Unfed Insects Research Articles

Exploring the role of glycoprotein hormone GPA2/GPB5 in the medically important insect, Rhodnius prolixus

Glycoprotein hormones are formed by the heterodimerization of alpha and beta subunits. In vertebrates, there are five glycoprotein hormones, four of which have a common alpha subunit (GPA1) bound to a specific beta subunit (GPB1, GPB2, GPB3, or GPB4), and the fifth, thyrostimulin, is formed by the dimerization of GPA2 and GPB5 subunits. These hormones mediate physiological events such as development, metabolism, and reproduction, although the functional role of thyrostimulin in vertebrates has not been fully elucidated. Recent reports in invertebrates, specifically in holometabolous insects, suggest that GPA2/GPB5 plays a critical role in development, diuresis, and reproduction. In this study, we clone and characterize the transcripts for the glycoprotein hormone GPA2/GPB5 and its receptor (LGR1) in fifth instar Rhodnius prolixus, a hemimetabolous insect vector of Chagas disease. Sequence analyses reveals considerable identity and similarity between GPA2/GPB5 and LGR1 and those reported in other arthropod species. Quantitative PCR (qPCR) shows that both subunit transcripts, GPA2 and GPB5, and LGR1 transcripts are present in a variety of tissues, with greatest expression of the subunits in the central nervous system (CNS) and highest LGR1 expression in the Malpighian tubules (MT). Results from temporal qPCR analyses reveal a decrease in transcript expression 24 h after feeding, followed by an increase as the days post-feeding advance. Using immunohistochemistry, we show that GPB5 is expressed throughout the CNS, and importantly is present in neurosecretory cells in the brain and abdominal neuromeres and their neurohemal organs, indicating a neurohormonal role for this signaling pathway. A reduction in LGR1 transcript expression (via RNA interference) led to a greater weight loss and mortality rate in unfed insects. In addition, when a blood meal is offered, the insects with reduced LGR1 consume a significantly smaller blood meal and have higher mortality rates as the days post-feeding advance. Overall, the results suggest that the GPA2/GPB5 signaling pathway may play roles during a prolonged unfed state and in feeding-related events.

Identification and characterization of the SIFamide receptor in the hemimetabolous Chagas disease vector, Rhodnius prolixus Stål, 1859, (Hemiptera, Reduviidae, Triatominae)

Within arthropods, the SIFamide family of neuropeptides appears to be involved in the modulation of a range of physiological and behavioral events. In Rhodnius prolixus, we have previously shown the presence of SIFamidergic-like processes in neurohemal release sites and provided evidence for a role for Rhopr-SIFa in modulating heartbeat frequency and feeding behaviors. Here, the R. prolixus SIFamide receptor (RhoprSIFR) has been identified, cloned, and sequenced. Sequence analyses show high similarity and identity between the RhoprSIFR and other cloned SIFamide receptors. Quantitative PCR shows that the RhoprSIFR transcript is found in a variety of tissues, including those involved in feeding and reproduction. In unfed insects, high transcript expression is observed in the central nervous system and midgut, suggesting a role of Rhopr-SIFa in various processes related to feeding and digestion. Expression of the RhoprSIFR transcript changes between unfed, 24 h post-fed, and 7 d post-fed conditions. Expression of the RhoprSIFR transcript significantly increases in the anterior midgut and posterior midgut 7 d post-feeding and knockdown of the RhoprSIFR transcript significantly reduces the size of blood meal consumed. This data suggests a possible role for Rhopr-SIFa in regulating long-term post-feeding osmotic balance and digestion of the blood meal. Lastly, transcript expression of Rhopr-SIFa and RhoprSIFR also varies temporally in relation to the reproductive stage, suggesting an involvement of this signaling pathway in reproductive activities. Identification of the RhoprSIFR and its expression profile now provide tools for a more detailed understanding into the precise coordination of feeding and other physiological processes in R. prolixus.

"Urate and NOX5 Control Blood Digestion in the Hematophagous Insect Rhodnius prolixus".

