Laboratory Colony Research Articles

Morphological Aspects of Antennal Sensilla of the Rhodnius brethesi Matta, 1919 (Hemiptera: Reduviidae) from the Negro River, Amazon Region of Brazil.

Studies conducted in river Ererê located in the left margin of Negro River, municipality of Barcelos, state of Amazonas, have confirmed that Rhodnius brethesi has as its natural habitat the palm tree Leopoldinia piassaba. By scanning electron microscopy, sensillum type was studied on the antennae of R. brethesi. The specimens used come from the field and laboratory colony. No differences were observed between R. brethesi and other Triatominae studied. In the R. brethesi antennas, differences were observed only between the antennal segments and in the dorsal and ventral portions. Trichobothria sensilla show a difference with a lamellar base, suggesting that this conformation of the base of the sensilla is a synapomorphic feature of the genus. Another important observation is that, considering that R. brethesi is a specialist, infesting only one type of palm tree, trichoidea sensilla may be involved with plant-derived odorants. The knowledge of such functions could benefit the understanding of the likely biological role of these structures in chemical communication and also provide basic information for future studies of niche recognition, since this species of triatomine is only found in the L. piassaba palm.

Innate trypanolytic factors in triatomine hemolymph against Trypanosoma rangeli and T. cruzi: a comparative study in eight Chagas disease vectors

There is limited information about the innate immunity of triatomines against Trypanosoma rangeli, an infectious, non-pathogenic human parasite, and T. cruzi, the causative agent of Chagas’ disease. This study aimed at addressing this gap by studying the in vitro trypanolytic hemolymph activity from insects not infected by T. rangeli or T. cruzi. Eight triatomine species were examined including Rhodnius prolixus, R. robustus, R. colombiensis, R. pallescens, R. pictipes, Triatoma dimidiata, T. maculata, and Panstrongylus geniculatus. The hemolymph of R. prolixus and R. robustus laboratory colonies demonstrated strong trypanolytic activity during the first 14 hours of parasite incubation with 81% lysis for the T. rangeli C genotype, 90% for the E genotype, 95% for T. cruzi discrete taxonomic unit (DTU) TcII, 94% for TcV, 96% for TcVI, 94% for Tcbat, and 90% for Tcmarinkellei. No lysis activity was detected 14 or 24 hours after parasite incubation with the hemolymph of insects from R. pictipes R. pallescens, P. geniculatus, and T. maculata colonies. Identical results (absence of lysis) were observed using hemolymph from R. colombiensis and T. dimidiata laboratory colonies and specimens captured in sylvatic environments. The hemolymph lytic activity against recently obtained cultures (95% decrease in live parasite count) and long-term T. cruzi TcII ones (96% decrease) was similar. Lytic activity was similar in hemolymph from R. prolixus nymphs, males and females and insects fed on chicken or mouse blood. This is the first comparative study of the trypanolytic activity of hemolymph from different triatomine species against T. rangeli and T. cruzi genotypes. Although the chemical structures of such lysis factors and the mechanisms determining their expression have not been fully determined, their identification furthers our understanding of triatomines’ innate immunity and their role in Trypanosoma transmission.

Loss of pyrethroid resistance in newly established laboratory colonies of Aedes aegypti.

BackgroundResistance to pyrethroid insecticides in Aedes aegypti has become widespread after almost two decades of the frequent use of these pesticides to reduce arbovirus transmission. Despite this resistance, pyrethroids continue to be used because they are relatively inexpensive and have low human toxicity. Resistance management has been proposed as a way to retain the use of pyrethroids in natural populations. A key component of resistance management is the assumption that negative fitness is associated with resistance alleles such that resistance alleles will decline in frequency when the insecticides are removed. At least three studies in Ae. aegypti have demonstrated a decrease in pyrethroid resistance once the insecticide has been removed.Methods/Principal findingsThe present study aims to evaluate variation in the loss of pyrethroid resistance among newly established laboratory populations of Ae. aegypti from Mexico. Eight field collections were maintained for up to eight generations, and we recorded changes in the frequencies of the mutations at the V1,016I locus and at the F1,534C locus in the voltage-gated sodium channel gene (VGSC). I1,016 and C1,534 confer resistance. We also examined resistance ratios (RR) with type 1 and 2 pyrethroids.Conclusions/SignificanceWe demonstrate that, in general, the frequency of the Ae. aegypti pyrethroid-resistance alleles I1,016 and C1,534 decline when they are freed from pyrethroid pressure in the laboratory. However, the pattern of decline is strain dependent. In agreement with earlier studies, the RR was positively correlated with the frequencies of the resistance allele I1,016 and showed significant protection against permethrin, and deltamethrin, whereas F1,534C showed protection against permethrin but not against deltamethrin.

