Cellular localisation of aminopeptidase in the midgut of Rhodnius prolixus Stål (Hemiptera:Reduviidae) during blood digestion

Summary By use of the artificial substrate leucyl--naphthylamide, aminopeptidase was localised in the midgut cells of the haematophagous insect Rhodnius prolixus before and at various times up to 25 days after a meal of rabbit blood. The enzyme was primarily associated with the membranes of the microvilli, with extracellular membrane layers and with the lysosomes of the midgut cells. Aminopeptidase activity was also detected on the rough endoplasmic reticulum and at the periphery of intracellular storage vesicles. The absence of aminopeptidase on the microvilli of the crop supports the conclusion that the crop is not involved in the digestion of blood-meal proteins and that protein digestion is restricted to the intestine. The sites of localisation are in accordance with models for the spatial separation of digestive enzymes in the midgut of several non-haematophagous insects, and this suggests that aminopeptidase plays a major role in the terminal digestion of the blood meal. The changes in enzyme localisation during the digestive period correlate with previously described cycles of digestive-enzyme activity and changes in midgut ultrastructure. A model for blood protein digestion in R. prolixus is described.     

The effect of severing abdominal nerves on egg production in Rhodnius prolixus Stål

Severing the two large abdominal nerves which serve segment VI and the internal genitalia in Rhodnius does not prevent the increase in egg production which follows mating so long as the transport of semen into the spermathecae is unaffected. It is concluded that there is no nervous component in the transmission of the mating stimulus from the genitalia to the central nervous system. Severing the remaining abdominal nerves (i.e. those to segments II–V) on one side reduces the egg production of mated females but does not affect that of virgin females. These results are interpreted in terms of interrupting an inhibitory innervation of the abdominal neurosecretory organs which secrete an antigonadotropin.     

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.     

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.     

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.     

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.     

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.     

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).     

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.     

Patterns of acoustic variation in Cicada barbara Stål (Hemiptera, Cicadoidea) from the Iberian Peninsula and Morocco

Field recordings of the calling song and of an amplitude modulated signal produced by males of Cicada barbara from North Africa and the Iberian Peninsula were analysed in order to assess the geographical acoustic variation and the potential usefulness of acoustic data in the discrimination of subspecies and populations. Sound recordings were digitized and the frequency and temporal properties of the calls of each cicada were analysed. In all regions studied, peak frequency, quartiles 25, 50 and 75% and syllable rate showed low coefficients of variation suggesting inherent static properties. All frequency variables were correlated with the latitude, decreasing from south to north. In addition, most acoustic variables of the calling song showed significant differences between regions, and PCA and DFA analyses supported a partitioning within this species between Iberian Peninsula+Ceuta and Morocco, corroborating mtDNA data on the same species. Therefore, the subspecific division of C. barbara into C. barbara barbara from Morocco and C. barbara lusitanica from Portugal, Spain and Ceuta finds support from the present acoustic analyses, a result which is also reinforced by molecular markers.     

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.     

Characterization and functional study of juvenile hormone diol kinase gene in Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

The brown planthopper, Nilaparvata lugens (Stål) is an important pest in rice. It has been widely recognized that the juvenile hormone (JH) is regulated by its hydrolase, which includes juvenile hormone esterase (JHE), juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone diol kinase (JHDK). In this paper, we cloned the gene of Jhdk and the gene expression at different stages of N. lugens was analysed, and the relationship with Jhe and Jheh was studied after silencing the jhdk gene of N. lugens (Nljhdk) through double-stranded RNA (dsRNA) feeding. We also explored the expression of the three JH hydrolase after indoxacarb treatments. RT-PCR was used to amplify the full length Jhdk cDNA, and the Nljhdk gene was expressed throughout all the development periods tested and showed the lowest level at the 4th instar and the highest in the 5th instar. The expression level of Nljhdk in male adults was higher than that of female adults. Through feeding, dsRNA against Nljhdk successfully knocked down the target gene, which had no significant effect on the expression of the jhe gene of N. lugens (Nljhe), while the expression of Nljheh was upregulated. Indoxacarb could inhibit N. lugens reproduction, and the expression level of Nljhe and Nljhdk increased with the increasing of indoxacarb concentration, but the expression of the jheh gene of N. lugens (Nljheh) was reduced. These studies provide a line of experimental evidence in N. lugens to support that Nljhdk encodes the functional protein involved in JH degradation and further showed the relationship of the three hydrolases and the mechanism of indoxacarb inhibition of the fecundity of N. lugens.     

