Potential of laticifer fluids for inhibiting Aedes aegypti larval development: evidence for the involvement of proteolytic activity

It has been shown previously that the laticifer fluid of Calotropis procera (Ait.) R.Br. is highly toxic to the egg hatching and larval development of Aedes aegypti L. In the present study, the larvicidal potential of other laticifer fluids obtained from Cryptostegia grandiflora R.Br., Plumeria rubra L. and Euphorbia tirucalli L. was evaluated. We attempted to correlate larvicidal activity with the presence of endogenous proteolytic activity in the protein fraction of the fluids. After collection, the fluids were processed by centrifugation and dialysis to obtain the soluble laticifer protein (LP) fractions and eliminate water insoluble and low molecular mass molecules. LP did not visibly affect egg hatching at the doses assayed. LP from Cr. grandiflora exhibited the highest larval toxicity, while P. rubra was almost inactive. E. tirucalli was slightly active, but its activity could not be correlated to proteins since no protein was detected in the fluid. The larvicidal effects of LP from C. procera and Cr. grandiflora showed a significant relationship with the proteolytic activity of cysteine proteinases, which are present in both materials. A purified cysteine proteinase (papain) from the latex of Carica papaya (obtained from Sigma) was similarly effective, whereas trypsin and chymotrypsin (both serine proteinases) were ineffective. The results provide evidence for the involvement of cysteine proteinase activity in the larvicidal action of some laticifer fluids. C. procera is an invasive species found in areas infested with Ae. aegypti and thus could prove useful for combating mosquito proliferation. This is the first report to present evidence for the use of proteolytic enzymes as chemical agents to destroy Ae. aegypti larvae.

Larval recovery of Toxocara cati in experimentally infected Rattus norvegicus and analysis of the rat as potential reservoir for this ascarid

Toxocara cati is a common feline parasite transmitted by the ingestion of embryonated eggs, by the transmammary route or by predation of paratenic hosts harbouring third-stage larvae in their bodies. In the present study, the larval distribution of T. cati in tissues and organs of Rattus norvegicus experimentally infected with 300 embryonated eggs was analysed. Third-stage larvae were recovered from livers, lungs, kidneys, eyes, brains and carcasses of infected rats, following tissue digestion with HCl 0.5% for 24 h at 37°C. Some differences from the known larval distribution of Toxocara canisin the same rodent species were found.

Classification of immature mosquito species according to characteristics of the larval habitat in the subtropical province of Chaco, Argentina

To classify mosquito species based on common features of their habitats, samples were obtained fortnightly between June 2001-October 2003 in the subtropical province of Chaco, Argentina. Data on the type of larval habitat, nature of the habitat (artificial or natural), size, depth, location related to sunlight, distance to the neighbouring houses, type of substrate, organic material, vegetation and algae type and their presence were collected. Data on the permanence, temperature, pH, turbidity, colour, odour and movement of the larval habitat's water were also collected. From the cluster analysis, three groups of species associated by their degree of habitat similarity were obtained and are listed below. Group 1 consisted of Aedes aegypti. Group 2 consisted of Culex imitator, Culex davisi, Wyeomyia muehlensi and Toxorhynchites haemorrhoidalis separatus. Within group 3, two subgroups are distinguished: A (Psorophora ferox, Psorophora cyanescens, Psorophora varinervis, Psorophora confinnis, Psorophora cingulata, Ochlerotatus hastatus-oligopistus, Ochlerotatus serratus, Ochlerotatus scapularis, Culex intrincatus, Culex quinquefasciatus, Culex pilosus, Ochlerotatus albifasciatus, Culex bidens) and B (Culex maxi, Culex eduardoi, Culex chidesteri, Uranotaenia lowii, Uranotaenia pulcherrima, Anopheles neomaculipalpus, Anopheles triannulatus, Anopheles albitarsis, Uranotaenia apicalis, Mansonia humeralis and Aedeomyia squamipennis). Principal component analysis indicates that the size of the larval habitats and the presence of aquatic vegetation are the main characteristics that explain the variation among different species. In contrast, water permanence is second in importance. Water temperature, pH and the type of larval habitat are less important in explaining the clustering of species.