Low levels of reactive oxygen species (ROS) are now recognized as essential players in cell signaling. Here, we studied the role of two conserved enzymes involved in redox regulation that play a critical role in the control of ROS in the digestive physiology of a blood-sucking insect, the kissing bug Rhodnius prolixus. RNAi-mediated silencing of RpNOX5 and RpXDH induced early mortality in adult females after a blood meal. Recently, a role for RpNOX5 in gut motility was reported, and here, we show that midgut peristalsis is also under the control of RpXDH. Together with impaired peristalsis, silencing either genes impaired egg production and hemoglobin digestion, and decreased hemolymph urate titers. Ultrastructurally, the silencing of RpNOX5 or RpXDH affected midgut cells, changing the cells of blood-fed insects to a phenotype resembling the cells of unfed insects, suggesting that these genes work together in the control of blood digestion. Injection of either allopurinol (an XDH inhibitor) or uricase recapitulated the gene silencing effects, suggesting that urate itself is involved in the control of blood digestion. The silencing of each of these genes influenced the expression of the other gene in a complex way both in the unfed state and after a blood meal, revealing signaling crosstalk between them that influences redox metabolism and nitrogen excretion and plays a central role in the control of digestive physiology.

Sulfakinin inhibits activity of digestive enzymes in the brown planthopper, Nilaparvata lugens

• Two maltase genes and one lipase gene were identified in the brown planthopper. • Refed induces digestive enzyme secretion in Nilaparvata lugens . • Sulfakinin injection reduces digestive enzyme activity. • Sulfakinin injection decrease transcriptional levels of digestive enzyme genes. In animals, feeding can regulate release of digestive enzymes. Digestive enzymes are produced and released in response to specific ratios of nutrients, so the quality and quantity of food ingested are important factors in the secretion and activity of digestive enzymes. In general, the enzyme activity and secretion in the fed insects are relatively higher than that in the unfed insects. Neuropeptides and peptide hormones are important regulators of enzyme activity. In several insects, the neuropeptide sulfakinin (SK) is known to be a regulator of feeding and digestion similar to cholecystokinin in mammals. However, the roles of diet and SK in regulation of activity of digestive enzymes in the important pest insect, the brown planthopper ( Nilaparvata lugens ), are unknown. In this study, we identified six genes encoding different digestive enzymes and cloned three of these. We found that enzymatic activity and transcriptional levels of digestive enzymatic activity genes were upregulated by refeeding animals for 5 h after 24 h starvation. Furthermore, injection of N. lugens SK reduces digestive enzyme activity and leads to a downregulation of digestive enzyme gene transcripts. This study provides new views into the action of diet and SK in regulation of digestive enzymes in (hemimetabolous) insects. Taken together with the roles of SK in inducing satiety, our data strongly suggest that SK signaling is important in regulation of food ingestion and processing.

Transcriptomic analysis of regulatory pathways involved in female reproductive physiology of Rhodnius prolixus under different nutritional states

The triatomine Rhodnius prolixus, a vector of the etiological agent of Chagas disease, has long been used as model to understand important aspects of insect physiology. Despite this history, the impact of the nutritional state on regulatory pathways associated with reproductive performance in triatomines has never been studied. The insulin-like peptide/target of rapamycin (ILP/ToR) signaling pathway is typically responsible for detecting and interpreting nutrient levels. Here, we analyzed transcriptomes from the central nervous system, fat bodies and ovaries of adult females in unfed and fed conditions, with a focus on the ILP/ToR signaling. The results show an up-regulation of transcripts involved in ILP/ToR signaling in unfed insects. However, we demonstrate that this signaling is only activated in tissues from fed insects. Moreover, we report that FoxO (forkhead box O) factor, which regulates longevity via ILP signaling, is responsible for the up-regulation of transcripts related with ILP/ToR signaling in unfed insects. As a consequence, we reveal that unfed females are in a sensitized state to respond to an increase of ILP levels by rapidly activating ILP/ToR signaling. This is the first analysis that correlates gene expression and protein activation of molecules involved with ILP/ToR signaling in R. prolixus females in different nutritional states.

PH control in the midgut of Aedesaegypti under different nutritional conditions.

