Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately

Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin separately to detect cryptic Bti-resistance evolving in field mosquito populations. Although no resistance to Bti was detected in none of the three mosquito species tested (Aedes rusticus, Aedes sticticus and Aedes vexans), an increased tolerance to Cry4Aa (3.5-fold) and Cry11Aa toxins (8-fold) was found in one Ae. sticticus population compared to other populations of the same species, suggesting that resistance to Bti may be arising in this population. This study confirms previous works showing a lack of Bti resistance in field mosquito populations treated for decades with this bioinsecticide. It also provides a first panorama of their susceptibility status to individual Bti Cry toxins. In combination with bioassays with Bti, bioassays with separate Cry toxins allow a more sensitive monitoring of Bti-resistance in the field.

Serial measurement of the circulating levels of tumour necrosis factor and its soluble receptors 1 and 2 for monitoring leprosy patients during multidrug treatment

Leprosy is an infectious and contagious spectral disease accompanied by a series of immunological events triggered by the host response to the aetiologic agent, Mycobacterium leprae . The induction and maintenance of the immune/inflammatory response in leprosy are linked to multiple cell interactions and soluble factors, primarily through the action of cytokines. The purpose of the present study was to evaluate the serum levels of tumour necrosis factor (TNF)-α and its soluble receptors (sTNF-R1 and sTNF-R2) in leprosy patients at different stages of multidrug treatment (MDT) in comparison with non-infected individuals and to determine their role as putative biomarkers of the severity of leprosy or the treatment response. ELISA was used to measure the levels of these molecules in 30 healthy controls and 37 leprosy patients at the time of diagnosis and during and after MDT. Our results showed increases in the serum levels of TNF-α and sTNF-R2 in infected individuals in comparison with controls. The levels of TNF-α, but not sTNF-R2, decreased with treatment. The current results corroborate previous reports of elevated serum levels of TNF-α in leprosy and suggest a role for sTNF-R2 in the control of this cytokine during MDT.

Oviposition behaviour of Phlebotomus argentipes - A laboratory-based study

The breeding habitat of sandflies is a little studied and poorly understood phenomenon. More importantly, oviposition behaviour is a largely neglected aspect of sandfly biology and this knowledge gap further undermines our understanding of the biology of sandflies. Pheromones released by the eggs play an important role in identifying good sites for oviposition by female insects. Several recent studies have examined the oviposition pheromone. The present study provides a preliminary report on the oviposition behaviour of Phlebotomus argentipes, the only vector of kala-azar (or visceral leishmaniasis) on the Indian sub-continent. Sandflies prefer to oviposit their eggs on surfaces that contain organic substances, especially substances with an odour of decaying animal products and the remains of conspecific eggs. The results presented here suggest that the odour released by the organic substances of old sandfly colony remains that contain dead flies, old unhatched eggs, larval food containing vertebrate faeces, frass and other organic matter serves as an attractant for the ovipositing females of P. argentipes and hence greatly increases the number of oviposited eggs compared to eggs deposited in controlled oviposition pots. This result will be helpful in maintaining an efficient colony of P. argentipes and may be a promising tool for monitoring and controlling the target insect as part of a synergistic approach.

Acoustic communication in insect disease vectors

Acoustic signalling has been extensively studied in insect species, which has led to a better understanding of sexual communication, sexual selection and modes of speciation. The significance of acoustic signals for a blood-sucking insect was first reported in the XIX century by Christopher Johnston, studying the hearing organs of mosquitoes, but has received relatively little attention in other disease vectors until recently. Acoustic signals are often associated with mating behaviour and sexual selection and changes in signalling can lead to rapid evolutionary divergence and may ultimately contribute to the process of speciation. Songs can also have implications for the success of novel methods of disease control such as determining the mating competitiveness of modified insects used for mass-release control programs. Species-specific sound “signatures” may help identify incipient species within species complexes that may be of epidemiological significance, e.g. of higher vectorial capacity, thereby enabling the application of more focussed control measures to optimise the reduction of pathogen transmission. Although the study of acoustic communication in insect vectors has been relatively limited, this review of research demonstrates their value as models for understanding both the functional and evolutionary significance of acoustic communication in insects.

