["Farmer Field School", a participatory educational approach for improving the fight against malaria vectors in irrigated rice-growing areas in Benin]. / « Champ-École Paysan ¼, une approche pédagogique participative pour l'amélioration de la lutte contre les vecteurs du paludisme en zone de riziculture irriguée au Bénin.

Background & rationale: Malaria is a major health problem in Benin where it is the main cause of morbidity and mortality, particularly among children under 5 and pregnant women. Although the vast majority of malaria cases occurs in rural and agricultural areas and are often associated with development projects, very few interventions target the agro-ecosystem. In Benin, irrigated rice growing is expanding to meet the increasing demand of the population. However, continuous flooding and tillage systems induce the development and proliferation of malaria and other diseases vectors. Intermittent flooding of rice plots and minimal tillage can reduce significantly the proliferation of mosquitoes including Anopheles in rice fields. However, the dissemination and implementation of these agricultural practices require community-wide action for greater effectiveness. As part of strengthening the capacity of farming communities in the fight against malaria vectors, the "Farmer Field School" appears to be an innovative approach. This learning by doing method promotes interactions between groups of producers to disseminate proven technologies. This study aims to disseminate among rice producers the agricultural practices of intermittent flooding and minimal tillage, likely to reduce the proliferation of malaria vectors in the rice fields. Materials & methods: This study was carried out in the rice-growing perimeter of Malanville, Benin (11° 52' 5" North, 3° 22' 59" East) which covers an area of 516 hectares. Farmer Field Schools were set up after a basic survey at producer level. This survey was carried out through in-depth interview, focus group discussions and direct field observation with producers. Focus groups discussions and interviews made it possible to understand the perception of rice farmers on the link between rice production and the transmission of malaria. In order to disseminate new agricultural practices such as intermittent flooding and minimal tillage among producers, twelve plots have been set up. Farmer Field Schools were monitored weekly with rice producers accompanied by a facilitator and a medical entomologist (learning facilitator or moderator) helping the farmers with the collection and identification of mosquito larvae. According to the different stages of rice development (transplanting, tillering, maturation), the mosquito larvae were collected in the test and control plots from 10 a.m. to 2 p.m. by the dipping method. Then the water in the test compartments (intermittent flooding) was emptied. A cycle of 7 days of flooding and 2 days of drying was carried out for intermittent flooding. Mosquito larvae were identified morphologically using the identification key and Anopheles genus larvae were isolated in plastic cups. The impacts of intermittent flooding and minimum tillage in reducing breeding sites and larval densities were established by determining and comparing the larval densities of mosquitoes and of Anopheles between the test and control plots. Results: Direct observations in the field allowed to identify three tillage systems, which include the use of tiller (28%), plow (66%) and hoe (6%) on the rice field. Continuous flooding was the only irrigation system used by farmers. The water used for irrigation comes either from boreholes installed individually or from the Niger River. The volume of water used varies with the seasons, the size of the farms and the variety of rice grown. Farmers observe that the nuisance of mosquitoes increases during the rice production period with an outbreak of malaria cases, especially among children, which leads to crowds in health centers. The preventive measures against malaria among farmers are the use of impregnated mosquito nets distributed free of charge by the national program against malaria, and of insecticide bombs or spirals. Considering the development stages of the rice, the larval densities varied according to the treatments. Overall, minimal tillage applied to intermittent flooding significantly reduced the density of mosquito larvae of all species. The reduction rates were 10.5, 5.4 and 2.5 during transplanting, tillering and maturation, respectively. Considering only the Anopheles larvae, minimal tillage applied to intermittent flooding reduced their density by 16, 5.5 and 4 respectively during transplanting, tillering and maturation. Discussion/conclusion: The rice-growing area of Malanville has many favorable conditions for rice production, including the presence of water supply sources such as the Niger River located near the rice field and numerous boreholes. The availability of water pockets for mosquito breeding during irrigation appeared to contribute to the extension of malaria transmission. The present study showed that the intermittent flooding coupled with minimal tillage could reduce the proliferation of malaria vectors. The results suggested that with technical support to farmers through the "Farmer Field School", the malaria incidence could be reduced in the farming community.

Larval Rearing and Nutrition of the Polyphagous Tephritid Pest Anastrepha ludens on Artificial Diets with Calcium Alginate, Agar, or Carrageenan as Gelling Agents at Various Concentrations and across Extreme Larval Density Conditions.

