Ultrastructural localisation of cathepsin B in the midgut of Rhodnius prolixus Stål ( Hemiptera : Reduviidae) during blood digestion

The artificial substrate N-benzoyl-dl-arginine-β-naphthylamine, was used to localise cathepsin B in midgut cells of the haematophagous insect, Rhodnius prolixus Stål (Hemiptera : Reduviidae), during blood digestion. Cathepsin B was localised primarily in the lysosomes of cells from all 3 midgut regions and in Golgi vesicles of the digestive intestinal regions, but not in association with any other cellular structures. The timing of localisation correlated with previously described cycles of endoproteinase activity and with known ultrastructural modifications to the midgut cells. Secretory vesicles, which originated from the Golgi complexes, were present only in the intestinal regions, and in the anterior intestine, they showed a strong positive correlation (r = 0.939, P = 0.01) with post-feeding cathepsin B activity. Cathepsin B plays a major role in primary extracellular digestion of blood proteins, and is active in the midgut lumen and lysosomes rather than in association with the microvilli.     

Characterization of an acidic proteinase from the posterior midgut of Rhodnius prolixus stål (hemiptera: Reduviidae)

Rhodnius prolixus Stål contained cathepsin D in the posterior midgut to breakdown ingested blood proteins. Cathepsin D can be separated from cathepsin B, a second endoprotein contained within midgut preparations, using DEAE ion exchange chromatography. Maximum haemoglobin hydrolysis occurred at pH 2.8 and this substrate was more rapidly hydrolyzed than albumin. Identification of the partially purified proteinase as cathepsin D was based on the pH optimum, substrate preference and inhibition of haemoglobin hydrolysis by pepstatin, diazoacetyl norleucine methyl ester and serum. The presence of cathepsin D as an extracellular digestive proteinase is consistent with the presence of cathepsin B and lysosomal carboxypeptidase B, that have also been detected in the posterior midgut of R. prolixus and other blood sucking Hemiptera.     

Activity cycles and the control of four digestive proteinases in the posterior midgut of Rhodnius prolixus Stål (Hemiptera: Reduviidae)

Proteinase levels in the posterior midgut of fifth-instar and adult Rhodnius prolixus follow a cyclic pattern after ingestion of the bloodmeal. In the fifth instar, cathepsin B showed two peaks: the first occurred 6 days after ingestion and the second at the time of ecdysis. Cathepsin D, cathepsin B and lysosomal carboxypeptidase B reached maximal levels 6 days after ingestion. At this time the highest levels of these proteinases were found in mated females, the lowest in males and intermediate levels in virgin females. Maximal levels of aminopeptidase occurred later than catheptic enzymes, and the decline, after maximal levels were achieved, was much more gradual.Catheptic-proteinase levels within the posterior midgut in fifth-instar larvae and adults correlated positively with the amount of protein contained in this gut region. This indicates that production of these proteinases is controlled by a secretagogue mechanism. Aminopeptidase levels were controlled in a different manner. The mated state or sex of adults altered the proteinase levels by changing the amount of protein that was passed into the digestive midgut from the crop.     

Anti-diuresis in the blood-feeding insect Rhodnius prolixus Stål: antagonistic actions of cAMP and cGMP and the role of organic acid transport

Secretion of primary urine by upper Malpighian tubules of the blood-sucking insect Rhodnius prolixus has recently been shown to be inhibited by cyclic GMP (cGMP). In the present work, we have demonstrated that cGMP has effects antagonistic to those of cAMP in Rhodnius tubules and have further characterized the effects of cGMP on tubular secretion. Cyclic GMP inhibited secretion at all concentrations from 5x10(-6) to 10(-3)M, though this inhibition was partially or wholly reversed by large (2mM) doses of cAMP. While sub-maximal concentrations of cGMP did not significantly alter [K(+)] and [Na(+)] of secreted fluid, high external [cGMP] reduced secretion to minimal levels and caused [K(+)] and [Na(+)] to approach pre-stimulation levels. Cyclic GMP does not appear to affect the permeability of the lower Malpighian tubule to water. Both cAMP and cGMP likely enter tubule cells by way of an organic acid transporter whose activity is induced by feeding. Sensitivity of the tubules to exogenous cGMP and cAMP, which is assumed to be a function of transport activity, reaches a peak approximately 5 days after the blood meal and declines rapidly thereafter. Transport of anions into upper tubules involves at least two different transporters: one for acylamides (e.g., p-aminohippuric acid) and another for sulphonates (e.g., amaranth, phenol red). Amaranth and phenol red blocked the actions of both cGMP and cAMP, whereas p-aminohippuric acid was without effect. This suggests that cyclic nucleotides enter by way of the sulphonate transporter.     

