the ability to induce heme aggregation is a feature of the PMM, possibly representing a pre-adaptation of the hemipterans to feeding on blood.

Heme crystallization in the midgut of triatomine insects.

Hemozoin (Hz) is a heme crystal produced by several blood-feeding organisms in order to detoxify free heme released upon hemoglobin (Hb) digestion. Here we show that heme crystallization also occurs in three species of triatomine insects. Ultraviolet-visible and infrared light absorption spectra of insoluble pigments isolated from the midgut of three triatomine species Triatoma infestans, Dipetalogaster maximus and Panstrongylus megistus indicated that all produce Hz. Morphological analysis of T. infestans and D. maximus midguts revealed the close association of Hz crystals to perimicrovillar membranes and also as multicrystalline assemblies, forming nearly spherical structures. Heme crystallization was promoted by isolated perimicrovillar membranes from all three species of triatomine bugs in vitro in heat-sensitive reactions. In conclusion, the data presented here indicate that Hz formation is an ancestral adaptation of Triatominae to a blood-sucking habit and that the presence of perimicrovillar membranes plays a central role in this process.

Infestation pattern and parasitic castration of the crustacean Riggia paranensis (Crustacea: Cymothoidea) on the fresh water fish Cyphocharax gilbert (Teleostei: Curimatidae)

Cyphocharax gilbert infested by Riggia paranensis shows parasitic castration. The prevalence of parasitism in C. gilbert varied among different environments, being higher in the middle rio Itabapoana. Fish were collected monthly using two cast nets (thrown 30 times during the day) and gillnets kept in the river during 12 hour, from sunset to sunrise, between September 1997 and August 2000. Infestation pattern was investigated on 1358 specimens. Most of them were infested (57.9 percent), with one or two parasites; the majority (62.9 percent) was collected during the rainy season (spring-summer). The parasite did not show preference for sex or size of hosts. A total of 91.5 percent of the 511 examined parasites had a body size that represented 10.1 percent to 20 percent of host standard length. The reproductive condition of 311 specimens of R. paranensis was analyzed checking the presence of oocytes in the ovarian and eggs or embryos in the marsupium. Nearly 73 percent of them were at reproductive phase, and had a body size that represented 5.1 percent to 20 percent of host standard length. The size of the immature parasites varied from 0.1 percent to 5 percent of the host size. The results suggest that R. paranensis may adopt a fast growth rate strategy and increase the investment in reproduction when they occupy most of the host's pericardial space.

Cyphocharax gilbert infestado por Riggia paranensis apresenta castração parasitária. A prevalência do parasito varia entre diferentes ambientes, sendo maior no trecho médio do rio Itabapoana. Os peixes foram coletados mensalmente neste rio usando duas tarrafas (lançadas por 30 vezes durante o dia) e redes de espera por 12 horas, do crepúsculo ao amanhecer, entre setembro de 1997 e agosto de 2000. O padrão de infestação foi investigado em 1358 peixes. Peixes infestados apresentaram um ou dois parasitos, representando 57,9 por cento da população e a maioria deles (62,9 por cento) foi coletada no período de maior precipitação (primavera-verão). O parasito não apresentou preferência por sexo ou tamanho do hospedeiro. No total, 91,5 por cento de 511 parasitos examinados apresentaram um tamanho corpóreo que representou de 10,1 por cento a 20 por cento do comprimento padrão do hospedeiro. A condição reprodutiva em 311 espécimes R. paranensis foi analisada através da presença de oócitos no ovário e ovos ou embriões no marsúpio, sendo que cerca de 73 por cento estavam em fase de reprodução, apresentando um tamanho corporal que variou entre 5,1 por cento a 20 por cento do comprimento padrão do hospedeiro. O tamanho dos parasitos imaturos variou de 0,1 por cento a 5 por cento do tamanho do hospedeiro. Os resultados sugerem que R. paranensis pode adotar uma estratégia de rápido crescimento e deve aumentar o investimento em reprodução quando ocupa grande parte do espaço disponível na cavidade pericardial do hospedeiro.

The haemoxisome: a haem-iron containing structure in the Rhodnius prolixus midgut cells.

Rhodnius prolixus midgut was analysed using transmission electron microscopy and electron spectroscopic imaging in order to localize the cellular structures involved in haem metabolism. In the posterior midgut, special cellular electron-dense structures were observed. These structures are here designated haemoxisomes. Haemoxisomes are present in the epithelial cells at various time points after a blood meal. Several days after the blood meal, some of them become less electron-dense. By electron spectroscopic imaging, large amounts of iron and oxygen were detected in these cellular structures. The iron is probably bound to the porphyrin ring as an iron-protoporphyrin IX complex, as detected using the diaminobenzidine technique. An interesting observation was the presence of endoplasmic reticulum surrounding the haemoxisomes during some special periods. Iron content was monitored in the posterior midgut epithelium and was found to be constant at the initial days after a blood meal, but slightly higher at the end of the digestive process (from 13th up to 20th day). These results are in agreement with the observation that the appearance of the haemoxisomes changes at the end of the digestive process. The ability to degrade haem seems to depend on the presence of endoplasmic reticulum as observed using a haem degradation assay in the presence of an endoplasmic reticulum-enriched fraction. Taken together these results suggest that haemoxisomes may play a role in intracellular haem detoxification.

A new intracellular pathway of haem detoxification in the midgut of the cattle tick Boophilus microplus: aggregation inside a specialized organelle, the hemosome.