Research on larval rearing and nutrition of tephritid flies on artificial diets is key for the sterile insect technique. Here, we examined the effects of the type of gel (calcium alginate, agar, or carrageenan), at varying percentages in artificial diets for the polyphagous pest Anastrepha ludens, on the physicochemical and nutritional traits of the diets, and the effects of the type of gel, the gel content and the larval density (larvae/g of diet) used in production, quality parameters for mass-reared tephritids, diet removal (an indirect estimation of diet consumption), and nutritional traits of flies. Regardless of the gel content, calcium alginate diets were firmer and more resistant to penetration than the agar and carrageenan diets. The larval recovery, pupation, pupal weight, and flight ability of A. ludens were lower in calcium alginate diets than in agar and carrageenan diets. Diet removal was higher in calcium alginate diets; however, low levels of ammonium and high levels of uric acid in excretions from larvae on these diets suggest an alteration in protein metabolism. The firmness and penetration resistance characteristics of calcium alginate diets may have limited movement and feeding of larvae, but this could be overcome by the collective feeding of large groups of larvae. Our findings provide insights into the mechanism governing gel-diet rearing systems for A. ludens.

Water Physicochemical Parameters and Microbial Composition Distinguish Anopheles and Culex Mosquito Breeding Sites: Potential as Ecological Markers for Larval Source Surveillance.

The presence of mosquitoes in an area is dependent on the availability of suitable breeding sites that are influenced by several environmental factors. Identification of breeding habitats for vector surveillance and larval source management is key to disease control programs. We investigated water quality parameters and microbial composition in selected mosquito breeding sites in urban Accra, Ghana and associated these with abundance of Anopheles (Diptera: Culicidae) and Culex (Diptera: Culicidae) larvae. Physicochemical parameters and microbial composition explained up to 72% variance among the breeding sites and separated Anopheles and Culex habitats (P < 0.05). Anopheles and Culex abundances were commonly influenced by water temperature, pH, nitrate, and total hardness with contrasting impacts on the two mosquito species. In addition, total dissolved solids, biochemical oxygen demand, and alkalinity uniquely influenced Anopheles abundance, while total suspended solids, phosphate, sulphate, ammonium, and salinity were significant determinants for Culex. The correlation of these multiple parameters with the occurrence of each mosquito species was high (R2 = 0.99, P < 0.0001). Bacterial content assessment of the breeding ponds revealed that the most abundant bacterial phyla were Patescibacteria, Cyanobacteria, and Proteobacteria, constituting >70% of the total bacterial richness. The oligotrophic Patescibacteria was strongly associated with Anopheles suggestive of the mosquito's adaptation to environments with less nutrients, while predominance of Cyanobacteria, indicative of rich nutritional source was associated with Culex larval ponds. We propose further evaluation of these significant abiotic and biotic parameters in field identification of larval sources and how knowledge of these can be harnessed effectively to reduce conducive breeding sites for mosquitoes.

Efectos de la competencia larvaria intraespecífica de Aedes aegypti y Aedes albopictus en condiciones de laboratorio / Effects of intraspecific larval competence of Aedes aegypti and Aedes albopictus under laboratory conditions

RESUMEN Introducción: Aedes aegypti y Aedes albopictus constituyen dos de las especies más importantes de mosquitos, debido a que son vectores primarios de enfermedades emergentes y reemergentes que representan una amenaza significativa para la salud pública. Objetivo: Evaluar el efecto de la competencia larvaria intraespecífica causada por baja y alta densidad de dos poblaciones de Ae. aegypti y Ae. albopictus sobre aspectos de su ciclo de vida en condiciones de laboratorio. Métodos: Se utilizaron dos poblaciones de mosquitos, Ae. aegypti y Ae. albopictus, ambas colectadas en estado de larva en el Reparto Juan de Dios Fraga, La Lisa, La Habana, Cuba. Se establecieron condiciones de baja (70 larvas) y alta densidad larvaria (140 larvas) en cada réplica para cada una de las especies. Se emplearon larvas desde el primer estadio, que se siguieron hasta la formación de los adultos. Se evaluaron las variables duración del ciclo larvario, supervivencia larvaria, así como talla y supervivencia de hembras adultas, respectivamente. Resultados: Se encontró que las variables evaluadas no se afectaron en condiciones de baja densidad larvaria. Sin embargo, el efecto de la relación larvaria intraespecífica bajo condiciones de alta densidad presentada en los hábitats de Ae. aegypti y Ae. albopictus tuvo un efecto negativo en cada una de las variables del ciclo de vida evaluadas. Conclusiones: Estas alteraciones observadas son importantes dentro de los componentes de la capacidad vectorial, con implicaciones en la epidemiología de las enfermedades transmitidas por estos mosquitos. Los resultados pudieran extrapolarse al comportamiento de estos mosquitos en su entorno natural. Estas condiciones podrían aumentar los patrones de transmisión del virus del dengue, así como un mayor riesgo de infección para la población humana.

