Behavior of triatomines (Hemiptera: Reduviidae) vectors of Chagas' disease. I. Courtship and copulation of Panstrongylus megistus (Burm., 1835) in the laboratory

A study of the courtship and copulation behavior of Panstrongylus megistus was carried out in the laboratory. Fifty-five newly-fed virgin couples were used. Experiments were performed during the day (9:00 to 12:00 a.m.) and at night (7:00 to 10:00 p.m.). Behavior was recorded by direct observation and was found to consist of the following sequence of behavioral patterns: the male approached the female and jumped on her or mounted her; he took on a dorsolateral position and immobilized the female dorsally and ventrally with his three pairs of legs; the male genital was placed below those of the female; the paramers of the male immobilized the female's genitals; copulation started. The couple joined by the iniciative of the male. The female could be receptive and accept copulation, or nonreceptive and reject the male. Copulation occurred more often on the occasion of the first attempt by the male. Duration of copulation was X = 29.3 +/- 9.3 min (CV = 83%). No behavioral differences were observed between couples tested during the day or at night.     

Behavior of triatomines (Hemiptera: Reduviidae) vectors of Chagas' disease. III. Influence of the number of matings on the fecundity and fertility of Panstrongylus megistus (Burm., 1835) in the laboratory

A study of the effect of mating in the fecundity and fertility of females of P. megistus fed on pigeon blood every 14 days, was carried out in the laboratory. Two groups were constituted: I - females which mated only once; II - females which stayed always with the males. Only 56.7% of group I females laid fertile eggs, while as much as 90% of group II females laid fertile eggs. The duration of the fertile oviposition was greater in the females which stayed always with the males. Some females of this group were able to mate up to seven times throughout their life-span. This fact render useless sterile males in the control of these insects.     

Behavior of triatomines (Hemiptera:Reduviidae) vectors of Chagas' disease. IV. Fecundity, fertility and longevity of Panstrongylus megistus (Burm., 1835) pairs and virgin females starved under laboratory conditions

A laboratory study was conducted on the fecundity, fertility and life span of Panstrongylus megistus pairs and on the fecundity and life span of P. megistus virgin females submitted to starvation after the last moulting. Of the mated females, 22.2% laid eggs, 4.4% of which were fertile. Females resisted starvation more than males. Of the starved virgin females, only 10% laid eggs, with a low egg-laying rate (0.47) per female. Resistance to starvation was lower in virgin than in mated females.     

The hemocytes of Panstrongylus megistus (Hemiptera: Reduviidae)

Five hemocyte types were identified in the hemolymph of Panstrongylus megistus by phase contrast and common light microscopy using some histochemical methods. These are: Prohemocytes, small cells presenting a great nucleus/cytoplasm ratio; Plasmatocytes, the most numerous hemocytes, are polymorphic cells mainly characterized by a large amount of lysosomes; Granulocytes, hemocytes very similar to plasmatocytes which contain cytoplasmic granules and are especially rich in polysaccharides; Oenocytoids, cells presenting a small nucleus and a thick cytoplasm; they show many small round vacuoles when observed in Giemsa smears and many cytoplasmic granules under phase microscopy; Adipohemocytes, very large hemocytes, presenting many fat droplet inclusions which could correspond to free fat bodies which entered the hemolymph. Only prohemocytes and plasmatocytes can be clearly classified; all the other hemocyte types have a more ambiguous classification.     

Scanning electron microscopy of the dorsal vessel of Panstrongylus megistus (Burmeister, 1835) (Hemiptera: Reduviidae).

In this study we analyzed the microanatomy of the dorsal vessel of the triatomine Panstrongylus megistus. The organ is a tubule anatomically divided into an anterior aorta and a posterior heart, connected to the body wall through 8 pairs of alary muscles. The heart is divided in 3 chambers by means of 2 pairs of cardiac valves. A pair of ostia can be observed in the lateral wall of each chamber. A bundle of nerve fibers was found outside the organ, running dorsally along its major axis. A group of longitudinal muscular fibers was found in the ventral portion of the vessel. The vessel was found to be lined both internally and externally by pericardial cells covered by a thin laminar membrane. Inside the vessel the pericardial cells were disposed in layers and on the outside they formed clusters or rows.     