Aedes aegypti is one of the most important disease vectors in the world. Because their gut is the first site of interaction with pathogens, it is important to understand A. aegypti gut physiology. In this study, we investigated the mechanisms of pH control in the midgut of A. aegypti females under different nutritional conditions. We found that unfed females have an acidic midgut (pH∼6). The midgut of unfed insects is actively maintained at pH6 regardless of the ingestion of either alkaline or acidic buffered solutions. V-ATPases are responsible for acidification after ingestion of alkaline solutions. In blood-fed females, the abdominal midgut becomes alkaline (pH7.54), and the luminal pH decreases slightly throughout blood digestion. Only ingested proteins were able to trigger this abrupt increase in abdominal pH. The ingestion of amino acids, even at high concentrations, did not induce alkalinisation. During blood digestion, the thoracic midgut remains acidic, becoming a suitable compartment for carbohydrate digestion, which is in accordance with the higher alpha-glucolytic activity detected in this compartment. Ingestion of blood releases alkalising hormones in the haemolymph, which induce alkalinisation in ex vivo preparations. This study shows that adult A. aegypti females have a very similar gut physiology to that previously described for Lutzomyia longipalpis It is likely that all haematophagous Nematocera exhibit the same type of physiological behaviour.

Physiological development and dispersal ability of newly emerged Monochamus galloprovincialis

Novel associations between exotic pathogens and native insects may result in major ecological and economical losses. In Europe, Monochamus galloprovincialis (Olivier) (Coleoptera: Cerambycidae) is the only known vector of the exotic pine wood nematode Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle (Nematoda: Aphelenchoididae), the causal agent of pine wilt disease (PWD). Transportation of goods containing nematode-infested beetles is the main pathway for the spread of the disease. In this scenario, management actions involving early detection and eradication are critical to stop further spread by the vector. Although dispersal of mature M. galloprovincialis has been successfully tracked using commercial baits and traps, dispersal ability of immature individuals is poorly understood. Sexual maturation and other physiological traits related to dispersal were studied for newly emerged M. galloprovincialis after different shoot-feeding spans. Sexual maturation was complete after on average 12 (males) or 13 (females) feeding days. Monochamus galloprovincialis adults emerged with an average of 10% lipid and 23.8% (males) or 29.9% (females) pterothorax muscle content, and these percentages did not change significantly during shoot feeding. Microtomography images of wing muscle structures at different maturation stages confirmed these results. Emerged adults that were kept unfed survived an average of 12 days in the lab. The ability of unfed immature insects to fly through hostless terrain was studied by marking and releasing newly emerged insects from a crop area located up to 3 km away from two small pine stands. The longest flown distance recorded was 2 km. Fitted regressions indicated that immature insects could have travelled up to 3 109 m to reach the experimental stands. We found that M. galloprovincialis emerge with well-developed thoracic muscles and energy reserves that enable them to sustain long flights over non-forested areas. These findings should aid managers and policy makers in devising sound procedures in areas where the risk of introducing PWD is high.

Identification of the first insulin-like peptide in the disease vector Rhodnius prolixus: Involvement in metabolic homeostasis of lipids and carbohydrates

Insulin-like peptides (ILPs) are functional analogs of insulin and have been identified in many insect species. The insulin cell signaling pathway is a conserved regulator of metabolism, and in insects, as well as in other animals, can modulate physiological functions associated with the metabolism of lipids and carbohydrates. In the present study, we have identified the first ILP from the Rhodnius prolixus genome (termed Rhopr-ILP) and investigated its involvement in energy metabolism of unfed and recently fed fifth instars. We have cloned the cDNA sequence and analyzed the expression profile of the transcript, which is predominantly present in neurosecretory cells in the brain, similar to other insect ILPs. Using RNAi, we have reduced the expression of this peptide transcript by 90% and subsequently measured the carbohydrate and lipid levels in the hemolymph, fat body and leg muscles. Reduced levels of Rhopr-ILP transcript induced increased carbohydrate and lipid levels in the hemolymph and increased lipid content in the fat body, in unfed insects and recently fed insects. Also their fat bodies displayed enlarged lipid droplets within the cells. On the other hand, the carbohydrate content in the fat body and in the leg muscles of unfed insects were decreased when compared to control insects. Our results indicate that Rhopr-ILP is a modulator of lipid and carbohydrate metabolism, probably through signaling the presence of available energy and nutrients in the hemolymph.