Production of TNF-α, nitric oxide and hydrogen peroxide by macrophages from mice with paracoccidioidomycosis that were fed a linseed oil-enriched diet

Omega-3 polyunsaturated fatty acids (n-3 PUFA) can modulate the immune system and their primary effect is on macrophage function. Paracoccidioidomycosis (PCM) is an endemic systemic mycosis in Latin America that is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). Macrophages are the main defence against this pathogen and have microbicidal activity that is dependent on interferon-Γ and tumour necrosis factor (TNF)-α. These cytokines stimulate the synthesis of nitric oxide (NO) and hydrogen peroxide (H2O2), leading to the death of the fungus. To study the effect of n-3 PUFA on the host immune response during experimental PCM, macrophages that were obtained from animals infected with Pb18 and fed a diet enriched by linseed (LIN) oil were cultured and challenged with the fungus in vitro. The macrophage function was analysed based on the concentrations of TNF-α, NO and H2O2. LIN oil seems to influence the production of TNF-α during the development of disease. A diet enriched with LIN oil influences the microbicidal activity of the macrophages by inducing the production of cytokines and metabolites such as NO and H2O2, predominantly in the chronic phase of infection.

Proteolytic activity in the adult and larval stages of the human roundworm parasite Angiostrongylus costaricensis

Angiostrongylus costaricensis is a nematode that causes abdominal angiostrongyliasis, a widespread human parasitism in Latin America. This study aimed to characterize the protease profiles of different developmental stages of this helminth. First-stage larvae (L1) were obtained from the faeces of infected Sigmodon hispidus rodents and third-stage larvae (L3) were collected from mollusks Biomphalaria glabrata previously infected with L1. Adult worms were recovered from rodent mesenteric arteries. Protein extraction was performed after repeated freeze-thaw cycles followed by maceration of the nematodes in 40 mM Tris base. Proteolysis of gelatin was observed by zymography and found only in the larval stages. In L3, the gelatinolytic activity was effectively inhibited by orthophenanthroline, indicating the involvement of metalloproteases. The mechanistic class of the gelatinases from L1 could not be precisely determined using traditional class-specific inhibitors. Adult worm extracts were able to hydrolyze haemoglobin in solution, although no activity was observed by zymography. This haemoglobinolytic activity was ascribed to aspartic proteases following its effective inhibition by pepstatin, which also inhibited the haemoglobinolytic activity of L1 and L3 extracts. The characterization of protease expression throughout the A. costaricensis life cycle may reveal key factors influencing the process of parasitic infection and thus foster our understanding of the disease pathogenesis.

Characterisation of Culex quinquefasciatus (Diptera: Culicidae) larval habitats at ground level and temporal fluctuations of larval abundance in Córdoba, Argentina

The aims of this study were to characterise the ground-level larval habitats of the mosquito Culex quinquefasciatus, to determine the relationships between habitat characteristics and larval abundance and to examine seasonal larval-stage variations in Córdoba city. Every two weeks for two years, 15 larval habitats (natural and artificial water bodies, including shallow wells, drains, retention ponds, canals and ditches) were visited and sampled for larval mosquitoes. Data regarding the water depth, temperature and pH, permanence, the presence of aquatic vegetation and the density of collected mosquito larvae were recorded. Data on the average air temperatures and accumulated precipitation during the 15 days prior to each sampling date were also obtained. Cx. quinquefasciatus larvae were collected throughout the study period and were generally most abundant in the summer season. Generalised linear mixed models indicated the average air temperature and presence of dicotyledonous aquatic vegetation as variables that served as important predictors of larval densities. Additionally, permanent breeding sites supported high larval densities. In Córdoba city and possibly in other highly populated cities at the same latitude with the same environmental conditions, control programs should focus on permanent larval habitats with aquatic vegetation during the early spring, when the Cx. quinquefasciatus population begins to increase.

A comparison of larval, ovitrap and MosquiTRAP surveillance for Aedes (Stegomyia) aegypti

In Brazil, the entomological surveillance of Aedes (Stegomyia) aegypti is performed by government-mandated larval surveys. In this study, the sensitivities of an adult sticky trap and traditional surveillance methodologies were compared. The study was performed over a 12-week period in a residential neighbourhood of the municipality of Pedro Leopoldo, state of Minas Gerais, Brazil. An ovitrap and a MosquiTRAP were placed at opposite ends of each neighbourhood block (60 traps in total) and inspections were performed weekly. The study revealed significant correlations of moderate strength between the larval survey, ovitrap and MosquiTRAP measurements. A positive relationship was observed between temperature, adult capture measurements and egg collections, whereas precipitation and frequency of rainy days exhibited a negative relationship.