Circadian rhythms in insect disease vectors

Organisms from bacteria to humans have evolved under predictable daily environmental cycles owing to the Earth’s rotation. This strong selection pressure has generated endogenous circadian clocks that regulate many aspects of behaviour, physiology and metabolism, anticipating and synchronising internal time-keeping to changes in the cyclical environment. In haematophagous insect vectors the circadian clock coordinates feeding activity, which is important for the dynamics of pathogen transmission. We have recently witnessed a substantial advance in molecular studies of circadian clocks in insect vector species that has consolidated behavioural data collected over many years, which provided insights into the regulation of the clock in the wild. Next generation sequencing technologies will facilitate the study of vector genomes/transcriptomes both among and within species and illuminate some of the species-specific patterns of adaptive circadian phenotypes that are observed in the field and in the laboratory. In this review we will explore these recent findings and attempt to identify potential areas for further investigation.

Genetic basis of triatomine behavior: lessons from available insect genomes

Triatomines have been important model organisms for behavioural research. Diverse reports about triatomine host search, pheromone communication in the sexual, shelter and alarm contexts, daily cycles of activity, refuge choice and behavioural plasticity have been published in the last two decades. In recent times, a variety of molecular genetics techniques has allowed researchers to investigate elaborate and complex questions about the genetic bases of the physiology of insects. This, together with the current characterisation of the genome sequence of Rhodnius prolixus allows the resurgence of this excellent insect physiology model in the omics era. In the present revision, we suggest that studying the molecular basis of behaviour and sensory ecology in triatomines will promote a deeper understanding of fundamental aspects of insect and, particularly, vector biology. This will allow uncovering unknown features of essential insect physiology questions for a hemimetabolous model organism, promoting more robust comparative studies of insect sensory function and cognition.

Serological based monitoring of a cohort of patients with chronic Chagas disease treated with benznidazole in a highly endemic area of northern Argentina

This study aimed to evaluate well-documented diagnostic antigens, named B13, 1F8 and JL7 recombinant proteins, as potential markers of seroconversion in treated chagasic patients. Prospective study, involving 203 patients treated with benznidazole, was conducted from endemic areas of northern Argentina. Follow-up was possible in 107 out of them and blood samples were taken for serology and PCR assays before and 2, 3, 6, 12, 24 and 36 months after treatment initiation. Reactivity against Trypanosoma cruzi lysate and recombinant antigens was measured by ELISA. The rate of decrease of antibody titers showed nonlinear kinetics with an abrupt drop within the first three months after initiation of treatment for all studied antigens, followed by a plateau displaying a low decay until the end of follow-up. At this point, anti-B13, anti-1F8 and anti-JL7 titers were relatively close to the cut-off line, while anti-T. cruzi antibodies still remained positive. At baseline, 60.8% (45/74) of analysed patients tested positive for parasite DNA by PCR and during the follow-up period in 34 out of 45 positive samples (75.5%) could not be detected T. cruzi DNA. Our results suggest that these antigens might be useful as early markers for monitoring antiparasitic treatment in chronic Chagas disease.

Avaliação da infestação de insetos-praga associados à batata (Solanum tuberosum L.) sob efeito de nutrientes nitrogenados e potássicos e teores acumulados de aminoácidos livres nas cultivares Achat e Monalisa

Evaluation of insect-pest infestation associated to potato (Solanum tuberosum L.) under effect of nitrogen and potassium fertilizers and the accumulated amount of free aminoacids in Achat and Monalisa cultivars. The objective of this work was to evaluate the occurence of insect-pests on potato plants influenced by dosages of nitrogen and potassium accumulated in plant organs. A total of 169 plants of the Achat and Monalisa cultivars were evaluated to determine the presence-absence of Diabrotica speciosa Germar, 1824 and Agrotis ipsilon Hüfnagel, 1767. The experiment was carried out and executed at the Universidade Federal Fluminense, and the delineation was complete randomized block design, with four replication and nine treatments, using three fertilization level (0; 75 and 150 Kg/ha) with N-urea + KCl. The aminoacid levels were adjusted by the Leucine standard-curve (µg/l), using the Ninhydrin method, at 570 nm. The results showed that the tubercles of Monalisa accumulated high free aminoacid levels with 7,95% in the treatment N1K2 and 7,75% in the N2K1.These treatments, induced the infestation by D. speciosa larvae in 27,03%, when the aminoacid level was 2,01 ± 0,58% (X ± EP), with probability of 0,0196