Spatio‐temporal analysis of the role of faecal depositions in aggregation behaviour of the triatomine Rhodnius prolixus

AbstractSeveral triatomine species aggregate over conspecific faeces. In some domestic species, this behaviour might help individuals to find their refuges because faeces are usually deposited around such places. In the present study, the defaecation behaviour of the triatomine bug Rhodnius prolixus Stål (Heteroptera: Reduviidae) is described in detail, including the temporal pattern of aggregation of nymphs with different nutritional status to faeces of different post‐deposition times. First, using an experimental arena, the spatial distribution of faecal deposition spots released by nymphs, in relation to the position of a refuge, is recorded. The results show that a high density of faeces is deposited inside the refuge, and few and distant spots, in relation to the position of the refuge, are dropped outside. Second, whether the aggregation behaviour of nymphs is altered by the time elapsed from deposition of the faeces, and by the nutritional status of the insects, is determined. The post‐deposition time of the faeces strongly modulates the aggregation response of the insects. Differences in aggregation between fed and unfed insects are only marginally significant; however, different temporal windows of aggregation according to the post‐deposition times of the faeces are demonstrated between bugs of different nutritional status. The results are discussed with respect to the relevance of the aggregation signal in relation to the characteristics of the natural refuges used by R. prolixus.

Temperature-Dependent Survival of Adult Lygus hesperus (Hemiptera: Miridae).

The western tarnished plant bug, Lygus hesperus Knight (Hemiptera: Miridae), is a key pest of many horticultural and agronomic crops in the western United States. Despite its well documented pest status, many aspects of the basic biology, including overwintering ecology, of L. hesperus are poorly understood. We examined the influence of eight constant temperatures from 10 to 35°C on survival of nondiapausing adult L. hesperus held with or without food, and the consequences of exposure to an extended period at 10°C on subsequent reproduction. Survival analyses indicated that, on average, fed insects tended to live longer than unfed insects, females lived longer than males, and the survival time decreased with increasing temperature. Nonlinear regressions indicated that median survival for insects grouped by gender and feeding status declined exponentially with increasing temperature. Survival functions for combinations of insect class (gender and feeding status) and temperature were adequately described by the respective two-parameter logistic functions. When adults were held for 9 d at 27°C with food after a 33-d period at 10°C either with or without food, no deleterious effects of prior starvation on propensity to mate or fecundity were demonstrated. These findings indicate that when temperatures are low, nondiapausing L. hesperus adults are capable of extended host-free survival with little or no impact on subsequent reproduction. Our findings suggest the current understanding of L. hesperus overwintering dynamics is incomplete. In addition, our results provide quantitative baseline information to facilitate more comprehensive investigation of the ecology of L. hesperus overwintering.