She’s a femme fatale: low-density larval development produces good disease vectors

Two hypotheses for how conditions for larval mosquitoes affect vectorial capacity make opposite predictions about the relationship of adult size and frequency of infection with vector-borne pathogens. Competition among larvae produces small adult females. The competition-susceptibility hypothesis postulates that small females are more susceptible to infection and predicts frequency of infection should decrease with size. The competition-longevity hypothesis postulates that small females have lower longevity and lower probability of becoming competent to transmit the pathogen and thus predicts frequency of infection should increase with size. We tested these hypotheses for Aedes aegypti in Rio de Janeiro, Brazil, during a dengue outbreak. In the laboratory, longevity increases with size, then decreases at the largest sizes. For field-collected females, generalised linear mixed model comparisons showed that a model with a linear increase of frequency of dengue with size produced the best Akaike’s information criterion with a correction for small sample sizes (AICc). Consensus prediction of three competing models indicated that frequency of infection increases monotonically with female size, consistent with the competition-longevity hypothesis. Site frequency of infection was not significantly related to site mean size of females. Thus, our data indicate that uncrowded, low competition conditions for larvae produce the females that are most likely to be important vectors of dengue. More generally, ecological conditions, particularly crowding and intraspecific competition among larvae, are likely to affect vector-borne pathogen transmission in nature, in this case via effects on longevity of resulting adults. Heterogeneity among individual vectors in likelihood of infection is a generally important outcome of ecological conditions impacting vectors as larvae.

New classification of natural breeding habitats for Neotropical anophelines in the Yanomami Indian Reserve, Amazon Region, Brazil and a new larval sampling methodology

Here we present the first in a series of articles about the ecology of immature stages of anophelines in the Brazilian Yanomami area. We propose a new larval habitat classification and a new larval sampling methodology. We also report some preliminary results illustrating the applicability of the methodology based on data collected in the Brazilian Amazon rainforest in a longitudinal study of two remote Yanomami communities, Parafuri and Toototobi. In these areas, we mapped and classified 112 natural breeding habitats located in low-order river systems based on their association with river flood pulses, seasonality and exposure to sun. Our classification rendered seven types of larval habitats: lakes associated with the river, which are subdivided into oxbow lakes and nonoxbow lakes, flooded areas associated with the river, flooded areas not associated with the river, rainfall pools, small forest streams, medium forest streams and rivers. The methodology for larval sampling was based on the accurate quantification of the effective breeding area, taking into account the area of the perimeter and subtypes of microenvironments present per larval habitat type using a laser range finder and a small portable inflatable boat. The new classification and new sampling methodology proposed herein may be useful in vector control programs.

Larval endoparasitoids (Hymenoptera) of frugivorous flies (Diptera, Tephritoidea) reared from fruits of the cerrado of the State of Mato Grosso do Sul , Brazil

This paper presents a five years survey of endoparasitoids obtained from the larvae of frugivorous Tephritidae and Lonchaeidae flies. The insects were reared from cultivated and wild fruits collected in areas of the cerrado in the State of Mato Grosso do Sul, Brazil. The flies obtained from 14 host fruit species were eight Anastrepha species, Ceratitis capitata (Wiedemann, 1824) (Tephritidae); Dasiops sp. and Neosilba spp. (Lonchaeidae). Eleven parasitoid species were collected: Braconidae - Asobara anastrephae (Muesebek, 1958), Doryctobracon areolatus (Szépligeti, 1911), D. fluminensis (Costa Lima, 1938), Opius bellus Gahan, 1930 and Utetes anastrephae (Viereck, 1913); Figitidae - Aganaspis nordlanderi Wharton, 1998, Lopheucoila anastrephae (Rhower, 1919), Odontosema anastrephae (Borgmeier, 1935) and Trybliographa infuscata Gallardo, Díaz & Uchôa-Fernandes, 2000 and, Pteromalidae - Spalangia gemina Boucek, 1963 and S. endius Walker, 1839. In all cases only one parasitoid emerged per puparium. D. areolatus was the most abundant and frequent parasitoid of fruit fly species, as was L. anastrephae in Neosilba spp. larvae. This is the first record of A. nordlanderi in the midwestern Brazilian region.

Estudo da dispersão larval radial pós-alimentar em Chrysomya megacephala (Fabricius) (Diptera, Calliphoridae)

Blowflies utilize discrete and ephemeral sites for breeding and larval nutrition. After the exhaustion of food, the larvae begin dispersing to search for sites to pupate or to additional food source, process referred as postfeeding larval dispersal. Some of the most important aspects of this process were investigated in Chrysomya megacephala, utilizing a circular arena to permit the radial dispersion of larvae from the center. To determinate the localization of each pupa, the arena was split in 72 equal sectors from the center. For each pupa, distance from the center of arena, weight and depth were determined. Statistical tests were performed to verify the relation among weight, depth and distance of burying for pupation. It was verified that the larvae that disperse farther are those with higher weights. The majority of individuals reached the depth of burying for pupation between 7 and 18 cm. The study of this process of dispersion can be utilized in the estimation of postmortem interval (PMI) for human corpses in medico-criminal investigations.