ABSTRACT Introduction: Aedes aegypti and Aedes albopictus are two of the most important mosquito species, since they are the main vector of emerging and re-emerging diseases that represent a significant threat to public health. Objective: To evaluate the effect of intraspecific larval competence caused by low and high density in two populations of Ae. aegypti and Ae. albopictus on aspects of their life cycles under laboratory conditions. Methods: Two mosquitos populations were used, Ae. aegypti and Ae. albopictus, both collected during their larval stage in Juan de Dios Fraga town, La Lisa, Havana, Cuba. Low (70 larvae) and high (140 larvae) density conditions were established in each replication for each species. Larvae from the first stage were used and follow-up was established until adults emerged. Variables such as: larval cycle duration, larval survival, and adult female size and survival were evaluated. Results: The variables evaluated were not affected under low larval density conditions. However, the intraspecific larval relationship under high density conditions in Ae. aegypti and Ae. albopictus habitats had a negative effect in each of the variables of the life cycles. Conclusions: The observed alterations are important within the components of vectorial capacity, with implications for the epidemiology of these mosquitos-borne diseases. The results could be extended to the behavior of these mosquitoes in their natural environment. These conditions could increase the transmission patterns of dengue virus and the risk of infection for the human population.

Diversity of larval habitats of Anopheles mosquitoes in urban areas of Benin and influence of their physicochemical and bacteriological characteristics on larval density.

BACKGROUND: The implementation of anti-larval strategies in the fight against malaria vectors requires fundamental knowledge of their oviposition sites. The aim of this study was to assess the spatial and temporal distribution of Anopheles breeding sites as well as the influence of abiotic and biotic factors on the proliferation of larvae in urban and non-urban areas of Benin. METHODS: Sampling of Anopheles larvae was carried out during the rainy and dry seasons in urbanized and non-urbanized areas of the cities of Cotonou, Bohicon, Parakou, and Natitingou in Benin. The Anopheles larval breeding sites were georeferenced and characterized by their nature, type, physicochemical (pH, temperature, dissolved oxygen, conductivity, turbidity, salinity) and biological attributes (larval density and coliform density). RESULTS: A total of 198 positive breeding sites for Anopheles larvae were identified, comprising 163 (82.3%) in the rainy season and 35 (17.7%) in the dry season. Out of these larval habitats, 61.9% were located in urbanized areas, and were predominantly puddles. Principal component analysis revealed a high positive correlation of larval density with temperature and dissolved oxygen, and with salinity in the coastal zone. In addition, cross-sectional analysis of the microbiological results with larval density showed a significant negative correlation between larval productivity and faecal coliform load. CONCLUSIONS: This study indicated the presence of multiple larval habitats of Anopheles in the urban areas which were created through human activities, and associations between larval density and intrinsic factors of the habitats such as temperature, dissolved oxygen and faecal coliform load. This type of information may be useful for the implementation of appropriate control strategies in urban areas, including regulation of the human activities that lead to the creation of breeding sites, proper environmental management and targeted larvicidal use.

Black Soldier Fly Larvae Influence Internal and Substrate Bacterial Community Composition Depending on Substrate Type and Larval Density.

Saprophagous fly larvae interact with a rich community of bacteria in decomposing organic matter. Larvae of some species, such as the black soldier fly, can process a wide range of organic residual streams into edible insect biomass and thus produce protein as a sustainable component of livestock feed. The microbiological safety of the insects and substrates remains a point of concern. Substrate-associated bacteria can dominate the larval gut microbiota, but the larvae can also alter the bacterial community in the substrate. However, the relative importance of substrate type and larval density in bacterial community dynamics is unknown. We investigated four larval densities (0 [control], 50, 100, or 200 larvae per container [520 mL; diameter, 75 mm]) and three feed substrates (chicken feed, chicken manure, and camelina substrate [50% chicken feed, 50% camelina oilseed press cake]) and sampled the bacterial communities of the substrates and larvae at three time points over 15 days. Although feed substrate was the strongest driver of microbiota composition over time, larval density significantly altered the relative abundances of several common bacterial genera, including potential pathogens, in each substrate and in larvae fed chicken feed. Bacterial communities of the larvae and substrate differed to a higher degree in chicken manure and camelina than in chicken feed. This supports the substrate-dependent impact of black soldier fly larvae on bacteria both within the larvae and in the substrate. This study indicates that substrate composition and larval density can alter bacterial community composition and might be used to improve insect microbiological safety. IMPORTANCE Black soldier fly larvae can process organic side streams into nutritious insect biomass, yielding a sustainable ingredient of animal feed. In processing such organic residues, the larvae impact the substrate and its microbiota. However, their role relative to the feed substrate in shaping the bacterial community is unknown. This may be important for the waste management industry to determine whether pathogens can be controlled by manipulating the larval density and the timing of harvest. We investigated how the type of feed substrate and the larval density (number of larvae per container) interacted to influence bacterial community composition in the substrates and larvae over time. Substrate type was the strongest driver of bacterial community composition, and the magnitude of the impact of the larvae depended on the substrate type and larval density. Thus, both substrate composition and larval density may be used to improve the microbiological safety of the larvae as animal feed.

Crowding does not affect monarch butterflies' resistance to a protozoan parasite.

