Jutta Linss

Structure and expression of two Trypanosoma cruzi genes encoding antigenic proteins bearing repetitive epitopes

Trypanosoma cruzi genes were cloned in lambda gt11 and screened with an anti-trypomastigote antiserum. Two out of twelve clones were selected in view of their reactivity with human chagasic sera. One clone encodes a flagellar antigen (FRA) of more than 300 kDa, whereas the other corresponds to a roughly 225-kDa cytoplasmic antigen (CRA). The flagellar antigen is present in both epimastigotes and trypomastigotes, but the cytoplasmic antigen is not found in trypomastigotes. The CRA clone is entirely composed of at least 23 copies of a 42-bp repeat and the FRA gene contains at least 14 copies of a 204-bp motif. The FRA gene hybridizes to a RNA of about 10 kb, while the CRA gene detects a transcript of 5.2 kb.

Assessing the Effects of Aedes aegypti kdr Mutations on Pyrethroid Resistance and Its Fitness Cost

Pyrethroids are the most used insecticide class worldwide. They target the voltage gated sodium channel (NaV), inducing the knockdown effect. In Aedes aegypti, the main dengue vector, the AaNaV substitutions Val1016Ile and Phe1534Cys are the most important knockdown resistance (kdr) mutations. We evaluated the fitness cost of these kdr mutations related to distinct aspects of development and reproduction, in the absence of any other major resistance mechanism. To accomplish this, we initially set up 68 crosses with mosquitoes from a natural population. Allele-specific PCR revealed that one couple, the one originating the CIT-32 strain, had both parents homozygous for both kdr mutations. However, this pyrethroid resistant strain also presented high levels of detoxifying enzymes, which synergistically account for resistance, as revealed by biological and biochemical assays. Therefore, we carried out backcrosses between CIT-32 and Rockefeller (an insecticide susceptible strain) for eight generations in order to bring the kdr mutation into a susceptible genetic background. This new strain, named Rock-kdr, was highly resistant to pyrethroid and presented reduced alteration of detoxifying activity. Fitness of the Rock-kdr was then evaluated in comparison with Rockefeller. In this strain, larval development took longer, adults had an increased locomotor activity, fewer females laid eggs, and produced a lower number of eggs. Under an inter-strain competition scenario, the Rock-kdr larvae developed even slower. Moreover, when Rockefeller and Rock-kdr were reared together in population cage experiments during 15 generations in absence of insecticide, the mutant allele decreased in frequency. These results strongly suggest that the Ae. aegypti kdr mutations have a high fitness cost. Therefore, enhanced surveillance for resistance should be priority in localities where the kdr mutation is found before new adaptive alleles can be selected for diminishing the kdr deleterious effects.

Physiological and Morphological Aspects of Aedes aegypti Developing Larvae: Effects of the Chitin Synthesis Inhibitor Novaluron

Population control of the dengue vector mosquito, Aedes aegypti, is difficult due to many reasons, one being the development of resistance to neurotoxic insecticides employed. The biosynthesis of chitin, a major constituent of insect cuticle, is a novel target for population control. Novaluron is a benzoylphenylurea (BPU) that acts as a chitin synthesis inhibitor, already used against mosquitoes. However, information regarding BPU effects on immature mosquito stages and physiological parameters related with mosquito larval development are scarce. A set of physiological parameters were recorded in control developing larvae and novaluron was administered continuously to Ae. aegypti larvae, since early third instar. Larval instar period duration was recorded from third instar until pupation. Chitin content was measured during third and fourth instars. Fourth instars were processed histochemically at the mesothorax region, stained with hematoxylin and eosin (HE) for assessment of internal tissues, and labeled with WGA-FITC to reveal chitinized structures. In control larvae: i) there is a chitin content increase during both third and fourth instars where late third instars contain more chitin than early fourth instars; ii) thoracic organs and a continuous cuticle, closely associated with the underlying epidermis were observed; iii) chitin was continuously present throughout integument cuticle. Novaluron treatment inhibited adult emergence, induced immature mortality, altered adult sex ratio and caused delay in larval development. Moreover, novaluron: i) significantly affected chitin content during larval development; ii) induced a discontinuous and altered cuticle in some regions while epidermis was often thinner or missing; iii) rendered chitin cuticle presence discontinuous and less evident. In both control and novaluron larvae, chitin was present in the peritrophic matrix. This study showed quantitatively and qualitatively evidences of novaluron effects on Ae. aegypti larval development. To our knowledge, this is the first report describing histological alterations produced by a BPU in immature vector mosquitoes.