Rhodnius respiration depends on multiple factors

Sucking great lungfuls of air into the body is the main response of most mammals to physical exertion. As their metabolic rates rise, they inhale more deeply to maintain power. However, for insects, breathing is more complex. Equipped with a network of fine ventilation tubes that permeate every tissue in the body, insects have to tightly regulate the passage of oxygen into the body as their metabolic demands increase, by altering their breathing patterns. Timothy Bradley from the University of California, Irvine, USA, notes that respiratory patterns were thought to be principally determined by metabolic rate. But bugs don't just ramp up their metabolism in response to exercise; their metabolic rates increase as temperatures rise and during reproduction and digestion, so it wasn't clear whether other activities might result in different respiratory patterns. ‘There was some evidence that the respiratory control mechanism might be temperature sensitive’, says graduate student Erica Heinrich. She and Bradley began investigating the effects of feeding and temperature on the blood-sucking insect Rhodnius prolixus (p. 2752).‘Rhodnius prolixus provides certain valuable features as a model insect for studying respiratory control’, says Heinrich, who explains that the insect's metabolic rate rockets after dining on blood. This allowed her to measure the metabolic rates and respiratory patterns of the insects as they exhaled CO2 after a satisfying rabbit blood meal, in addition to monitoring the effects of temperature on the unfed insects as she varied the temperature between 18 and 38°C.Sure enough, as the temperature rose, the insects' metabolic rate increased 3.5 times above their resting metabolic rate and, as their metabolic demands increased, they initially opened and closed their spiracle valves (breathed discontinuously) until the metabolic rate became high enough and the spiracles remained open continuously.Next, the duo measured the metabolic rates of the well-fed insects and they were amazed to see the bugs' metabolic rates hurtle to almost 14 times their resting metabolic rates. However, when they analysed the bugs' CO2 exhalation pattern, they found, surprisingly, that instead of holding the spiracles open and exhaling CO2 continuously, they continued closing the spiracles intermittently, even though the insects' metabolic rate was four times higher than that at the hottest temperature.The duo also monitored the amount of CO2 released by the insects during each exhalation burst and found that they exhaled less CO2 per breath as the temperature rose. This was in contrast to the digesting insects, which exhaled more CO2 each time they opened their spiracles as their metabolic rates rose.Having shown that the insects' respiratory pattern was different under the two situations, Bradley says, ‘It is overly simplistic to attribute respiratory pattern to metabolic rate alone’, and the duo suspects that the insects' respiratory system is temperature sensitive. Explaining that if the respiration system was not sensitive to temperature, insects should always exhale the same amount of CO2 each time that open their spiracles, Heinrich says, ‘However, we found that as the temperature increases, the volume of CO2 released in a burst decreases’. She and Bradley suspect that this temperature sensitivity may result from changes in the pH of water in the insect's body. ‘Increased temperature decreases the pH of water’, explains Heinrich. She adds, ‘If the CO2 threshold that triggers spiracle opening is sensed via pH, then exposure to high temperatures will trigger premature spiracle opening… and less CO2 would need to accumulate via metabolism to reach the pH threshold. This will result in reduced volumes of CO2 release during spiracle opening’.

Expression analysis and molecular characterization of aquaporins in Rhodnius prolixus

Aquaporins (AQPs) are water channels responsible for transport of water and, in some cases, transport of small solutes such as urea and glycerol across lipid bilayer membranes. Hematophagous insects, such as Rhodnius prolixus, ingest large volumes of fluid and must rapidly eliminate the excess of water and salts from the blood meal within the gut. In order to deal with this increase in body fluid volume, a hormone-controlled diuresis is activated, during which a high rate of water and salt absorption occurs across the anterior midgut, followed by secretion of water and salts by the Malpighian tubules (MTs). Previously, one member of the MIP family (major intrinsic protein that includes the AQP family) was identified in the MTs of R. prolixus, and named RpMIP. We have described here that the RpMIP gene has different variants, and is present in tissues other than MTs. In addition, we have characterized a new AQP (RhoprAQP1) found in different tissues of R. prolixus. The expression of these transcripts in unfed insects as well as blood fed insects was evaluated using real-time quantitative PCR. Molecular models of the predicted proteins were constructed and the characteristics of their pores evaluated. A yeast complementation assay was used to validate that the products of these transcripts were bona fide AQPs. Both RhoprAQP1 and RhoprMIP-A were capable of transporting water whereas RhoprMIP-A was also capable of transporting H2O2. Taken together, these analyses suggest that RhoprMIP is probably an aquaglyceroporin, while RhoprAQP1 appears to be a strict aquaporin that transports only water.

Adult Survival ofDelphastus catalinae(Coleoptera: Coccinellidae), a Predator of Whiteflies (Hemiptera: Aleyrodidae), on Diets of Whiteflies, Honeydew, and Honey

Delphastus catalinae (Horn) is a coccinellid predator that is commercially sold for the management of whiteflies. A study was conducted to assay the effect of selected diets on the survival of adult D. catalinae. Treatments of water (as a control), 10% honey, honeydew, and whiteflies [Bemisia tabaci (Gennadius)] were provided to the beetles in laboratory assays. Newly emerged, unfed adult insects were used at the start of a survival experiment with trials lasting 50 d. Another survival experiment used mixed-aged adults from a greenhouse colony, and the trials lasted 21 d. Survival was poor on a diet of solely water; ~1% survived beyond a week at 26°C. Survival using the newly emerged insects was similar between those fed honeydew and honey diets, but those on the whitefly diet had the greatest survival (~60% on day 50). However, in the experiment with mixed-aged beetles, adults on honey, and whitefly diets performed the same over a 21-d experiment. Excluding those on the water diet, survival of beetles on the various diets ranged from ~50-80% after 21 d. In an open choice assay across 7 h, D. catalinae adults were found on the whitefly diet in a much greater incidence than on the other diets, and the number of beetles found on the whitefly diet increased over time. The data supports that when D. catalinae are employed in greenhouses or fields for whitefly management, during low prey populations, honeydew from the whitefly can help sustain the population of this predator. Moreover, a supplemental food such as a honey solution can help sustain the population of D. catalinae when the prey is decreased to low numbers. These results may help in the development of strategies to enhance the utility of predators for the management of whiteflies.