Host density is an important factor when it comes to parasite transmission and host resistance. Increased host density can increase contact rate between individuals and thus parasite transmission. Host density can also cause physiological changes in the host, which can affect host resistance. Yet, the direction in which host density affects host resistance remains unresolved. It is also unclear whether food limitation plays a role in this effect. We investigated the effect of larval density in monarch butterflies, Danaus plexippus, on the resistance to their natural protozoan parasite Ophryocystis elektroscirrha under both unlimited and limited food conditions. We exposed monarchs to various density treatments as larvae to mimic high densities observed in sedentary populations. Data on infection and parasite spore load were collected as well as development time, survival, wing size, and melanization. Disease susceptibility under either food condition or across density treatments was similar. However, we found high larval density impacted development time, adult survival, and wing morphology when food was limited. This study aids our understanding of the dynamics of environmental parasite transmission in monarch populations, which can help explain the increased prevalence of parasites in sedentary monarch populations compared to migratory populations.

A sustainable and economic strategy to reduce risk antibiotic resistance genes during poultry manure bioconversion by black soldier fly Hermetia illucens larvae: Larval density adjustment.

Black soldier fly (Hermetia illucens) larvae (BSFL) are common insects that are known for bioconversion of organic waste into a sustainable utilization resource. However, a strategy to increase antibiotic resistance gene (ARG) elimination in sustainable and economic ways through BSFL is lacking. In the present study, different larval densities were employed to assess the mcr-1 and tetX elimination abilities, and potential mechanisms were investigated. The application and economic value of each larval density were also analyzed. The results showed that the 100 larvae cultured in 100 g of manure group had the best density because the comprehensive disadvantage evaluation ratio was the lowest (14.97%, good bioconversion manure quality, low ARG deposition risk and reasonable larvae input cost). Further investigation showed that mcr-1 could be significantly decreased by BSFL bioconversion (4.42 ×107 copies/g reduced to 4.79 ×106-2.14 ×105 copies/g)(P＜0.05); however, mcr-1 was increasingly deposited in the larval gut with increasing larval density. The tetX abundance was stabilized by BSFL bioconversion, except that the abundance at the lowest larval density increased (1.22 ×1010 copies/g increase, 34-fold). Escherichia was the host of mcr-1 and tetX in all samples, especially in fresh manure; Alcaligenes was the host of tetX in bioconversion manure; and the abundance of Alcaligenes was highly correlated with the pH of bioconversion manure. The pH of bioconversion manure was extremely correlated with the density of larvae. Klebsiella and Providencia were both hosts of tetX in the BSF larval gut, and Providencia was also the host of mcr-1 in the BSF larval gut. The density of larvae influenced the bioconversion manure quality and caused the ARG host abundance to change to control the abundance of ARGs, suggesting that larval density adjustment was a useful strategy to manage the ARG risk during BSFL manure bioconversion.

Effects of Rearing Density on Developmental Traits of Two Different Biotypes of the Gypsy Moth, Lymantria Dispar L., from China and the USA.

The life-history traits of the gypsy moth, Lymantria dispar L. (Lepidoptera: Erebidae), have been observed to vary with larval population density, which can increase significantly during an outbreak of this pest. Laboratory studies on density-dependent variation in gypsy moth development have focused on single populations and were limited to comparing solitary larvae with groups of larvae reared at a single density. To evaluate how density-dependent impacts on development vary with different populations and subspecies of L. dispar, we compared the effects of rearing larvae of a European gypsy moth (L. dispar dispar L.) population from Connecticut, USA; and larvae of two populations of the Asian gypsy moth (L. dispar asiatica Vnukovskij) from Guizhou and Hebei provinces in China. Larvae were reared on an artificial diet at densities of one, three, five, seven, and nine larvae per 115 mL container, and the duration of larval development, percentage of surviving larvae, and the rates of pupation and emergence were measured at each density. A two-tailed response to density variation with values falling away on both sides from a peak or climbing from a base was observed for all three populations tested, with the most rapid larval development and the highest values of survival, pupation, and emergence observed at a density of five larvae/container. Although differences in larval development time, survival, pupation and emergence were observed among the different populations under the conditions of our study, our findings indicate that density-dependent effects on the development of different gypsy moth subspecies and populations follow the same trends.

Neglecting larval rearing conditions in Drosophila melanogaster can negatively impact research on ageing.

The developmental conditions of Drosophila melanogaster flies can modify the phenotypic traits of adults. However, the control of these conditions is neglected by some authors in their articles and the readers are unaware, for instance, whether flies developed in crowded cultures or fed on a new or used medium. Controlling developmental conditions allows to know precisely the viability of flies, their duration of development and sex-ratio, which can be warning signals of bad rearing conditions. As developmental conditions can modify the results of experiments on the effects of ageing it is necessary to strictly control them.

Effects of Larval Population Density and Food Type on the Life Cycle of Musca domestica (Diptera: Muscidae).

