Evaluating the effects of anticoagulants on Rhodnius prolixus artificial blood feeding

Abstract

Blood-sucking insects are responsible for the transmission of several important disease-causing organisms such as viruses, bacteria, and protozoans. The hematophagous hemipteran Rhodnius prolixus is one of the most important vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. Due to the medical importance of this insect, it has been used as a study model in physiology and biochemistry since the 1930s. Artificial feeding has been recognized as a feasible and a more ethical alternative method of feeding these hematophagous insects. To prevent clotting after blood collection defibrination or treatment with anticoagulants are necessary. Although anticoagulants have been routinely used for stabilizing the collected blood, there is a gap in demonstration of the effects of using anticoagulants on the feeding and development of the hematophagous insect Rhodnius prolixus. In this study, we compared the survival rate, molting efficiency, fertility, and infection development between insects that were fed on blood containing three different anticoagulants (citrate, EDTA, and heparin). We observed that fifth instar nymphs that were fed on blood containing EDTA and citrate could not perform digestion properly, which resulted in molting inefficiency. Adult insects that were fed on EDTA-containing blood laid lower number of eggs, and also had a diminished egg hatch percentage. When we delivered T. cruzi parasites in blood containing citrate or EDTA to the insects, a lower number of parasites and metacyclic trypomastigotes was observed in the intestine compared to the group fed on heparin-containing blood. Since heparin could potentially inhibit DNA polymerase activity in DNA samples extracted from the intestine, we analyzed different heparin concentrations to determine which one is the best for use as an anticoagulant. Concentrations ranging between 2.5 and 5 U/mL were able to inhibit coagulation without severely impairing DNA polymerase activity, thus indicating that this should be considered as the range of use for feeding experiments. Our results suggest that among the three anticoagulants tested, heparin can be recommended as the anticoagulant of choice for R. prolixus feeding experiments.

Digestive α-L-fucosidase activity in Rhodnius prolixus after blood feeding: effect of secretagogue and nutritional stimuli

“…Citrated blood was used by different groups in several contexts, especially for studies of T. cruzi infection ( Silva-Cardoso et al, 2018 ). However, citrate can alter the course of digestion or development, due to the presence of digestive enzymes or other proteins that depend on calcium ions for their activity.…”

“…However, citrate can alter the course of digestion or development, due to the presence of digestive enzymes or other proteins that depend on calcium ions for their activity. A recent report showed an important negative impact of citrate on R. prolixus physiology, with impairment of oviposition and egg hatching ( Silva-Cardoso et al, 2018 ). Our results showed a strong reduction of midgut α-L-fucosidase after feeding with citrated blood, but it is not clear if this is caused by direct inhibition of the enzyme or by indirect physiological effects resulting in lower levels of α-L-fucosidase.…”

“…In our experiments, heparinated blood resulted in the highest α-L-fucosidase activities. Furthermore, heparin has been recommended as the anticoagulant of choice for R. prolixus feeding experiments, due to minimal physiological impacts and no interference in biochemical and molecular biology assays ( Silva-Cardoso et al, 2018 ). However, despite differences between the general digestive patterns of R. prolixus fed in these different blood treatments are not expected, this issue was never investigated in detail.…”

Deficiency of Acetyl-CoA Carboxylase Impairs Digestion, Lipid Synthesis, and Reproduction in the Kissing Bug Rhodnius prolixus

“…Alternatively, females were fed on blood supplemented with heparin (5 U/ml; Hepamax-S, Blau Farmacêutica, São Paulo, Brazil) (Silva-Cardoso et al, 2018) in an artificial feeder (Garcia et al, 1975), and their body mass was measured at days 1 and 10 after feeding to determine weight loss resulting from blood digestion.…”

Development of insecticide-impregnated polyester/cotton blend fabric and assessment of their repellent characteristics against Cimex lectularius and dengue vectors Aedes albopictus and Aedes aegypti

“…These ectoparasites have a long history of having a profound detrimental effect on human civilization. They are known to spread viruses, bacteria, and protozoans, three essential disease-causing organisms [ 19 , 20 ]. All racial and socio-economic categories are affected by their infestations.…”