Utilização de bioensaios e marcadores moleculares para detecção da resistência de coleópteros de produtos armazenados a inseticidas

The qualitative and quantitative losses caused by stored product insects are of great concern, and since there is only a few active ingredients available for their control it is very important to have a frequent insect resistance monitoring. The objective of this research is to evaluate combination of bioassays and molecular marker techniques to detect insecticide resistance in stored product beetles. The Coleoptera species used for the tests were Sitophilus oryzae (L.) (Curculionidae), Rhyzopertha dominica (F.) (Bostrichidae) and Oryzaephilus surinamensis (L.) (Silvanidae). For the bioassays it was used the impregnated filter paper technique, applying 1 mL of deltamethrin (K-Obiol 25 CE TM) using four concentrations and five replicates, including a control with solvent only. Ten adults of each species were liberated separately on each dish. The mortality was evaluated after 24 h and resistance determined by probit analysis. The samples used for the PCR-RAPD were either in vivo or preserved in 70% ethanol, kept in -18°C freezer. After extraction, quantification and DNA quality analysis, the 25 µL samples had the DNA amplified and tested with six primers. The bioassays showed a crescent mortality proportional to insecticide concentration. The resistance factor for R. dominica, S. zeamais and S. oryzae were: 2,2; 3,2 and 9,2, respectively, compared to the susceptible populations of each species. The PCR-RAPD analysis revealed bands which indicate inter and intraspecific variability in the populations, but it was not possible to correlate them to resistance. The association of bioassay and PCR-RAPD represents a precise and valuable tool for resistance management of stored product insects, but more populations and primers should be tested.

Development of a control alternative for the citrus fruit borer, Ecdytolopha aurantiana (Lepidoptera, Tortricidae): from basic research to the grower

All research steps, developed from 1995 to 2000, to synthesize the sex pheromone of Ecdytolopha aurantiana (Lima, 1927) are described, in order to monitoring this pest that causes losses in the order of 50 million dollars per year to citriculture in the State of São Paulo. The basic researches conducted are described, including the development of an artificial diet for the insect, the study of its temperature and humidity requirements, behavioral studies, and synthesis of the male-attracting substance up to the formulation and distribution of the pheromone to the grower, by means of its commercialization. It is a case of success, at a cost of 50 thousand dollars, involving inter- and multidisciplinary researches, which can be adopted to other insect pests in the country.

Ants and their effects on an insect herbivore community associated with the inflorescences of Byrsonima crassifolia (Linnaeus) H.B.K. (Malpighiaceae)

The effects of ants on the insect community on inflorescences of Byrsonima crassifolia (Malpighiaceae) were tested in an ant exclusion experiment in a cerrado vegetation in southeastern Brazil. Forty-four species of insects (23 families) and nine species of ants (6 genera and 3 subfamilies) were found on the inflorescences of B. crassifolia. The exclusion of ants, primarily Camponotus sericeiventris and Camponotus spp., reduced the treehopper population to 20% of the original abundance. Ant exclusion and time influenced the abundance of chewing (Exclusion, P<0.001; Time, P<0.002), and sucking insects (Exclusion, P<0.02; Time, P<0.01). Twice as many chewing and sucking insects were found on ant-excluded inflorescences as compared to control inflorescences (P<0.001). One and half more sucking insects were found on ant-excluded than on control inflorescences. Only time significantly influenced the richness of chewing and sucking insects associated with B. crassifolia inflorescences. Inflorescences on control branches were significantly less attacked by herbivores than inflorescences on ant-excluded branches (P<0.001). Therefore, these results suggest that the presence of ants alters the structure of insect herbivore community associated with B. crassifolia.