Insulin receptors regulate the fecundity of Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

Two InR (insulin receptor) genes have been identified in the Nilaparvata lugens. In this study, we used RNA interference (RNAi) to investigate the role of InR genes in the fecundity of N. lugens. The expression of NLInR1 and NLInR2 genes was simultaneously silenced with mixture of dsInR1 and dsInR2 (dsInRs) injection. Our results showed that larvae RNAi against both NLInR1 and NLInR2 reduced the number of eggs laid by N. lugens and some eggs as well as ovaries were abnormal. In addition, the relative expression of Vg (vitellogenin) and VgR (vitellogenin receptor) was significantly reduced on the 4th and 6th days after insects treated with larvae RNAi reached the adult stage. We also determined the relative expression levels of insulin/insulin-like signaling (IIS) related genes in RNAi-treated larvae and found that the expression levels of Chico (homologous receptor substrate), Akt (protein kinase B), PI3K (phosphoinositide 3-kinase), and PTEN (phosphatase and tensin homolog) genes decreased whereas FOXO (forkhead box O) and GSK3 (glycogen synthase kinase-3) levels increased on the 4th and 6th days after insects reached the adult stage. These results indicate that silencing of NLInR1 and NLInR2 reduces the fecundity of N. lugens through the IIS pathway.     

Rhodnius prolixus and Rhodnius robustus–like (Hemiptera, Reduviidae) wing asymmetry under controlled conditions of population density and feeding frequency

Habitat change in Rhodnius spp may represent an environmental challenge for the development of the species, particularly when feeding frequency and population density vary in nature. To estimate the effect of these variables in stability on development, the degree of directional asymmetry (DA) and fluctuating asymmetry (FA) in the wing size and shape of R. prolixus and R. robustus–like were measured under laboratory controlled conditions. DA and FA in wing size and shape were significant in both species, but their variation patterns showed both inter-specific and sexual dimorphic differences in FA of wing size and shape induced by nutrition stress. These results suggest different abilities of the genotypes and sexes of two sylvatic and domestic genotypes of Rhodnius to buffer these stress conditions. However, both species showed non-significant differences in the levels of FA between treatments that simulated sylvan vs domestic conditions, indicating that the developmental noise did not explain the variation in wing size and shape found in previous studies. Thus, this result confirm that the variation in wing size and shape in response to treatments constitute a plastic response of these genotypes to population density and feeding frequency.     

First report of Triatoma nigromaculata (Stål, 1859) (Hemiptera, Reduviidae, Triatominae) for Colombia

The presence of Triatoma nigromaculata was recorded for the first time in Colombia in the rural village La Playa in the province of Cauca. A single female was captured from an outdoor area near a household. The presence of Trypanosoma parasites in the specimen was not established due to the poor condition of the specimen. The epidemiological importance of this finding is discussed with respect to the potential for transmission of Chagas disease in the province of Cauca and, more broadly, in Colombia.     