Larval density is an important factor modulating larval resource-acquisition, influencing development of insects. This study aimed to evaluate the effect of larval density and substrate content on some life-history parameters of Musca domestica Linnaeus, 1758 (Diptera: Muscidae). This research was carried out from March 2019 through September 2019 at Animal Physiology Laboratory of Ondokuz Mayis University, Samsun, Turkey. Groups of 25, 100, 200, and 400 newly hatched M. domestica larvae were transferred to a polyethylene cup filled with different substrates (i.e., wheat bran, poultry meal, soybean meal) and kept at 25°C, 62% RH with a photoperiod of 12:12 (L:D) h. A two-way analysis of variance (Two way ANOVA) was used to analyze the data on the percentage of pupal and larval survival development time, pupal, and adult weight to evaluate the effect of density and rearing substrate. In this study, increasing larval density and nutrient content of food led to changes in the larval and pupal development time of M. domestica. The results also indicated that the weight of pupae and adult survival was negatively affected by increasing larval density. The wheat bran diet was superior to the other diets for all parameters tested. Our study indicated that life history parameters of Musca domestica are affected by the rearing conditions.

Effects of Larval Density on Plutella xylostella Resistance to Granulosis Virus.

It has been reported that some phase-polyphenic insects from high-density conditions are more resistant to pathogens than those from low-density conditions. This phenomenon is termed "density-dependent prophylaxis" (DDP). However, whether non phase-polyphenic insects exhibit DDP has rarely been elucidated. The diamondback moth (DBM), Plutella xylostella, one of the most destructive insect pests affecting cruciferous crops, is non phase-polyphenic. In this study, the resistance of DBM larvae to P. xylostella granulosis virus (Plxy GV) and their immune response to the virus when reared at densities of 1, 2, 5, 10, 15, and 20 larvae per Petri dish were investigated under laboratory conditions. Compared with larvae reared at lower densities, larvae reared at moderate density showed a significantly higher survival rate, but the survival rate significantly decreased with further increases in rearing density. Furthermore, the phenoloxidase, lysozyme and antibacterial activity and total hemocyte count in the hemolymph of the larvae, regardless of whether they were challenged with the virus, from different larval densities corresponded to the observed differences in resistance to Plxy GV. These results demonstrated that P. xylostella larvae exhibited DDP within a certain limited density. This study may help to elucidate the biocontrol effect of different density populations of P. xylostella by granulosis virus and guide improvements in future management strategy.

Larval density affects phenotype and surrounding bacterial community without altering gut microbiota in Drosophila melanogaster.

Larval crowding represents a complex stressful situation arising from inter-individual competition for time- and space-limited resources. The foraging of a large number of individuals may alter the chemical and bacterial composition of food and in turn affect individual's traits. Here we used Drosophila melanogaster to explore these assumptions. First, we used a wide larval density gradient to investigate the impact of crowding on phenotypical traits. We confirmed that high densities increased development time and pupation height, and decreased viability and body mass. Next, we measured concentrations of common metabolic wastes (ammonia, uric acid) and characterized bacterial communities, both in food and in larvae, for three contrasting larval densities (low, medium and high). Ammonia concentration increased in food from medium and high larval densities, but remained low in larvae regardless of the larval density. Uric acid did not accumulate in food but was detected in larvae. Surprisingly, bacterial composition remained stable in guts of larvae whatever their rearing density, although it drastically changed in the food. Overall, these results indicate that crowding deeply affects individuals, and also their abiotic and biotic surroundings. Environmental bacterial communities likely adapt to altered nutritional situations resulting from crowding, putatively acting as scavengers of larval metabolic wastes.

Characterization and Expression of Genes Encoding Superoxide Dismutase in the Oriental Armyworm, Mythimna separata (Lepidoptera: Noctuidae).

Superoxide dismutase (SOD) is an antioxidant metalloenzyme that catalyzes the dismutation of the superoxide anion O2- to O2 and H2O2. Many studies have focused on the role of SOD in response to abiotic stress, but its role during biotic stress, such as changes in organismal population density, has rarely been investigated. The oriental armyworm, Mythimna separata, is an economically important pest that exhibits phenotypic changes in response to population density. Solitary and gregarious phases occur at low and high population density, respectively. To examine the role of SODs in response to population density stress, we cloned two genes encoding SOD, MsCuZnSOD and MsMnSOD, and compared their expression in solitary and gregarious phases of M. separata. The MsCuZnSOD and MsMnSOD ORFs were 480 and 651 bp and encoded predicted protein products of 159 and 216 amino acids, respectively. The two SODs contained motifs that are typical of orthologous proteins. Real-time PCR indicated that the two SOD genes were expressed throughout developmental stages and were significantly upregulated in more mature stages of gregarious M. separata. Expression of the two SOD genes in various tissues of sixth-instar larvae was higher in gregarious versus solitary insects. Furthermore, expression of the SOD genes was significantly upregulated in response to crowding in solitary individuals, but suppressed in gregarious insects subjected to isolation. Collectively, these results suggest that population density may be key factor in the induction of SOD genes in M. separata.

Effect of larval density and substrate quality on the wing geometry of Stomoxys calcitrans L. (Diptera: Muscidae).