Optimization of Aedes albopictus rearing procedures for combined sterile insect techniques (SIT) and Wolbachia-based laboratory studies in Sri Lanka

The establishment of a laboratory colony is an essential first step for conduction of laboratory studies on the biology and control of Aedes albopictus mosquitoes. Therefore, with the objective of generating high quality mosquitoes as research material for utilization in ongoing vector control studies and to disseminate authenticated, high-quality Ae. albopictus mosquito rearing information to the research community, maintenance of an Ae. albopictus mosquito colony was initiated at the Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka. A self-mating colony was established from eggs of the F1 progeny of individuals collected as free-living larvae in Narahenpita (Western Province), Sri Lanka. The mean temperature of 28 °C (± 2 °C) and relative humidity of 80% (± 5%) was constantly maintained inside the insectary. Lighting was provided by fluorescent lights, regulated with 12:12 h continuous dark and light period. Pest insects were controlled manually. Mosquitoes were maintained on bovine blood provided via an artificial membrane feeding system and a continuous supply of 10% sugar supplements. Larvae were maintained in deoxygenate water and fed with International Atomic Energy Agency (IAEA) recommended diet of tuna meal, bovine liver powder, and brewery yeast in a ratio of 37.5:27:10.5 in 1 L. Data on fecundity, fertility, larvae death, pupation, adult emergence, adult mosquito longevity were recorded. Adhering to bio-safety, all discarded materials were boiled thoroughly and incinerated if required. This report on the establishment and maintenance of a laboratory colony of Ae. albopictus will be of value for identifying the critical requirements essential under artificial conditions.

Insecticidal and Cholinesterase Activity of Dichloromethane Extracts of Tithonia diversifolia on Atta cephalotes Worker Ants (Formicidae: Myrmicinae).

Leaf-cutter ants are agricultural and urban pests that defy chemical control methods. Laboratory and field studies have revealed repellent and insecticidal activity by the extracts of Tithonia diversifolia (Asteraceae), known as Mexican sunflower, as a promising alternative for the control of the leaf-cutter ant Atta cephalotes. This study evaluated the effects of different extracts (non-polar and polar) of T. diversifolia dry leaves on worker ants from laboratory colonies of A. cephalotes through ingestion and contact. In addition, the biological activity of the extracts as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was evaluated. A dichloromethane extract at 1000 ppm presented the highest insecticidal activity through ingestion, causing 70% and 90% worker ant mortality after five and seven days of treatment, respectively. The acetylcholinesterase inhibition values showed that the dichloromethane presented the best AChE concentration of inhibition (IC50) at 73.9 ± 11.06 μg/mL, compared to its fractions, which demonstrates that its activity is potentiated when the crude extract is used. Our results can be attributed to the existence of terpenes and sesquiterpene lactones, which are likely inhibitors of AChE, in T. diversifolia.

Wolbachia Endosymbiont of the Horn Fly (Haematobia irritans irritans): a Supergroup A Strain with Multiple Horizontally Acquired Cytoplasmic Incompatibility Genes.