Resistance of Sugarcane Cultivars to Mahanarva fimbriolata (Stål) (Hemiptera: Cercopidae)

The spittlebug Mahanarva fimbriolata (Stål) (Hemiptera: Cercopidae) is one of the most important pests of the sugarcane crop in Brazil. Despite of its importance, there is currently a lack of information regarding sugarcane cultivars’ resistance to the spittlebug. Therefore, our objective was to evaluate the response of sugarcane genotypes to this species. Two experiments were carried out under laboratory conditions using a random block design with treatments in a factorial arrangement of 2?×?13 (experiment 1) and 2?×?12 (experiment 2), with six replicates. The first factor included two levels of infestation (infested and noninfested plants with spittlebugs), while the second consisted of the cultivars. Nymph survival varied from 47.9 to 84.5%, indicating that there are different levels of antibiosis to M. fimbriolata among the tested cultivars. The highest degree of antibiosis was found in cultivars IACSP96-7586 and IACSP96-2008, in which nymph survival was close to 48%. IACSP96-7586 also presented some degree of tolerance, but IACSP96-7569 and IACSP97-6682 stood out as the most tolerant cultivars to the pest, showing the lowest reduction in weight of aboveground biomass. On average, spittlebug infestations caused a significant reduction in relative leaf chlorophyll content and aboveground biomass weight.     

Influence of Mahanarva fimbriolata (Stål) (Hemiptera: Cercopidae) injury on the quality of cane juice

Mahanarva fimbriolata (St?l) is an important pest in Latin America and causes significant reduction in sugarcane productivity. There is no information regarding the effect of this pest on the quality of cane juice used for sugar and alcohol production. This work aimed at evaluating the quality of sugarcane juice from plants attacked by spittlebugs. The experiment was arranged in a completely randomized design with 15 replications, and comprised two treatments: control and chemical treatment with thiamethoxam. An average of 9.2 +/- 4.44 spittlebug nymphs m-1 were found in the plots prior to insecticide application. Nymphs were counted 18, 35, 55, and 82 days after the initial sampling (december/2003). During the mid growing season (July 2004), the juice was extracted from stalks and analyzed for Brix, Pol, RS, pH, fiber, purity, TRS, dextran, starch, and total phenolic compounds. Stalk yield was also measured. Chemical treatment was efficient in reducing spittlebug population, and elevated both stalk yield and juice pH. The accumulated infestation expressed as insect-days was significantly and negatively correlated to yield, Pol, pH, and purity. The concentration of phenolic compounds increased with pest infestation, while dextran and starch levels were not affected. The infestation of 2.4 and 7.3 nymphs m-1 day-1 caused reductions of 8.3% and 29.8% in yield; 1.9% and 5.8% in Pol; 0.4% and 1.1% in pH and 0.4% and 1.2% in purity, respectively, in comparison to areas where the pest population was extremely low (< 0.1 nymphs m-1).     

The food requirements of Phonoctonus nigrofasciatus St?l. (Hemiptera, Reduviidae)

The food requirements of Phonoctonus nigrofassiatus Stål. when fed on nymphs of Dysdercus were investigated. Whithin an instar the conversion ratio varies with the weight of food consumed. Thus, for the first instar nymph the conversion ratio wsa 62.6% when food was limited and 44.3% when food was plentiful. It is suggested that the relationship between conversion ratio and weight food consumed may follow an S-shaped curve. The earlier instars appear to be more efficient than the later ones. This is not merely a further reflection of differing food consumption. The Predatory Value is a variable index which must be used with care when comparing different instars. It is thought that 20–22 fifth instar Dysdercus are necessary to provide the food required for the development of a Phonoctonus from first instar to adult.

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Zusammenfassung Die Nahrungsbedürfnisse von Phonoctonus nigrofasciatus Stål. bei Fütterung mit Dysdercus-Larven wurden untersucht. Innerhalb eines Stadiums variiert die Umsatzrate mit dem Gewicht der aufgenommenen Nahrung. So betrug die Ausnutzungsrate 62.6%, wenn die Nahrungsmenge begrenzt war, und 44.3%, wenn reichlich Futter geboten wurde. Es wird vermutet, daß die Beziehung zwischen Umsatzverhältnis und Gewicht der Nahrung einer S-förmigen Kurve folgt. Die frühen Stadien scheinen wirkungsvoller als die späteren zu sein. Das ist nicht nur eine Folge des unterschiedlichen. Nahrungsverbrauchs. Der Prädator-Wert (Wert als Räuber) ist ein verinderlicher Index, der mit Vorsicht angewendet werden muß, wenn verschiedene Entwicklungsstadien verglichen werden. Es wird angenommen, daß 20 bis 22 fünfte Larvenstadien von Dysdercus erforderlich sind, um den Nahrungsbedarf für die Entwicklung eines Phonoctonus vom ersten Larvenstadium bis zur Imago zu decken.