BACKGROUND: In insects, oviposition decisions may lead to egg deposition in substrates with different larval density and nutritional levels. Individuals developing in such substrates may present plasticity in their phenotype. Here, we investigated the effect of two factors related to oviposition decisions, namely larval density and substrate quality, on the wing size and wing shape of the stable fly, Stomoxys calcitrans L. (Diptera: Muscidae). METHODS: We reared S. calcitrans larvae at different densities (5, 15 and 25) and on different substrates (camel, cow, donkey and sheep dung). For each fly that emerged, we recorded body weight, and detached, slide-mounted and photographed the right wing. Next, we collected 15 landmarks on each photographed wing, and applied geometric morphometric analysis to assess variation in wing size and wing shape of S. calcitrans across the different larval densities and substrate types. RESULTS: We observed that wing size and wing shape of S. calcitrans were affected by larval density and the nature of the developmental substrate. Flies reared in a group of 5 had larger wing centroid size, wing length, wing width, wing area and wing loading compared with those reared in a group of 25. Also, flies developed in donkey and sheep dung had larger wing centroid size, wing length, wing width, wing area and wing loading in comparison with those grown in camel and cow dung. Canonical variate analysis followed by discriminant analysis revealed significant wing shape variation in S. calcitrans across the different densities and substrates. Wing size had a significant but weak positive effect on wing shape. CONCLUSIONS: This study demonstrates the high sensitivity of S. calcitrans wings to variation in larval density and developmental substrate, and that use of landmark-based geometric morphometric analysis could improve our understanding of how flies of veterinary importance respond to environmental variability.

The Role of Tyramine ß-Hydroxylase in Density Dependent Immunityof Oriental Armyworm (Mythmina separata) Larva.

High population density alters insect prophylactic immunity, with density-dependent prophylaxis (DDP) being reported in many polyphonic insects. However, the molecular mechanism for DDP remains unclear. In current study, the role of tyramine ß-hydroxylase (Tßh) in the immune response of M. separata larvae that were subject to different rearing densities conditions was investigated. The tyramine ß-hydroxylase activity of larvae from high density treatments (10 and 30 larvae per jar) was significantly higher than that of the larvae from low density treatments (one, two, and five larvae/jar). A tyramine ß-hydroxylase (designated MsTßh) containing a 1779 bp open reading frame was identified. Multiple sequence alignment and phylogenetic analysis indicated that MsTßh was orthologous to the Tßh that was found in other lepidopterans. Elevated MsTßh expression was observed in larvae under high density (10 larvae per jar). Silencing MsTßh expression by the injection of dsRNA in larvae from the high density treatment produced a 25.1% reduction in octopamine levels, while at the same time, there was a significant decrease in phenoloxidase (PO) and lysozyme activity, total haemocyte counts, and survival against Beauveria infection 56.6%, 88.5%, 82.0%, and 55.8%, respectively, when compared to control larvae. Our findings provide the first insights into how MsTßh mediates the octopamine level, which in turn modulates the immune response of larvae under different population densities.

Did microbial larviciding contribute to a reduction in malaria cases in eastern Botswana in 2012-2013?

Setting: Larviciding has potential as a component of integrated vector management for the reduction of malaria transmission in Botswana by complementing long-lasting insecticide nets and indoor residual sprays. Objective: To evaluate the susceptibility of local Anopheles to commonly used larvicides. Design: This field test of the efficacy of Bacillus thuringiensis subsp. israliensis vs. Anopheles was performed by measuring larval density before treatment and 24 h and 48 h after treatment in seven sites of Bobirwa district, eastern Botswana, in 2012 and 2013. Vector density and malaria cases were compared between Bobirwa and Ngami (northwestern Botswana), with no larviciding in the control arm. Results: Larviciding reduced larval density by 95% in Bobirwa in 2012, with two cases of malaria, while in 2013 larval density reduction was 81%, with 11 cases. Adult mosquito density was zero for both years in Robelela village (Bobirwa), compared to respectively four and 26 adult mosquitoes per room in Shorobe village (Ngami) in 2012 and 2013. There were no cases of malaria in Robelela in either year, but in Shorobe there were 20 and 70 cases, respectively, in 2012 and 2013. Conclusion: Larviciding can reduce the larval density of mosquitoes and reduce malaria transmission in Botswana. Large-scale, targeted implementation of larviciding in districts at high risk for malaria is recommended.