The horn fly, Haematobia irritansirritans, is a hematophagous parasite of livestock distributed throughout Europe, Africa, Asia, and the Americas. Welfare losses on livestock due to horn fly infestation are estimated to cost between $1 billion and $2.5 billion (U.S. dollars) annually in North America and Brazil. The endosymbiotic bacterium Wolbachia pipientis is a maternally inherited manipulator of reproductive biology in arthropods and naturally infects laboratory colonies of horn flies from Kerrville, TX, and Alberta, Canada, but it has also been identified in wild-caught samples from Canada, the United States, Mexico, and Hungary. Reassembly of PacBio long-read and Illumina genomic DNA libraries from the Kerrville H. i. irritans genome project allowed for a complete and circularized 1.3-Mb Wolbachia genome (wIrr). Annotation of wIrr yielded 1,249 coding genes, 34 tRNAs, 3 rRNAs, and 5 prophage regions. Comparative genomics and whole-genome Bayesian evolutionary analysis of wIrr compared to published Wolbachia genomes suggested that wIrr is most closely related to and diverged from Wolbachia supergroup A strains known to infect Drosophila spp. Whole-genome synteny analyses between wIrr and closely related genomes indicated that wIrr has undergone significant genome rearrangements while maintaining high nucleotide identity. Comparative analysis of the cytoplasmic incompatibility (CI) genes of wIrr suggested two phylogenetically distinct CI loci and acquisition of another cifB homolog from phylogenetically distant supergroup A Wolbachia strains, suggesting horizontal acquisition of these loci. The wIrr genome provides a resource for future examination of the impact Wolbachia may have in both biocontrol and potential insecticide resistance of horn flies.IMPORTANCE Horn flies, Haematobia irritans irritans, are obligate hematophagous parasites of cattle having significant effects on production and animal welfare. Control of horn flies mainly relies on the use of insecticides, but issues with resistance have increased interest in development of alternative means of control. Wolbachia pipientis is an endosymbiont bacterium known to have a range of effects on host reproduction, such as induction of cytoplasmic incompatibility, feminization, male killing, and also impacts vector transmission. These characteristics of Wolbachia have been exploited in biological control approaches for a range of insect pests. Here we report the assembly and annotation of the circular genome of the Wolbachia strain of the Kerrville, TX, horn fly (wIrr). Annotation of wIrr suggests its unique features, including the horizontal acquisition of additional transcriptionally active cytoplasmic incompatibility loci. This study provides the foundation for future studies of Wolbachia-induced biological effects for control of horn flies.

Uneven substrates constrain walking speed in ants through modulation of stride frequency more than stride length.

Natural terrain is rarely flat. Substrate irregularities challenge walking animals to maintain stability, yet we lack quantitative assessments of walking performance and limb kinematics on naturally uneven ground. We measured how continually uneven 3D-printed substrates influence walking performance of Argentine ants by measuring walking speeds of workers from laboratory colonies and by testing colony-wide substrate preference in field experiments. Tracking limb motion in over 8000 videos, we used statistical models that associate walking speed with limb kinematic parameters to compare movement over flat versus uneven ground of controlled dimensions. We found that uneven substrates reduced preferred and peak walking speeds by up to 42% and that ants actively avoided uneven terrain in the field. Observed speed reductions were modulated primarily by shifts in stride frequency instead of stride length (flat R2: 0.91 versus 0.50), a pattern consistent across flat and uneven substrates. Mixed effect modelling revealed that walking speeds on uneven substrates were accurately predicted based on flat walking data for over 89% of strides. Those strides that were not well modelled primarily involved limb perturbations, including missteps, active foot repositioning and slipping. Together these findings relate kinematic mechanisms underlying walking performance on uneven terrain to ecologically relevant measures under field conditions.

Experimental infections and co-infections with Leishmania braziliensis and Leishmania infantum in two sand fly species, Lutzomyia migonei and Lutzomyia longipalpis

Leishmaniases are neglected tropical diseases and Leishmania (Leishmania) infantum and Leishmania (Viannia) braziliensis are the most important causative agents of leishmaniases in the New World. These two parasite species may co-circulate in a given endemic area but their interactions in the vector have not been studied yet. We conducted experimental infections using both single infections and co-infections to compare the development of L. (L.) infantum (OGVL/mCherry) and L. (V.) braziliensis (XB29/GFP) in Lutzomyia longipalpis and Lutzomyia migonei. Parasite labelling by different fluorescein proteins enabled studying interspecific competition and localization of different parasite species during co-infections. Both Leishmania species completed their life cycle, producing infective forms in both sand fly species studied. The same happens in the co infections, demonstrating that the two parasites conclude their development and do not compete with each other. However, infections produced by L. (L.) infantum reached higher rates and grew more vigorously, as compared to L. (V.) braziliensis. In late-stage infections, L. (L.) infantum was present in all midgut regions, showing typical suprapylarian type of development, whereas L. (V.) braziliensis was concentrated in the hindgut and the abdominal midgut (peripylarian development). We concluded that both Lu. migonei and Lu. longipalpis are equally susceptible vectors for L. (L.) infantum, in laboratory colonies. In relation to L. (V.) braziliensis, Lu. migonei appears to be more susceptible to this parasite than Lu. longipalpis.