The hard tick Boophilus microplus ingests large volumes of cattle blood, as much as 100 times its own mass before feeding. Huge amounts of haem are produced during haemoglobin digestion, which takes place inside acidic lysosomal-type vacuoles of the digest cells of the midgut. Haem is a promoter of free radical formation, so haemoglobin digestion poses an intense oxidative challenge to this animal. In the present study we followed the fate of the haem derived from haemoglobin hydrolysis in the digest cells of the midgut of fully engorged tick females. The tick does not synthesize haem, so during the initial phase of blood digestion, absorption is the major route taken by the haem, which is transferred from the digest cells to the tick haemocoel. After this absorptive period of a few days, most of the haem produced upon haemoglobin degradation is accumulated in the interior of a specialized, membrane-delimited, organelle of the digest cell, herein called hemosome. Haem accounts for 90% of the hemosome mass and is concentrated in the core of this structure, appearing as a compact, non-crystalline aggregate of iron protoporphyrin IX without covalent modifications. The unusual FTIR spectrum of this aggregate suggests that lateral propionate chains are involved in the association of haem molecules with other components of the hemosome, which it is proposed is a major haem detoxification mechanism in this blood-sucking arthropod.

Expression and immunolocalization of a Boophilus microplus cathepsin L-like enzyme.

Efforts are being undertaken to control tick infestations that cause important economic losses. A cathepsin L-like endopeptidase of Boophilus microplus was expressed in Escherichia coli; the recombinant enzyme was capable of hydrolysing gelatin, tick vitellin and bovine haemoglobin. In this paper we focus on the expression and local of synthesis of this enzyme in the tick. RT-PCR experiments showed that this endopeptidase is transcribed in the gut of partially engorged tick females. In immunoblotting, polyclonal antibodies against the recombinant enzyme reacted with proteins of larvae older than 5 days, of fully and partially engorged female gut. In immunolocalization experiments the enzyme was localized in probable secretory cells of the gut. Based on our findings we postulate that BmCL1 may be involved in haemoglobin degradation in the B. microplus gut. This enzyme may be used as target for the control of this parasite.

Rhodnius prolixus vitellin is composed of three different populations: comparison with vitellogenin.

Rhodnius prolixus oocyte extracts were chromatographed on an ion exchange column in order to purify vitellin (VT). Three VT heterogeneous populations were identified and named VT(1), VT(2), and VT(3) according to their order of elution from the column. The phosphate content of each population was determined, after lipid extraction, and a heterogeneous distribution was found: VT(1) being the less phosphorylated (50 mol P/mol protein) and VT(3) the heavily phosphorylated population (281 mol P/mol protein). Analysis of radioactivity associated with each VT population purified from animals fed with (32)Pi showed the same phosphorylation profile. Due to the fact that vitellogenin is the known precursor of VT, we have also chromatographed 32P-VG in the same way as we purified VT. Only one VG's population was detected and resembled to VT(3) with respect to its elution profile. All VT populations contain the same neutral lipids, but they were heterogeneous with respect to phospholipid composition. VT(1) presents phosphatidylcholine and phosphatidylethanolamine whereas VT(2) and VT(3) also showed cardiolipin and probably phosphatidylserine. Sugar composition of VT(2) and VT(3) includes mannose as the main associated carbohydrate but VT(1) also contains glucose resembling VG. Although VG and VT are similar with respect to the elution profile, their sugar composition is different. These results suggest a post-endocytosis processing on VG molecule. The possible biological function of VT heterogeneous populations is discussed.

Aedes aegypti peritrophic matrix and its interaction with heme during blood digestion.

A large amount of heme is produced upon digestion of red cell hemoglobin in the midgut of mosquitoes. The interaction between heme and the peritrophic matrix (PM) was studied in Aedes aegypti. By light microscopy, the PM appeared as a light brownish layer between the intestinal epithelium and the alimentary bolus. This natural color can be attributed to the presence of heme bound to the matrix. In histochemical studies, a diffuse peroxidase activity of the heme molecules was clearly observed between the erythrocytes and the PM at 14 h after the blood meal. This activity tends to increase and concentrate in the PM reaching its maximum thickness at 24 h after feeding. Most of the heme of the PM was found associated to with enormous number of small electron-dense granules. The amount of heme bound to the PM increased in parallel with the progression of digestion, reaching a maximum at 48 h after feeding, when 18 nmol of heme were found in an individual matrix. The association of heme with PM from insects fed with plasma is saturable, suggesting the existence of specific binding sites for hemin in the PM. Taken all together, our data indicate that the PM performs a central role in heme detoxification in this insect.

On the pro-oxidant effects of haemozoin.

Haemozoin (Hz) is a haem aggregate produced in some blood-feeding organisms. There is a general belief that Hz formation would be a protective mechanism against haem toxicity. Here we show that when aggregated into Hz, haem is less deleterious than its free form. When haem was added to phosphatidylcholine (PC) liposomes, there was an intense stimulation of oxygen consumption, which did not occur when Hz was incubated with the same preparation. Evaluation of oxygen radical attack to lipids, by measurement of thiobarbituric acid reactive substances (TBARS), showed significantly lower levels of lipid peroxidation in samples containing PC liposomes incubated with Hz than with haem. However, TBARS production induced by Hz was much higher when using 2-deoxyribose (2-DR) as substrate, than with PC liposomes. Spin-trapping analysis by electron paramagnetic resonance (EPR) of Hz and tert-butylhydroperoxide (tert-BuOOH) showed that production of methoxyl and tert-butoxyl radicals was only slightly reduced compared to what was observed with haem. Interestingly, when large Hz crystals were used in 2-DR TBARS assays and tert-BuOOH EPR experiments, the pro-oxidant effects of Hz were strongly reduced. Moreover, increasing concentrations of Hz did not induce erythrocyte lysis, as occurred with haem. Thus, the reduced capacity of Hz to impose radical damage seems to result from steric hindrance of substrates to access the aggregated haem, that becomes less available to participate in redox reactions.