Contexte : Les opérations larvicides ont, en tant qu'élément de la gestion intégrée des vecteurs, le potentiel de réduire la transmission du paludisme au Botswana en complétant les moustiquaires imprégnées d'insecticide rémanent et la pulvérisation d'insecticide a effet rémanent. Objectif : Evaluer la sensibilité des Anophèles locaux aux larvicides généralement utilisés.Schéma : Le test de terrain de l'efficacité du Bacillus thuringiensis sous-espèce israeliensis a été réalisé vis-à-vis d'Anopheles en mesurant la densité larvaire avant traitement et 24 h et 48 h après traitement dans sept sites du district de Bobirwa (est du Botswana) en 2012 et 2013. La densité vectorielle et les cas de paludisme ont été comparés à Bobirwa et à Ngami (nord-ouest du Botswana), le district témoin sans opérations larvicides.Résultats : Les opérations larvicides ont réduit la densité larvaire de 95%, avec deux cas en 2012, tandis qu'en 2013 la réduction de la densité larvaire a été de 81%, avec 11 cas à Bobirwa. La densité de moustiques adultes a été de zéro pour les deux années dans le village de Robelela (Bobirwa), comparé à quatre et à 26 adultes par pièce dans le village de Shorobe (Ngami) en 2012 et 2013, respectivement. Il n'y a pas eu de cas de paludisme à Robelela au cours des deux années, mais respectivement 20 et 70 cas sont survenus à Shorobe en 2012 et 2013.Conclusion: Les opérations larvicides peuvent réduire la densité larvaire des moustiques et réduire la transmission du paludisme au Botswana. La mise en œuvre à grande échelle et ciblée d'opérations larvicides dans les districts à haut risque de paludisme est recommandée.

Marco de referencia: La aplicación de larvicidas podría convertirse en un componente del plan integrado de control de los vectores destinado a disminuir la transmisión del paludismo en Botswana, que complemente la utilización de mosquiteros impregnados de insecticidas de larga duración y la fumigación de interiores con insecticidas de acción residual.Objetivo: Evaluar la susceptibilidad de los anófeles locales a los larvicidas más utilizados.Método: Se llevó a cabo un ensayo sobre el terreno de la eficacia de Bacillus thuringiensis subespecie israliensis contra el género Anopheles, mediante la medición de la densidad larvaria antes del tratamiento y 24 h y 48 h después del mismo, en siete lugares del distrito de Bobirwa (Botswana oriental) en el 2012 y el 2013. Se compararon la densidad del vector y los casos de paludismo en Bobirwa y en Ngami (Botswana nororiental), que constituyó la rama testigo del estudio sin tratamiento larvicida.Resultados: La aplicación del larvicida en Bobirwa disminuyó un 95% la densidad larvaria y se presentaron dos casos de paludismo en el 2012, pero en el 2013 esta disminución fue del 81% y se presentaron 11 casos. La densidad de mosquitos adultos fue cero en ambos años en la localidad de Robelela (Bobirwa), en comparación con cuatro a 26 adultos por pieza en Shorobe (Ngami) en el 2012 y el 2013, respectivamente. No se presentaron casos de paludismo en Robelela en estos dos años, pero en Shorobe ocurrieron 20 casos en el 2012 y 70 en el 2013.Conclusión: La aplicación de larvicidas puede disminuir la densidad de mosquitos y la transmisión del paludismo en Botswana. Se recomienda una aplicación de larvicida dirigida en gran escala en los distritos con alto riesgo de transmisión de paludismo.

Hormesis-like effect of mild larval crowding on thermotolerance in Drosophila flies.

Crowding is a complex stress that can affect organisms' physiology, especially through decreased food quality and accessibility. Here, we evaluated the effect of larval density on several biological traits of Drosophila melanogaster An increasing gradient, from 1 to 1000 eggs per milliliter of food, was used to characterize life-history traits variations. Crowded conditions resulted in striking decreases of fresh mass (up to 6-fold) and viability, as well as delayed development. Next, we assessed heat and cold tolerance in L3 larvae reared at three selected larval densities: low (LD, 5 eggs ml-1), medium (MD, 60 eggs ml-1) and high (HD, 300 eggs ml-1). LT50 values of MD and, to a lesser extent, HD larvae were repeatedly higher than those from LD larvae, under both heat and cold stress. We investigated potential physiological correlates associated with this density-dependent thermotolerance shift. No marked pattern could be drawn from the expression of stress-related genes. However, a metabolomic analysis differentiated the metabotypes of the three density levels, with potential candidates associated with this clustering (e.g. glucose 6-phosphate, GABA, sugars and polyols). Under HD, signs of oxidative stress were noted but not confirmed at the transcriptional level. Finally, urea, a common metabolic waste, was found to accumulate substantially in food from MD and HD larvae. When supplemented in food, urea stimulated cold tolerance but reduced heat tolerance in LD larvae. This study highlights that larval crowding is an important environmental parameter that induces drastic consequences on flies' physiology and can affect thermotolerance in a density-specific way.