Ultrastructural studies of the hemocytes of Panstrongylus megistus (Hemiptera: Reduviidae)

Ultrastructural analyses revealed the presence of six hemocyte types in the hemolymph of Panstrongylus megistus, partially confirming our previous results obtained through light microscopy. Prohemocytes: small, round hemocytes with a thin cytoplasm layer, especially rich in free ribosomes and poor in membranous systems. Plasmatocytes: polymorphic cells, whose cytoplasm contains many lysosomes and a well developed rough endoplasmic reticulum (RER). They are extremely phagocytic. Sometimes, they show a large vacuolation. Granulocytes: granular hemocytes whose granules show different degrees of electrodensity. Most of them, have an internal structuration. Coagulocytes: oval or elongated hemocytes, which show pronounced perinuclear cisternae as normally observed in coagulocytes. The cytoplasm is usually electrodense, poor in membranous systems and contains many labile granules. Oenocytoids: large and very stable hemocytes, whose homogeneous cytoplasm is rich in loose ribosomes and poor in membranous systems. Adipohemocytes: large cells, containing several characteristic lipid droplets. The cytoplasm is also rich in glycogen, RER and large mitochondria. The total and differential hemocyte count (THC and DHC) were also calculated for this reduviid. THC increases from 2,900 hemocytes/mm3 of hemolymph in the 4th instar to 4,350 in the 5th and then, decreases to 1,950 in the adults. Plasmatocytes and coagulocytes are the predominant hemocyte types.     

Effect of sequential cold shocks on survival and molting incidence in Panstrongylus megistus (Burmeister) (Hemiptera, Reduviidae)

The survival and molting incidence were studied in the insect, Panstrongylus megistus, following sequential cold shocks in which a milder shock at 0 or 5 degrees C for 1 h preceded a more severe shock (0 degrees C, 12 h). The shocks were separated by intervals of 8, 18, 24, and 72 h at 28 degrees C. The survival rate after sequential shocks was identical to that of unshocked controls. Cold-shock tolerance differed from heat-shock tolerance since the latter varied with the time between shocks and was much more transient. Sequential cold shocks produced a higher molting incidence when the first shock was given at 0 compared to 5 degrees C. This response was more rapid than that to sequential heat shocks. Cold-shock tolerance in P. megistus may involve heat-shock proteins, although other protective mechanisms may also occur concurrently. This is the first report of cold-shock tolerance in a blood-sucking hemipteran.     

Flight metabolism in Panstrongylus megistus (Hemiptera: Reduviidae): the role of carbohydrates and lipids

The metabolism of lipids and carbohydrates related to flight activity in Panstrongylus megistus was investigated. Insects were subjected to different times of flight under laboratory conditions and changes in total lipids, lipophorin density and carbohydrates were followed in the hemolymph. Lipids and glycogen were also assayed in fat body and flight muscle. In resting insects, hemolymph lipids averaged 3.4 mg/ml and significantly increased after 45 min of flight (8.8 mg/ml, P < 0.001). High-density lipophorin was the sole lipoprotein observed in resting animals. A second fraction with lower density corresponding to low-density lipophorin appeared in insects subjected to flight. Particles from both fractions showed significant differences in diacylglycerol content and size. In resting insects, carbohydrate levels averaged 0.52 mg/ml. They sharply declined more than twofold after 15 min of flight, being undetectable in hemolymph of insects flown for 45 min. Lipid and glycogen from fat body and flight muscle decreased significantly after 45 min of flight. Taken together, the results indicate that P. megistus uses carbohydrates during the initiation of the flight after which, switching fuel for flight from carbohydrates to lipids.     

Metabolic pathways for dietary lipids in the midgut of hematophagous Panstrongylus megistus (Hemiptera: Reduviidae)

The metabolism of dietary lipids in the anterior midgut of Panstrongylus megistus during blood digestion was studied. Fifth instar nymphs were fed a blood meal containing 7.1 +/- 0.4 mg of lipids, consisting mainly of triacylglycerol (TAG), and completed the overall process of digestion in about 20 days. Lipolysis of TAG and pathways for diacylglycerol (DAG) biosynthesis in the midgut were investigated by feeding the insects with [9,10-3H]-oleic acid-labeled triolein. Lumenal [3H]-triacylglycerol was hydrolyzed, generating mainly fatty acids (FA) and glycerol and to lesser extent, DAG. Almost no radioactivity associated with monoacylglycerol was found at any time. In midgut tissue, labeled fatty acids were incorporated into phosphatidic acid, DAG and TAG, whereas no significantly labeled monoacylglycerol was observed. In addition, the activities of enzymes related to DAG metabolism were assayed in non-blood fed midgut homogenates and at different times after feeding on a blood meal. Significant changes in the activities of phosphatidate phosphohydrolase (EC 3.1.3.4) and triacylglycerol lipase (EC 3.1.1.3) were observed during blood digestion, suggesting that these enzymes are important in regulating intracellular DAG synthesis and mobilization in midgut cells. Finally, the histological changes of lipid stores observed in anterior midgut confirmed the active process of uptake and trafficking of lipids performed by the enterocytes during blood digestion.     