The stress of being alone: Removal from the colony, but not social subordination, increases fecal cortisol metabolite levels in eusocial naked mole-rats

In many social species, hierarchical status within the group is associated with differences in basal adrenocortical activity. We examined this relationship in naked mole-rats (Heterocephalus glaber), eusocial rodents with arguably the most extreme social hierarchies of all mammals. This species lives in colonies where breeding is restricted to one socially dominant ‘queen’ and her male consorts, and all other individuals are reproductively suppressed ‘subordinates’. The relationship between cortisol and social status in naked mole-rats has not fully been elucidated, as prior results on this topic have been contradictory. We used non-invasive feces sampling to measure baseline cortisol levels in eight laboratory colonies of naked mole-rats, to either replicate or reject rank differences. First, we successfully validated an assay to measure fecal cortisol metabolites (FCMs). Removal from the colony for the validation experiment, either alone or with an opposite sex conspecific, induced prolonged elevation of FCM levels on a scale of days to weeks. This increase in cortisol did not prevent the removed animals from sexually maturing. In colony-housed animals, we found no relationship between rank in the social hierarchy and FCM levels. Further, queens, breeding males, and reproductively suppressed subordinates all had equivalent FCM levels. We conclude that this species shows little evidence of the ‘stress of dominance’ or ‘stress of subordination’ and that reproductive suppression in naked mole-rats is not driven by elevated cortisol levels.

Member species of the Anopheles gambiae complex can be misidentified as Anopheles leesoni

BackgroundAccurate Anopheles species identification is key for effective malaria vector control. Identification primarily depends on morphological analysis of field samples as well as molecular species-specific identifications. During an intra-laboratory assessment (proficiency testing) of the Anopheles funestus group multiplex PCR assay, it was noted that Anopheles arabiensis can be misidentified as Anopheles leesoni, a zoophilic member of the An. funestus group. The aim of this project was, therefore, to ascertain whether other members of the Anopheles gambiae complex can also be misidentified as An. leesoni when using the standard An. funestus multiplex PCR.MethodsThe An. funestus multiplex PCR was used to amplify DNA from An. gambiae complex specimens. These included specimens from the laboratory colonies and field samples from the Democratic Republic of Congo. Amplified DNA from these specimens, using the universal (UV) and An. leesoni species-specific primers (LEES), were sequence analysed. Additionally, An. leesoni DNA was processed through the diagnostic An. gambiae multiplex PCR to determine if this species can be misidentified as a member of the An. gambiae complex.ResultsLaboratory-colonized as well as field-collected samples of An. arabiensis, An. gambiae, Anopheles merus, Anopheles quadriannulatus, Anopheles coluzzii as well as Anopheles moucheti produced an amplicon of similar size to that of An. leesoni when using an An. funestus multiplex PCR. Sequence analysis confirmed that the UV and LEES primers amplify a segment of the ITS2 region of members of the An. gambiae complex and An. moucheti. The reverse was not true, i.e. the An. gambiae multiplex PCR does not amplify DNA from An. leesoni.ConclusionThis investigation shows that An. arabiensis, An. gambiae, An. merus, An. quadriannulatus, An. coluzzii and An. moucheti can be misidentified as An. leesoni when using An. funestus multiplex PCR. This shows the importance of identifying specimens using standard morphological dichotomous keys as far as possible prior to the use of appropriate PCR-based identification methods. Should there be doubt concerning field-collected specimens molecularly identified as An. leesoni, the An. gambiae multiplex PCR and sequencing of the internal transcribed spacer 2 (ITS2) can be used to eliminate false identifications.