On the immature stages and natural history of Gonioterma exquisita Duckworth (Lepidoptera, Elachistidae, Stenomatinae)

In the present work the natural history and immature stages of Gonioterma exquisita Duckworth, 1964 of the Brazilian cerrado region are described. The caterpillars are external folivorous feeders and present a diet restricted to pubescent-leaved host plants of the genus Byrsonima Rich. (Malpighiaceae). They are solitary caterpillars and they build a hard individual shelter that looks like a planorbid shell, made from silk and frass, covered with trichomes and silk, where they develop until the last larval instar on the host plant, and remain inside it until the adult emergence on herbaceous stratum. The larval development presented eight instars. Egg, larval head capsules, instar duration, pupa, shelter, and behavior are also described. Diapause is mentioned for the first time in this species.

The effect of sub-lethal doses of Azadirachta indica (Meliaceae) oil on the midgut of Spodoptera frugiperda (Lepidoptera, Noctuidae)

The fall armyworm, Spodoptera frugiperda, is one of the major field pests for maize production. It is mainly controlled by means of synthetic, and more recently by resistant cultivar of maize expressing Bt toxins. The neem tree, Azadirachta indica, is a plant that can potentially control insects with the advantage of being food and environmental safe. The aim of this study was to assess the effect of neem oil on the development and survival of S. frugiperda caterpillars by assessing histological alterations caused on their midgut. Newly hatched caterpillars were submitted to three neem oil concentrations: 0.006; 0.05; 0.4%, which were added to their artificial diet. Ten 3rd instar caterpillars, taken from each treatment, were submitted to histological analysis. The alimentary canals from the specimens were fixed in Baker for 12 hours, desiccated and diaphanized in alcohol/xylol (1:1) and xylol. After placing the samples in paraffin, they were sliced in 8 µm sections and stained with hematoxylin-eosin stain. The neem oil added to the diet of S. frugiperda caused total mortality at dose of 0.4% whilst still in the first instars, prolonged the larval and pupal stages, and reduced the pupal weight. Histo-physiological alterations such as degeneration of the epithelial lining of the midgut and in the peritrophic matrix were found at all concentrations of neem oil.

Bionomic data and larval density of Scarabaeidae (Pleurosticti) in sugarcane in the central region of Mato Grosso do Sul, Brazil

Bionomic data and larval density of Scarabaeidae (Pleurosticti) in sugarcane in the central region of Mato Grosso do Sul, Brazil. Phytophagous larvae of Scarabaeidae cause damage to diverse crops. Information on these pests is scarce; therefore, the objective of this study was to determine biological aspects and larval density of species occurring in an area of sugarcane. The studies were developed in Sidrolândia from April 2009 to March 2010. Scarab beetle larvae were collected in sugarcane roots every fifteen days, taken to the laboratory and reared to obtain the adults and determine biological parameters. A total of 2,656 larvae were collected, being 162 Liogenys fuscus, 120 Cyclocephala verticalis, 37 Cyclocephala forsteri, and 2337 Anomonyx sp. In January, 53.65 larvae m-2 were obtained, and the most abundant species was Anomonyx sp, representing 87.99% of the total larvae collected. From November to March, the greatest densities of Anomonyx were observed in the field. The adults of this species occurred from May to September, and egg laying from September to November. Eggs measured 1.1 x 1.7 mm, and incubation period last 15.4 days. First instar larvae were observed mainly in October; second instar larvae from November to April; and third instar from January to July. Pupae were observed from May to August. The most abundant scarab beetle, Anomonyx sp. in roots of sugarcane presents one generation per year in Sidrolândia, MS.

Nesting and use of pollen resources by Tetrapedia diversipes Klug (Apidae) in Atlantic Forest areas (Rio de Janeiro, Brazil) in different stages of regeneration

Nesting and use of pollen resources by Tetrapedia diversipes Klug (Apidae) in Atlantic Forest areas (Rio de Janeiro, Brazil) in different stages of regeneration. The nesting in trap-nests and use of pollen sources in larval food by Tetrapedia diversipes Klug, 1810 (Apidae) was compared between regenerating areas of Atlantic Forest. The study was conducted between April 2008 and October 2009 at União Biological Reserve, Rio de Janeiro, Brazil. T. diversipes nested in 66 trap-nests and showed a peak of nesting during the months of highest rainfall. The most frequent pollen type in brood cells during the wet season was Dalechampia sp. 1. During the dry season, the type Ludwigia sp. was the most frequent, followed by Dalechampia sp. 2. The high frequency of Dalechampia and Ludwigia species in the larval food, observed in both habitats and in the two seasons could be considered relevant for T. diversipes, suggesting highly selective diet based primarily on two plant species unrelated, but similar in size of pollen grains.