Biochemical and cellular characterization of lipophorin–midgut interaction in the hematophagous Panstrongylus megistus (Hemiptera: Reduviidae)

In order to better understand the metabolism of dietary lipids in hematophagous insects, we have performed a biochemical and cellular characterization of lipophorin (Lp)–midgut interaction in Panstrongylus megistus, a vector of Chagas' disease. The study was accomplished by solid-phase binding assays or with iodinated Lp (¹²⁵I-Lp), using midgut membranes from fifth instar nymphs after ecdysis and after insects received a blood meal. Results obtained from both physiological conditions indicated that Lp interacted specifically with the midgut, implying the participation of receptors. Binding capacity of lipophorin to membranes was dependent on the amount of membranes added in the system, reaching saturation at 0.1 μg/ml. However, membranes obtained after a blood meal exhibited higher binding activity. Saturation kinetics results using ¹²⁵I-Lp suggested a single binding site with high affinity for Lp in the midgut membranes (K_d = 5.1 ± 3.6 × 10^?8 M). The unrelated lipoprotein, human LDL, did not compete with Lp for its binding site in the midgut. The binding was dependent on pH and the treatment of membranes with trypsin or heat causes a significant inhibition of the binding. Midgut–Lp interaction was affected by changes in ionic strength and by suramin, but showed no requirement of calcium. Ligand blotting assays revealed two membrane proteins that specifically bound Lp (61 and 45 kDa). At cellular level, Lp binding sites were located mainly at the basal plasma membrane of isolated enterocytes. Labeled Lp with fluorescent probes directed to its proteins or its phospholipids fraction co-localized mainly at the basement membrane of the midgut. In addition, no intracellular Lp was observed at any condition. The lack of an endocytic pathway for Lp in the midgut of P. megistus is analyzed in the context of insect physiology.     

Effect of sequential heat and cold shocks on nuclear phenotypes of the blood-sucking insect,Panstrongylus megistus (Burmeister) (Hemiptera,Reduviidae)

Thermal shocks induce changes in the nuclear phenotypes that correspond to survival (heterochromatin decondensation, nuclear fusion) or death (apoptosis, necrosis) responses in the Malpighian tubules of Panstrongylus megistus. Since thermal tolerance increased survival and molting rate in this species following sequential shocks, we investigated whether changes in nuclear phenotypes accompanied the insect survival response to sequential thermal shocks. Fifth instar nymphs were subjected to a single heat (35 or 40 degrees C, 1 h) or cold (5 or 0 degrees C, 1 h) shock and then subjected to a second shock for 12 h at 40 or 0 degrees C, respectively, after 8, 18, 24 and 72 h at 28 degrees C (control temperature). As with specimen survival, sequential heat and cold shocks induced changes in frequency of the mentioned nuclear phenotypes although their patterns differed. The heat shock tolerance involved decrease in apoptosis simultaneous to increase in cell survival responses. Sequential cold shocks did not involve cell/nuclear fusion and even elicited increase in necrosis with advancing time after shocks. The temperatures of 40 and 0 degrees C were more effective than the temperatures of 35 and 5 degrees C in eliciting the heat and cold shock tolerances, respectively, as shown by cytological analysis of the nuclear phenotypes. It is concluded that different sequential thermal shocks can trigger different mechanisms of cellular protection against stress in P. megistus, favoring the insect to adapt to various ecotopes.     