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This work was carried out in the Department of Zoology and Applied Entomology at the Field Station of Imperial College, Sunninghill, Ascot, Berkshire.     

Development of genital plates in nymphs of Triatoma pallidipennis, Stål 1872, (Hemiptera: Reduviidae) and its application as sexing method

Searching for morphometric differences between sexes in immature forms, the development of genital plates in the exuviae of Triatoma pallidipennis St?l 1872 nymphs was studied. Differences were found in the form and size of the 9th genital urosternite, it is larger and wider in males as compared to females. This difference is reported in several South American Triatoma species. From our results it is possible to sex early stages from microscopic observation of genital plates in whole insects.     

Cell culture of the rice brown planthopper,Nilaparvata lugens Stål (Hemiptera: Delphacidae)

The rice brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the most destructive insect pests of rice in Asia. Although resistant rice varieties can be effective in managing planthopper populations, BPH has repeatedly been able to overcome resistant rice varieties. It is possible that BPH adaptation to resistant rice varieties may be related to its endosymbionts. We investigated methods for culturing BPH cells in order to study in-depth interactions between rice, BPH, and its endosymbionts. In this study, we tested EX-CELL? 405, EX-CELL? 420, Mitsuhashi and Maramorosch’s medium, and Kimura’s medium, for in vitro culture of BPH cells. Only Kimura’s medium was found to be suitable for BPH cell culture, and BPH embryos around blastokinetic stage were the best source for BPH cell culture. Cells from BPH embryonic tissues adhered to the plate substrate, and the migration of cells was observed within 24 h in primary culture. After 3 mo of subculture, various types of BPH cells were successfully maintained in the Kimura’s medium.     

First report on the occurrence of Trypanosoma rangeli Tejera, 1920 in the state of Ceará, Brazil, in naturally infected triatomine Rhodnius nasutus Stål, 1859 (Hemiptera, Reduviidae, Triatominae)

The aim of this work was to identify and report the occurrence of Trypanosoma rangeli and Trypanosoma cruzi in naturally infected Rhodnius nasutus (Hemiptera, Reduviidae, Triatominae) in the state of Ceará, Brazil. Triatomines feces, salivary glands, and hemolymph were collected for fresh examination, and specific detection of T. rangeli and T. cruzi DNA by polymerase chain reaction was carried out. The specific characterization of these two parasites showed the simultaneous presence of both parasites in two (7.7%) of the 26 positive insects. Our results provide further knowledge on the geographical distribution of T. rangeli in Brazil.     

Proteomic analysis of insecticide triazophos-induced mating-responsive proteins of Nilaparvata lugens Stål (Hemiptera: Delphacidae)

The brown planthopper, Nilaparvata lugens (St?l) (Hemiptera: Delphacidae), is a classic example of a resurgent pest induced by insecticides. It has been demonstrated that triazophos treatment causes an increase in the content of male accessory gland proteins (Acps) that can be transferred to females via mating, influencing female reproduction. However, the mechanism of this type of insecticide-induced Acps in males and the subsequent stimulation of reproduction in females are not well understood. To identify changes in the types of Acps and reproductive proteins in mated females, we conducted a comparative proteomic analysis. Six samples were categorized into four different groups: (1) untreated unmated males compared to treated unmated males (UUM vs TUM); (2) treated unmated males compared to treated mated males (TUM vs TMM); (3) untreated unmated females compared to treated unmated females (UUF vs TUF); (4) treated unmated females compared to treated mated females (TUF vs TMF). Protein expression changes among the four different groups were examined by two-dimensional gel electrophoresis (2-DE) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Of the 500-600 reproducibly detected protein spots on each gel, 107 protein spots were differentially expressed between the four different groups. Of the 63 proteins identified by LC-MS/MS analysis, 38 were up-regulated and 25 were down-regulated in the four different groups. Some novel proteins related to fecundity were observed including spermatogenesis-associated protein 5, testis development protein NYD-SP6, arginine kinase, actin-5C, vitellogenin, and ovarian serine protease nudel. The elevated expression of novel fecundity proteins in six samples of N. lugens females and males due to exposure to triazophos was confirmed by quantitative real-time PCR (qRT-PCR). The results suggest that these proteins may participate in the reproductive process of N. lugens adult females and males. Our findings fill a gap in understanding the relationship between insecticide-treated males and the stimulated reproduction of N. lugens females.     