Modeling of Equivalent Effective Temperature and its possible incidence on larval density of Anopheles mosquitoes (Diptera: Culicidae) in Villa Clara province, Cuba / Modelación de la Temperatura Efectiva Equivalente y posible incidencia en la densidad larval de mosquitos Anopheles (Diptera: Culicidae) en la provincia Villa Clara, Cuba

AbstractThe wind chill or Equivalent Effective Temperature (EET) is the thermic sensation that a person feels when being exposed to a certain combination of temperature from the air, relative humidity and wind velocity. The objective of this investigation was directed to determine the possible incidence of the EET upon the larval density of Anopheles mosquitoes in Villa Clara province, Cuba. The Climatological data were compiled from the Yabú station in Santa Clara, and a total of 5 370 measurements were included in a database every three hours, using the aggregate function of the Statistical Package of Social Science software version 13 (SPSS), from January 1st, 2011 to September 30th, 2013. A long term forecast (1 year of advance) was made to obtain EET and the Anopheles larval density in the locality of Santo Domingo was modelled. These entomological data were taken at the same time but monthly, so the EET data were converted to monthly scale to be correlated with the monthly data of the larval density. The result was a 97.1 % of variance with a standard error (SE) of 3.57 °C for the model of the EET with a year of anticipation; therefore, the tendency in time was significant. The modeling also included the Anopheles larval density of mosquitoes in Santo Domingo, Villa Clara province, observing an increase of the EET, while the Anopheles mosquito larvae also increased. The most important variables in the model were the EET that were back in 1, 2, 3, 4, 7, 16, 24, 40 for the previous year; that is to say 2 920, 2 921, and so on, which explained a strong contagion among the data. EET correlation compared with itself in previous year was high; therefore, it may be used as a predictable variable. The anophelinic density in Santo Domingo explained the 66 % of the variance, with a SE of 0.66 larvae.m-2. The tendency of the Anopheles larval density was to diminish. In conclusion, EET has an important impact in larval density of Anopheles with EET increase associated with larval density decrease.

ResumenLa Temperatura Efectiva Equivalente (TEE) es la sensación térmica que siente una persona frente a una determinada combinación de temperatura del aire, humedad relativa y velocidad del viento. El objetivo de la investigación fue determinar la posible incidencia de la TEE sobre la densidad larval de mosquitos del género Anopheles en la provincia Villa Clara, Cuba. Una base de datos recogió medidas climatológicos cada tres horas durante el periodo comprendido entre el 1 de enero 2011 hasta el 30 de septiembre 2013, proveyendo un total de 5 370 medidas de la estación Yabú de Santa Clara. Se realizó un pronóstico a largo plazo (1 año) para obtener la TEE y se modeló la densidad larvaria total de mosquitos en la localidad de Santo Domingo. Los datos entomológicos fueron recogidos en el mismo lapso temporal pero con una periodicidad mensual, por lo que los datos de TEE fueron convertidos a escala mensual para poder ser empleados con los datos de densidad larvaria. Para la modelación se utilizó la Metodología de Regresión Objetiva Regresiva que explicó el 97.1 % de varianza con un error estándar (SE) de 3.57 ºC para el modelo de TEE con un año de antelación; la tendencia en el tiempo fue significativa al aumento. Además, al modelar la densidad larvaria anofelínica en el municipio Santo Domingo, se observó que a medida que aumenta la TEE, disminuye la densidad larval anofelínica. La variable más importante en el modelo fue la TEE regresada en 1, 2, 3, 4, 7, 16, 24, 40, pero del año anterior, es decir 2 920, 2 921, y así sucesivamente, explicando un contagio muy fuerte entre los datos. Ello fue debido a que la correlación de TEE con ella misma en años anteriores fue alta, por lo que puede ser utilizada como variable predictora. El modelo de densidad larval anofelínica en Santo Domingo explicó el 66 % de la varianza, con un SE de 0.06 larvas/m2. La tendencia de la DLA fue a la disminución. En conclusión, la TEE tuvo una incidencia directamente proporcional en la densidad larval anofelínica, ya que a medida que aumentaba este indicador, disminuyó la densidad larval anofelínica.

Optimization of Methodology for Rearing Spodoptera albula on Artificial Diet.

Advances in techniques for rearing insects on artificial diets are fundamental to solving issues of basic and applied entomology. In this study, we evaluated the development of Spodoptera albula (Walker) (Lepidoptera: Noctuidae) on three artificial diets used for other species of Lepidoptera, at three larval densities, and two densities of adult couples housed in oviposition cages of two sizes, with the aim of optimizing methodology for rearing S. albula in the laboratory. Biological parameters were recorded from S. albula, and a fitness index was calculated based on the larval survival and duration and weight of pupae. The total and daily oviposition was recorded using 5 or 10 adult couples of S. albula housed in two cage sizes. Concentrations of total nitrogen and protein in the tested diets were determined. Development of S. albula was completed in all artificial diets; however, the diet used for rearing Anticarsia gemmatalis (Hübner) larvae was the most suitable for S. albula, yielding intermediate development time and higher survival relative to the other diets. Individualization of larvae favored S. albula development by producing overall greater weights of larvae and pupae, higher survival rates, and longer adult longevity. Cage size and number of couples per cage did not influence S. albula fecundity in the experiment conditions. Spodoptera albula can be satisfactorily reared on the artificial diet used for A. gemmatalis, using one larva per tube, and either density of adults at any cage size. Additional amendments are needed in the rearing methodology to achieve optimal conditions for larval development to adulthood.