Effect of fasting on the composition of the fat body lipid of Dipetalogaster maximus, Triatoma infestans and Panstrongylus megistus (Hemiptera: Reduviidae)

Modifications in content and lipid composition induced by fasting were examined in fat bodies from adults of Triatominae, Dipetalogaster maximus, Triatoma infestans and Panstrongylus megistus. With fasting, total lipid stores dropped approximately 50% for T. infestans and more than 70% for P. megistus. Total lipids analyzed by thin layer chromatography and fractionated by column chromatography on Unisil showed triacylglycerols as the main component in the three species, although P. megistus showed high levels of diacylglycerols (31–46%). Cholesterol amounted to 8–15%. In diacylglycerol fractions, C_16:0, C_18:1 and C_18:0 fatty acids were detected; their ratio varied with species but it was not dependent on nutritional status. In triacylglycerol fractions C_18:1 fatty acid was the major component at different times (48–68%); the ratio of monounsaturated to saturated in this fraction was 1.3, 2.6 and 1.2 for D. maximus, T. infestans and P. megistus respectively. The remarkable drop in lipid stores without noticeable changes in their relative composition would suggest that all types of lipid are used at similar rates. The higher content of diacylglycerols in P. megistus may be associated with the better flight performance of this species. Accepted: 4 August 1998     

Morphometric study of the genus Panstrongylus Berg, 1879 (Hemiptera, Reduviidae, Triatominae)

The subfamily Triatominae is actually represented by 137 species distributed among 6 tribes and 19 genera. Within this subfamily, the genus Panstrongylus, Berg 1879, is composed by 13 species widespread in sylvatic, peridomestic, and domestic habitats of Neotropical regions. These species are vectors of Chagas disease and consequently are found associated with its main hosts, such as birds and mammals. Interest in species of this genus has been increasing in the last few years. Reports of several authors indicate these Triatominae to invade and colonize houses, increasing their epidemiological significance. Morphometry was used in this study to investigate correlations among possible closely related species. We measured 224 specimens among 13 species through a set of metric variables of the head. The results indicated that the genus Panstrongylus seems to be homogeneous since 10 of the 14 species were shown to be closely related.     

Presence of Panstrongylus rufotuberculatus (Champion, 1899) (Hemiptera: Reduviidae: Triatominae) in Argentina.

Panstrongylus rufotuberculatus is reported for the first time in Argentina. Four adults were attracted by the light of human dwellings in Calilegua National Park, Jujuy, at 1150 masl within the subtropical humid forest. The individuals were similar to those described in the literature. The epidemiological significance of this species is very low in the area, although deforestation might increase its role in the transmission of Trypanosoma cruzi.     

The storage of nutritional resources during vitellogenesis of Panstrongylus megistus (Hemiptera: Reduviidae): the pathways of lipophorin in lipid delivery to developing oocytes

In this work, we have analyzed the pathways by which lipophorin (Lp) delivers its lipid cargo to developing oocytes of Panstrongylus megistus, a hematophagous vector of Chagas’ disease. Lp, vitellin, total lipids and proteins were measured in ovarian tissues at different stages of the reproductive cycle. Localization of Lp in developing oocytes, mainly at their cortical area, was demonstrated by immunofluorescence assays using an anti-Lp antibody labeled with FITC. In vivo approaches injecting fluorescently labeled Lp to follow the course of the entire particle (Lp-DiI or Lp-Oregon Green) or its lipid cargo (Lp-Bodipy-FA) were monitored by laser scanning confocal microscopy. Significant increases in the amounts of lipids, proteins and vitellin were observed in ovarian tissue with the progress of vitellogenesis. Unexpectedly, an increase in the amount of Lp was also observed. The experiments in vivo demonstrated that the uptake of fluorescent Lp labeled on its protein or lipid moiety by developing oocytes occurred very fast, being impaired at low temperatures. The co-injection of fluorescent Lp and vitellogenin (Vg) showed that both particles co-localized inside yolk bodies, confirming the endocytic pathway for Lp. When the fate of lipids transferred to oocytes was evaluated in vitellogenic females by co-injecting Lp-Bodipy-FA and Lp-DiI, the signal for Bodipy-FA was found in both lipid droplets and yolk bodies. In contrast, in injected females kept at 4 °C the fluorescence was reduced, being observed exclusively in lipid droplets, implying that lipid transfer to the oocyte was diminished but not abolished. Taken together, the results demonstrate that in the hematophagous P. megistus, the storage of lipid resources by developing oocytes occurs by the convergence of different pathways by which Lp maximizes the delivery of its lipid cargo. In addition, it was also shown that, to some extent, lipids stored in the oocyte lipid droplets can also originate from endocytosed Vg. The relevance of these events in the context of the physiology of reproduction in P. megistus is discussed.