Occurrence of Dictyla monotropidia Stål (Hemiptera: Tingidae) on Cordia verbenacea Al. DC in Brazil

Recently, Dictyla monotropidia St?l was observed feeding on plants of black sage (Cordia verbenacea Al. DC). The colonies of this insect were observed on abaxial surface of leaves, with nymphs and adults sucking the phloem sieve, causing spot, yellow aspect and leaf fall. D. monotropidia was already related as pest in other Cordia species in countries of Central and South America. Although, this is the first report of this insect attacking plants of C. verbenacea in Brazil.     

Effects of rice potassium level on the fecundity and expression of the vitellogenin gene of Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

Potassium (K) is a key component of plant nutrition, significantly influencing crop growth. Levels of this nutrient in plants can also influence a number of pest infestations. The brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is an important pest of rice in Asia. In this study, we examined K contents in rice grown in hydroponic solution, and its relationship to the fecundity and expression of the vitellogenin (Nlvg) gene of N. lugens which was reared on the rice. Our findings indicated that K contents in rice increased with the increasing K concentration within the hydroponic solution, but reduced at the highest K concentration (160 mg/L). The number of eggs laid by N. lugens which was reared on the rice varied significantly with K concentration, and increased by 0.12 and 0.22 fold under 20 mg/L and 160 mg/L K level compared to that of the control (40 mg/L), decreasing by 0.57 fold under 0 mg/L K. Nlvg mRNA expression increased 1.17 and 1.94 fold in individuals which were reared on rice grown in 20 mg/L and 160 mg/L K level, compared to that of the control before mating; and by 3.36 and 2.97 after mating, respectively. However, Nlvg mRNA expression fold decreased by 0.99 under 0 mg/L K level before mating and 0.91 after mating. The variation of eggs may be attributed to the change of Nlvg mRNA expression, because there was a positive correlation between the eggs and Nlvg mRNA expression. These results demonstrated low (20 mg/L) and highest K levels (160 mg/L) in hydroponic solution showed the lower K level in plants than the control, which facilitated the fecundity of N. lugens. The study of the effects of K levels on the fecundity should have significance for insect control.     

Induction of insect plant resistance to the spittlebug Mahanarva fimbriolata Stål (Hemiptera: Cercopidae) in sugarcane by silicon application

Changes in the agroecosystem with the increase of green cane harvesting in Brazil affected the insect populations associated to this crop, and secondary pests like the spittlebug Mahanarva fimbriolata St?l, became much more important. Many studies have demonstrated the active role played by silicon in plant defense against herbivory. The objective of this study was to evaluate the effects of silicon applications on the biology of the spittlebug reared on two resistant (SP79-1011 and SP80-1816) and one susceptible (SP81-3250) sugarcane cultivars. Sugarcane plants were grown under greenhouse conditions and submitted to different treatments: with and without silicon fertilizer in two different soil type (sandy and clay soil). The newly hatched nymphs were transferred to sugarcane roots and placed into boxes with lids, to keep a moistened and dark environment favoring their growth and maintenance of the root system, providing food access to the developing nymphs. After emergence, adult males and females were placed in cages for mating and oviposition. The silicon absorbed and accumulated in the plant caused an increase in nymphal mortality, and depending on the sugarcane cultivar tested this element also provided an increase in the duration of the nymphal stage and a decrease in the longevity of males and females. 'SP79-1011' presented the highest silicon content in leaves, and M. fimbriolata had the highest nymph mortality and the shortest female longevity. The pre-oviposition period, fecundity and egg viability were not affected by the silicon content in plants or the cultivar used.