Exposure of malaria vector larval habitats to domestic pollutants escalate insecticides resistance: experimental proof

Poor domestic waste management in unplanned urban areas have resulted into formation of polluted potential malaria vector larval habitats. Most of these larval habitats are contaminated with domestic pollutants such as hydrogen peroxide and detergents. The present study was designed to evaluate the susceptibility status of Anopheles gambie and Anopheles arabiensis to commonly used insecticides after being exposed to hydrogen peroxide and soap detergents in northern Tanzania. Anopheles gambiae s.s larvae were collected from the laboratory colony whereas those for An. arabiensis were sampled from the rice field in lower Moshi. The larvae were reared in two arms, larval habitats in arm one had water containing soaps while in arm two, the larval habitats contained hydrogen peroxide at different concentrations (0.001%, 0.0001%,0.00001% and 0.000001%). Emerging adult’s female of the two Anopheles species from the two larval habitats were exposed to 0.75% and 0.05% of permethrin and deltamethrin WHO Insecticides impregnated standard papers. Knockdown rates were observed for 1 h and mortality rates within 24 h after exposure. Regression analysis was performed using probit analysis to establish knockdown rates and lethal concentration for 50% and 95%. The wild and laboratory anopheles populations reared in detergent or hydrogen peroxide in different concentrations showed reduced susceptibility to permethrin and deltamethrin. The median knockdown time for 50% of the population (KDT50) ranged between 13.52 min (10.98–19.74) to 30.67 min (28.53–34.34) while the KDT95 raised between 32.01 min (29.47–38.23) to 111.84 min (109.70–115.54) for those reared in detergent and exposed to deltamethrin and permethrin. For those reared in hydrogen peroxide and exposed to deltamethrin and permethrin, KDT50 ranged between 8.24 min (6.92–13.51) to 24.28 min(22.70–27.46) while KDT95 ranged between 25.94 min(24.62–31.21) to 118.68 min(117.10–121.86). Mortality of wild population exposed to standard insectides treated papers after reared in detergent ranged between 40 to 95.3% while for those reared in hydrogen peroxide ranged between 67 to 95.3%. The findings of this study demonstrate that domestic waste pollutants such as hydrogen peroxide and soaps are partly associated with reduced susceptibility to insecticides among potential malaria vectors.

Fitness costs associated with thiamethoxam and imidacloprid resistance in three field populations of Diaphorina citri (Hemiptera: Liviidae) from Florida.

Insecticide resistance is an increasing problem in citrus production. The Asian citrus psyllid, Diaphornia citri Kuwayama, is recognized as one of the most important citrus pests worldwide and it has developed resistance in areas where insecticides have been overused. The development of insecticide resistance is often associated with fitness costs that only become apparent in the absence of selection pressure. Here, the fitness costs associated with resistance to thiamethoxam and imidacloprid were investigated in three agricultural populations of D. citri as compared with susceptible laboratory colonies. Results showed that all field populations had greater resistance than laboratory susceptible colonies. For both thiamethoxam and imidacloprid, a Candidatus Liberibacter asiaticus-positive (CLas+) colony was more susceptible than the CLas- colony. Resistance ratios ranged from 7.65-16.11 for imidacloprid and 26.79-49.09 for thiamethoxam in field populations as compared with a susceptible, CLas- laboratory strain. Among three resistant field populations, a significantly reduced net reproductive rate and finite rate of population increase were observed in a population from Lake Wales, FL as compared to both susceptible strains. The fecundity of field populations from Lake Wales, FL was statistically lower than both laboratory susceptible populations. Certain changes in morphological characteristics were observed among resistant, as compared, with susceptible strains. Our data suggest fitness disadvantages associated with insecticide resistance in D. citri are related to both development and reproduction. The lower fitness of D. citri populations that exhibit resistance to neonicotinoid insecticides should promote recovery of sensitivity when those populations are no longer exposed to thiamethoxam and/or imidacloprid in the field. The results are congruent with a strategy of insecticide rotation for resistance management.

The effects of host plant species on adult oviposition and larval performance of the aphid predator Aphidoletes aphidimyza

1. Although preference–performance relationships in insects are typically studied in a bi‐trophic context, it is well known that host plants can affect both the preference and performance of natural enemies of herbivorous insects.2. This study presents evidence from field and laboratory studies that two species of milkweeds, the putatively less defended Asclepias incarnata and the putatively more defended Asclepias syriaca, differentially affect adult oviposition and larval performance in Aphidoletes aphidimyza, an aphid‐feeding predatory midge, independent of aphid density.3. Host plant species affected predatory fly larvae abundance by a factor of 50 in the field and a factor of 8 in the laboratory. Larval and adult emergence rates in our laboratory studies provided strong evidence for reduced performance on A. syriaca. Oviposition in choice and no‐choice settings provided some evidence for preference for A. incarnata, and a potentially suppressive effect of A. syriaca.4. The results provide limited support for the hypothesis that natural selection can lead to positive correlations between adult oviposition preferences and larval performance upon various food sources, even when predatory insects oviposit onto host plants of their herbivorous prey.5. Preference and performance are not perfectly aligned in this system, however, because ovipositing females do not reject A. syriaca entirely. Potential explanations for mismatches between preference and performance in this system include the neural constraints associated with being a generalist, adaptive time‐limited foraging strategies, and unique evolutionary histories of laboratory colonies compared with wild insects.