Detection of Insect Immunity Substances (Lectins) in the Midgut Extracts from Larvae and Adult Red Palm Weevil Rhynchophorus ferrugineus (Olivier) in Al-Ahsa, Saudi Arabia

The hemagglutination activities of midgut (MG) fractions of red palm weevil, Rhynchophorus ferrugineus against mammalian erythrocytes (RBC) from man (ABO), rabbit, horse and sheep were studied. The highest titers were seen with rabbit RBC (p < 0.05) followed by human group B, human group O, horse, human group A, human group AB and sheep, respectively. Insect age and nutritional status significantly influenced the agglutinating activities. Some of the unfed insects were having low activities (p > 0.01) than the well fed insects (p < 0.05). The agglutination activities were linearly correlated with the insect age. The agglutination activities were higher in old insects than the young insects (p < 0.05). Further studies are needed to investigate the lectins in the hemolymph of the red palm weevil, Rhynchophorus ferrugineus beside demonstrating their agglutination activities against the natural entomopathogenes organisms of this economically important insect for use in its biological control.

Conditions for Short-Term Storage of Field-Collected Spined Soldier Bug, Podisus maculiventris (Say) (Heteroptera: Pentatomidae), Adults Prior to Augmentative Release1

The date at which spined soldier bugs, Podisus maculiventris (Say) (Heteroptera: Pentatomidae), can be trapped for augmentative release against overwintered Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), may be up to 5 wks earlier than the date of peak Colorado potato beetle emergence, necessitating storage of field-collected spined soldier bugs prior to release. The effects of temperature, photoperiod, and food on spined soldier bug survivorship and fecundity during and after a 5-wk storage period were investigated. In general, unfed spined soldier bugs had greater mortality and reduced fecundity compared with those fed string beans and mealworm larvae during storage. Temperature and photoperiod generally had no effect. However, female spined soldier bug survivorship was greater among unfed insects stored at 5°C compared with those stored at 15°C. Therefore, household refrigerators can be used for short-term storage of field-collected spined soldier bugs. Even when stored spined soldier bug females are fed, fecundity is low (14 nymphs per female originally collected). This may limit the use of field-collected spined soldier bugs for augmentative release against Colorado potato beetle to relatively small plantings of potatoes.

Presence of chitinase and beta-N-acetylglucosaminidase in the Aedes aegypti: a chitinolytic system involving peritrophic matrix formation and degradation

Measurement of the hydrolysis of specific fluorogenic substrates by spectrophotometry as well as the substrate activity–SDS–PAGE gel analysis of the chitinolytic activity in Aedes aegypti guts showed that both chitinase and β-N-acetylglucosaminidase are present and physiologically active. Both enzymes were present even in guts from unfed insects, but the activities increased rapidly after feeding on blood or an artificial protein-free diet. Chitinase activity was predominantly of the ‘endo’-type, reaching its maximum activity at 36 h and then declining to very low levels after the degradation of the peritrophic matrix (PM). Chitinase assay in gels after SDS–PAGE was a very sensitive method that allowed us to detect two chitinases with distinct molecular weights in the mosquito gut. Hydrolysis of a chitinase-specific substrate by chitinolytic activities in the mosquito guts was inhibited by allosamidin, a potent chitinase inhibitor. Allosamidin treatment led to the formation of an atypical thick PM, while the addition of exogenous chitinase completely blocked its formation. This chitinolytic system appears to operate both on the formation and degradation of the PM. Since the PM is involved in pathogen invasion, these results are important in facilitating a search for mechanisms that can block pathogen development in the mosquito vector.