Combination of diatomaceous earth and powder deltamethrin for insect control in stored corn

The use of diatomaceous earth (DE) is a very efficient insect control measure in stored grain IPM due to its low cost, easy application, reduction of active ingredient residues, lower environmental contamination and operator safety. The objective of this research was to evaluate the efficacy of different dosages of DE mixed with powder deltamethrin for controlling Sitophilus zeamais in stored corn. Samples of 100 g of clean and dry corn, in three replicates, were submitted to the following treatments: DE (Keepdry®), at the dosages of 500, 750 and 1000 g/t; powder deltamethrin (K-Obiol®) at 0,5 g a.i. /t and 1,0 g a.i. /t; and combinations of the lowest and highest DE dosages with the two dosages of deltamethrin. Thirty adults of S. zeamais were placed in each vial with the treated grains and kept in environment chambers at 25ºC. Mortality was evaluated from the 1st to the 28th day. In the treatments mixing DE with deltamethrin or deltamethrin alone, the mortalyti was registered since the first day. In the treatments using only DE, the first dead insects were recorded after the 3rd day, especially in the highest dosages. After the 7th day, however, there was no statistical difference among all treatments, except for the lowest dosage of DE which reached a satisfactory control level only by the 14th day. It was concluded that treatments using DE combined with low dosages of powder deltamethrin represent an efficient control measure against S. zeamais in stored corn because insect mortality is faster than in treatments using DE alone and residues of active ingredients are much lower than using the insecticide in high dosages.

Insect feeding preferences on Piperaceae species observed in São Paulo city, Brazil

Piperaceae species have been placed among the basal angiosperm and are adapted to a variety of habitats including moist forests, secondary vegetation and dry high lands. The major anatomical/morphology features are of small trees, vines, and shrubs for Piper species, while the epiphytic and succulent characteristics are predominant forms among Peperomia species. Their secondary chemistry can be mostly represented by amides, phenylpropanoids/lignoids, and chromenes in addition to a phletoria of biosynthetically mixed-origin secondary compounds. Although several amides and lignans are known as insecticides, several phytophagous insects, among which some considered pests of economic importance, have been observed feeding vigorously on Piperaceae species. Herein we describe the feeding preferences of fourteen phytophagous species of Coleoptera, Lepidoptera and Hemiptera over approximately fifty Piperaceae species observed in São Paulo, SP, Brazil, in a long-term basis.

Review of semiochemicals that mediate the oviposition of mosquitoes: a possible sustainable tool for the control and monitoring of Culicidae

The choice for suitable places for female mosquitoes to lay eggs is a key-factor for the survival of immature stages (eggs and larvae). This knowledge stands out in importance concerning the control of disease vectors. The selection of a place for oviposition requires a set of chemical, visual, olfactory and tactile cues that interact with the female before laying eggs, helping the localization of adequate sites for oviposition. The present paper presents a bibliographic revision on the main aspects of semiochemicals in regard to mosquitoes' oviposition, aiding the comprehension of their mechanisms and estimation of their potential as a tool for the monitoring and control of the Culicidae.

Pattern of attack of a galling insect reveals an unexpected preference-performance linkage on medium-sized resources

Pattern of attack of a galling insect reveals an unexpected preference-performance linkage on medium-sized resources. The Plant Vigor Hypothesis (PVH) predicts oviposition preference and higher offspring performance on longer and fast-growing shoots, and although several studies have tested its predictions, long-term studies concerning the patterns of host selection by galling species are still lacking. The PVH was tested in this study using Bauhinia brevipes (Fabaceae) as the host of a leaf gall midge, Asphondylia microcapillata (Diptera, Cecidomyiidae) during three consecutive years. Shoots were collected from the same 80 plants between 2001 and 2003 and shoot length, number of healthy and galled leaves, gall number, and mortality factors were recorded. Nearly 600 galls were found on the 5,800 shoots collected. Medium-sized shoots supported from 46 to 70% of all galls, with greater gall survival rate in 2002 and 2003. A decrease in parasitism rate coupled with an increase in gall predation lead to a constant similar gall survivorship rate in all years (x = 22.7%). Although gall abundance varied among years (122 in 2001, 114 in 2002 and 359 in 2003) preference for longer shoots was not observed because the percentage of galled shoots and galled leaves were higher on medium shoot length classes in all years. The observed distribution of gall abundance and galled shoots were always greater than the expected distribution on medium shoot length classes. These findings do not support the PVH, and show that A. microcapillata can maximize the female preference and larval performance on medium-sized shoots of B. brevipes.