Sand Fly Colony Crash Tentatively Attributed to Nematode Infestation.

Maintenance of laboratory colonies of insects and other arthropod pests offers significant research advantages. The availability, age, sex, housing conditions, nutrition, and relative uniformity over time of biological material for research facilitate comparison of results between experiments that would otherwise be difficult or impossible. A laboratory research colony of Phlebotomus papatasi (Scopoli), old world sand flies, was maintained with high-colony productivity for a number of years, but within a relatively short (4-6 mo) time period, colony productivity declined from over 10,000 flies per week to less than 100 per week. Mites and nematodes were both visible in the larval medium; however, the mites had been present throughout high productivity periods; therefore, it seemed reasonable to investigate the nematodes. PCR amplification of 18S rRNA yielded a clean cDNA sequence identified by BLAST search as Procephalobus sp. 1 WB-2008 (Rhabditida: Panagrolaimidae) small subunit ribosomal RNA gene, GenBank EU543179.1, with 475/477 nucleotide identities. Nematode samples were collected and identified as Tricephalobus steineri, (Andrássy, 1952) Rühm, 1956 (Rhabditida: Panagrolaimidae) based on morphological characteristics of the esophagus and the male copulatory apparatus. Mites (Tyrophagus putrescentiae [Acariformes: Acaridae]) may have played an additional predatory role in the loss of sand fly colony productivity. We hypothesized that the origin of the nematode infestation was rabbit dung from a local rabbitry used in preparation of the larval medium. Colony productivity was fully restored within 3 mo (two sand fly generational periods) by replacement of the rabbit dung from a clean source for use to prepare sand fly larval medium.

Inferring Termite Colony Size Using Wood Consumption in Subterranean Termites (Blattodea: Rhinotermitidae) in Laboratory-Rearing Conditions.

Termite colony size can influence its foraging activity, reproductive maturity, and, for pest species, potential for structural damage. Estimating colony size of subterranean termite species in field conditions has been challenging owing to their extensive foraging territory and their cryptic nesting habit and has primarily relied on mark-recapture methods. With laboratory-reared colonies in individual containers, determining colony size can be achieved by processing all termites from the nest material, which can be labor intensive and partially destructive. However, with the recent rise in the need of large laboratory colonies for use in colony-wide experimental protocols, there was an imperative to develop a procedure to estimate initial colony sizes without imposing a major stress on colonies before an experiment. In this study, the average daily wood consumption of whole colonies was used to infer the colony size of two Coptotermes, Wasmann (Blattodea: Rhinotermitidae) species and their hybrids in laboratory-rearing conditions. Correlations between the daily wood consumption and several demographic variables within colonies were established. Linear models varied across all species mating types with R2 values greater than 0.8 for all demographic variables. For colonies from all mating combinations, Pearson's correlation coefficient values were greater than 0.94 between their daily wood consumption and both the number of workers and total number of termites, and greater than 0.91 between daily wood consumption and colony mass. Therefore, in colonies with fixed laboratory conditions, their average daily wood consumption determination, which is nondestructive on colonies, can be used to infer colony size of subterranean termites.

The Behavioral Response of Blattella germanica (Blattodea: Ectobiidae) Exposed to DEET Varies Throughout Its Life Cycle.