Properties of a blood-meal-induced midgut lectin from the tsetse fly Glossina morsitans.

The properties of a blood-meal-induced lectin (agglutinin) from the midgut of Glossina morsitans capable of agglutinating Trypanosoma brucei were studied in vitro. The midgut homogenate from flies that had been fed twice had the highest agglutination activity, followed by that from the once-fed flies and that from the unfed insects. As compared with the bloodstream-form trypanosomes, a much lower concentration of the midgut homogenate was required for agglutination of the procyclic parasites. Furthermore, the agglutination process was specifically inhibited by D-glucosamine. Soybean trypsin inhibitor abrogated agglutination of the bloodstream-form parasites, whereas the procyclics were unaffected. The agglutination process was temperature-sensitive, with little activity being evident between 4 degrees and 15 degrees C. Similarly, heating the midguts to 60 degrees-100 degrees C led to loss of activity. When the midgut homogenate was separated by anion-exchange chromatography, the agglutination activity co-eluted with trypsin activity at approximately 50% NaCl. These results suggest a very close relationship between midgut trypsin-like enzyme and the agglutinin. Since successful agglutination of bloodstream-form trypanosomes requires protease activity, it may be that the enzyme cleaves off some surface molecules on the parasite surface, thus exposing the lectin-binding sites.

Comparative studies on the growth and reproductive performances of Rhodnius prolixus reared on different blood sources

Host blood source was found to affect both the development and the reproductive performance of Rhodnius prolixus. The insects were reared on citrated human, rabbit, chicken, sheep and horse blood sources, through a membrane feeder, during an entire life cycle, from eggs to adults. Development and reproduction in terms of the number of unfed insects, number of moulting, mortality intermoulting period, number of egg/female, conversion of blood into egg (mg meal/egg) and percentage of hatch as effective physiological parameters were investigated. Our results showed that human or rabbit blood meals were more nutritionally efficient than the other blood samples used because (i) the insects developed faster, presented low mortality and about 80% of them reached the adult stage; and (ii) females oviposited an average of at least 100% more eggs. The inefficiency of chicken and horse blood sources as diets for R. prolixus was manifested in (i) a decrease of the amount of ingested blood and (ii) only a reasonable nutritional quality. The inadequacy of sheep blood was observed by a mortality extremely high, poor moulting response and drastic reduction in egg production.

Changes in haemolymph serotonin levels associated with feeding in the blood-sucking bug, Rhodnius prolixus

The concentration of serotonin in the haemolymph of fifth-instar larvae of Rhodnius prolixus increase quickly after the onset of feeding. In insects fed on either blood or artificial diet the haemolymph serotonin concentration increases from approx. 7 nM in unfed insects to over 100 nM by 5 min after the onset of feeding whereafter it decreases again to low levels at 20 min. If insects are interrupted after 1 or 2 min of feeding the haemolymph serotonin concentration declines rather than following the predictable pattern of increase achieved when feeding is allowed to continue. One source of serotonin appears to be neurohaemal areas lying upon the abdominal nerves, since the intensity of serotonin-like immunoreactivity is greatly reduced in these areas following 15 min of feeding. We conclude that serotonin is a neurohormone in Rhodnius and that feeding is the natural stimulus responsible for its release and subsequent elevated concentration in the haemolymph.

Serotonergic supply to the epidermis of Rhodnius prolixus: Evidence for serotonin as the plasticising factor

Immunohistochemistry and high-performance liquid chromatography coupled with electrochemical detection has been used to demonstrate the presence of serotonin within the nerve supply to the epidermis of 5th-instar and adult Rhodnius prolixus. Serotonin is released from dorsal integument in vivo in response to feeding, and in vitro in response to high-potassium saline. Thus, the intensity of serotonin-like immunoreactive staining of the processes on the dorsal integument is much greater in unfed insects than in those examined 15 min after the onset of feeding. In addition the release of serotonin from dorsal integument into the perfusion medium is elevated in high-potassium saline in the presence of calcium. Serotonin-like immunoreactive processes are also found associated with salivary glands and digestive tract, as well as with skeletal muscle. The results support the notion that serotonin plays a key role in the integration of feeding activity and is probably the plasticising factor in Rhodnius.