Cultivar and insecticide applications affect the physiological development of the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

Nilaparvata lugens (St?l) (Hemiptera:Delphacidae) is a serious migratory pest of rice in Asia. Pest resurgence often occurs because of insecticide overuse. Using both susceptible (TN1) and moderately resistant (Xieyou 963) cultivars, we studied the effect of foliar insecticides on the percentage of brachypterous adults, female reproductive rate, and crude fat and soluble sugar contents in third- and fifth-instar nymphs and adults. The percentage of brachypterous adults and reproductive rate of adult females developed from nymphs that fed on insecticide-treated plants varied significantly with rice cultivar, type of insecticide, and its concentration. Feeding on susceptible plants increased the percentage of brachypterous adults and reproductive rate of adult females. Also, deltamethrin increased brachypterous production relative to imidacloprid and triazophos. The highest reproductive rate was on plants treated with triazophos. All insecticide treatments in both cultivars resulted in increase of soluble sugar contents in third- and fifth-instar nymphs and adults developed from nymphs feeding on insecticide-treated rice plants. This effect was stronger on the susceptible cultivar. Changes of crude fat content after N. lugens feeding on insecticide-treated plants were related to its feeding duration. Crude fat content in adult developed from nymphs feeding on treated plants was significantly higher that on control plants. These studies showed that plant and insecticide influences on physiological ecology of this planthopper will influence its population dynamics under commercial production of rice in Asia.     

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.     

Taxonomic review of the genus Tambinia Stål (Hemiptera, Fulgoromorpha, Tropiduchidae) with descriptions of four new species from the Pacific region

Four new species of Tambinia Stål (Hemiptera: Fulgoromorpha: Tropiduchidae), Tambinia conus sp. n. (Papua New Guinea), Tambinia macula sp. n. (Malaysia: Borneo), Tambinia robustocarina sp. n. (Malaysia: Sabah) and Tambinia sexmaculata sp. n. (Australia: Kuranda) are described and illustrated from the Pacific region. The diagnostic characters of this genus are redefined. A checklist and a key to the known species of Tambinia are provided.     

β-chain of ATP synthase as a lipophorin binding protein and its role in lipid transfer in the midgut of Panstrongylus megistus (Hemiptera: Reduviidae)

Lipophorin, the main lipoprotein in the circulation of the insects, cycles among peripheral tissues to exchange its lipid cargo at the plasma membrane of target cells, without synthesis or degradation of its apolipoprotein matrix. Currently, there are few characterized candidates supporting the functioning of the docking mechanism of lipophorin-mediated lipid transfer. In this work we combined ligand blotting assays and tandem mass spectrometry to characterize proteins with the property to bind lipophorin at the midgut membrane of Panstrongylus megistus, a vector of Chagas' disease. We further evaluated the role of lipophorin binding proteins in the transfer of lipids between the midgut and lipophorin. The β subunit of the ATP synthase complex (β-ATPase) was identified as a lipophorin binding protein. β-ATPase was detected in enriched midgut membrane preparations free of mitochondria. It was shown that β-ATPase partially co-localizes with lipophorin at the plasma membrane of isolated enterocytes and in the sub-epithelial region of the midgut tissue. The interaction of endogenous lipophorin and β-ATPase was also demonstrated by co-immunoprecipitation assays. Blocking of β-ATPase significantly diminished the binding of lipophorin to the isolated enterocytes and to the midgut tissue. In vivo assays injecting the β-ATPase antibody significantly reduced the transfer of [³H]-diacylglycerol from the midgut to the hemolymph in insects fed with [9,10-³H]-oleic acid, supporting the involvement of lipophorin-β-ATPase association in the transfer of lipids. In addition, the β-ATPase antibody partially impaired the transfer of fatty acids from lipophorin to the midgut, a less important route of lipid delivery to this tissue. Taken together, the findings strongly suggest that β-ATPase plays a role as a docking lipophorin receptor at the midgut of P. megistus.