cDNA cloning and expression of Rhodnius prolixus vitellogenin

It was shown that Rhodnius prolixus vitellogenin (Vg) is synthesized as precursors of 205 and 190 kDa. Each Vg subunit is antigenically related to a domain in the precursor molecules. Since Vg has been previously detected in R. prolixus male adults, protein synthesis by fat bodies from 5th instar male nymphs was investigated and no Vg synthesis could be detected. Also, a 6.1 Kb RNA is present in female adults but not in 5th instar male nymphs. Therefore, cDNAs from female adult and 5th instar male fat bodies were used for differential screening of a female fat body cDNA library leading to the isolation of several female specific clones. All the clones hybridizing to the female specific 6.1 Kb RNA species were identical. We also describe the construction of new expression vectors, pGex-A and pGex-B, derived from the previously described plasmid pGex-1N. The new vectors, together with pGex-3X, comprise a set of expression plasmids with cloning sites in all three possible reading frames that give a fusion polypeptide with the glutathione S-transferase. This carrier protein can be cleaved by digestion with factor Xa in all three plasmids; one of the Vg cDNA clones was subcloned in pGex-A. Antibodies affinity purified from the fusion protein Vg/glutathione S-transferase recognized both large Vg subunits, suggesting an antigenic relationship between them. Furthermore, the small Vg subunits were not recognized, indicating that they may be localized at the N-terminal region of Vg precursors.

Evidence for gene duplication in the voltage-gated sodium channel gene of Aedes aegypti

Herein, we show the first evidence of a duplication of the NaV gene of the mosquito Aedes aegypti, that might be involved in insecticide resistance. The duplicated haplotype is composed of one sequence with and another without a specific mutation, present in natural populations as a polymorphic trait.

Expression and polymorphism of a Trypanosoma cruzi gene encoding a cytoplasmic repetitive antigen.

The study of the expression of a Trypanosoma cruzi gene encoding a cytoplasmic repetitive antigen (CRA) during the metacyclogenesis process shows that this gene is not expressed in metacyclic trypomastigote forms of the parasite. However, a slight increase in CRA expression was observed following the nutritional stress of epimastigotes which precedes T. cruzi metacyclogenesis in vitro. The comparison of the expression of CRA in different T. cruzi strains shows that this gene is highly polymorphic: some strains display one and others display two polypeptides reacting with a CRA antiserum. The comparison of T. cruzi G-49 strain and Dm 28c clone shows that they display rather different Northern and Southern blot profiles when probed with a clone corresponding to the repetitive region of the CRA gene. A similar polymorphism was also observed for the gene encoding a flagellar repetitive antigen, suggesting that gene polymorphism might be a common feature of many T. cruzi genes.

The impact of insecticide applications on the dynamics of resistance: The case of four Aedes aegypti populations from different Brazilian regions

Background In the tropics, the utilization of insecticides is still an important strategy for controlling Aedes aegypti, the principle vector of dengue, chikungunya and Zika viruses. However, increasing insecticide resistance in Ae. aegypti populations might hinder insecticide efficacy on a long-term basis. It will be important to understand the dynamics and evolution of insecticide resistance by assessing its frequency and the mechanisms by which it occurs. Methodology/Principal findings The insecticide resistance status of four Brazilian Ae. aegypti populations was monitored. Quantitative bioassays with the major insecticides employed in the country was performed: the adulticide deltamethrin (a pyrethroid—PY) and the larvicides, temephos (an organophosphate) and diflubenzuron (a chitin synthesis inhibitor). Temephos resistance was detected in all populations although exhibiting a slight decrease over time probably due to the interruption of field use. All vector populations were susceptible to diflubenzuron, recently introduced in the country to control Ae. aegypti. Resistance against deltamethrin was extremely high in three populations. Molecular assays investigated substitutions in the voltage gated sodium channel (NaV), the PY target site, at positions 1011, 1016 and 1534. Elevated frequencies of substitutions Val1016Ile and Phe1534Cys related to high PY resistance levels were identified. Biochemical assays detected alterations in the activities of two detoxifying enzyme classes related to metabolic resistance, glutathion-S-transferases and esterases. The results obtained were evaluated in the context of both recent insecticide use and the records of dengue incidence in each locality. Conclusions/Significance The four Ae. aegypti populations evaluated were resistant to the neurotoxic insecticides, temephos and deltamethrin. However, they were still susceptible to diflubenzuron. A probable correlation between adult insect resistance to PY and the domestic application of insecticides is discussed, pointing to the need for awareness measures regarding the correct utilization by citizens. This work aims to contribute to the efficient and rational management of Ae. aegypti control of both larvae and adults.