The German cockroach, Blattella germanica L., is a hemimetabolous insect pest of economical and medical importance. N,N-diethyl-3-methylbenzamide (DEET) is an insect repellent whose effect on this species has received very little attention. The objective of this work was to determine whether the behavioral response of B. germanica to DEET varies along its life cycle. DEET repellence was assessed in small, medium, and large nymphs, and in adults of both sexes, all originated from the same laboratory colony (CIPEIN). The experimental arena consisted in a piece of filter paper treated with repellent on one half (195 µg/cm2) and solvent alone on the other half. A cockroach was placed on the filter paper, and its behavior was filmed. An image analyzer was used to quantify how long the insect spent on each side of the paper. As a control, a cockroach was exposed to a piece of filter paper treated with solvent (acetone) alone. Each assay was repeated independently six times. Distribution coefficient (DC) values were calculated, a parameter that ranges between 0 (attraction) and 1 (repellence). Small nymphs were more sensitive to DEET (mean DC = 0.93). The mean DC values of the other groups varied between 0.62 (medium nymphs) and 0.71 (male adults). The group of medium nymphs was the only one whose behavior was not significantly altered by exposure to DEET. The results show the importance of assessing insect repellents at different stages of the insect's life cycle in order to obtain a complete panorama of its effect.

The ghost of temperature past: interactive effects of previous and current thermal conditions on gene expression in Manduca sexta.

High temperatures can negatively impact the performance and survival of organisms, particularly ectotherms. While an organism's response to high temperature stress clearly depends on current thermal conditions, its response may also be affected by the temporal pattern and duration of past temperature exposures. We used RNA sequencing of Manduca sexta larvae fat body tissue to evaluate how diurnal temperature fluctuations during development affected gene expression both independently and in conjunction with subsequent heat stress. Additionally, we compared gene expression between two M. sexta populations, a lab colony and a genetically related field population that have been separated for >300 generations and differ in their thermal sensitivities. Lab-adapted larvae were predicted to show increased expression responses to both single and repeated thermal stress, whereas recurrent exposure could decrease later stress responses for field individuals. We found large differences in overall gene expression patterns between the two populations across all treatments, as well as population-specific transcriptomic responses to temperature; more differentially expressed genes were upregulated in the field compared with lab larvae. Developmental temperature fluctuations alone had minimal effects on long-term gene expression patterns, with the exception of a somewhat elevated stress response in the lab population. Fluctuating rearing conditions did alter gene expression during exposure to later heat stress, but this effect depended on both the population and the particular temperature conditions. This study contributes to increased knowledge of molecular mechanisms underlying physiological responses of organisms to temperature fluctuations, which is needed for the development of more accurate thermal performance models.

The effect of endophytic colonization of wheat plants by the fungus Beauveria bassiana on the development of the nymphs of the migratory and desert locusts

The experiments were aimed at evaluation of the effect of feeding with wheat colonized by the fungus Beauveria bassiana on the development of locusts. The seeds were treated with fungal conidia and 90 days after sowing, the plant parts were fed to II instar nymphs of laboratory colonies of migratory and desert locusts within the period of 18 days. The mortality levels between experimental and control (fed with untreated wheat) groups were not significantly different though a tendency of an increase in variants with contaminated fodder was observed. Two weeks after the beginning of the bioassay, insect mortality was 20-30% and 15% in control. As many as 50% of migratory locust and 40% of desert locust cadavers were covered by a fungal mycelium. Sequencing of two diagnostic DNA loci has confirmed attribution of the reisolate to the genotype of the initial Beauveria bassiana strain BBK-1. It can be therefore concluded that the endophyte fungus is capable of causing classical pathogenesis of mycosis in locust nymphs. Moreover, the nymphal development was retarded by feeding with the fungus-colonized plants.

Development of Ctenocephalides felis felis (Siphonaptera: Pulicidae) in different substrates for maintenance under laboratory conditions.

The aim of this study was to evaluate the efficiency of different substrates for larval development of Ctenocephalides felis felis during its biological cycle. Eight hundred eggs of C. felis felis from a flea maintenance colony were used. Different diets were formulated, in which the main substrates were meat flour, powdered milk, sugar, lyophilized bovine blood, tick metabolites and lyophilized egg. The flea eggs were placed in test tubes (10 per tube) and approximately 2 g of the diet to be tested was added to each tube. There were 10 replicates for each substrate. After 28 days, each tube was evaluated individually for the presence of pupae and emerged adults. The following percentages of the larvae completed the cycle to the adult stage: 67% in diets containing tick metabolites; 55%, meat flour; 39%, dehydrated bovine blood; 14%, powdered milk; and less than 1% in diets containing sugar, lyophilized bovine blood, lyophilized egg or wheat bran. It was concluded that among the diets tested, the one constituted by tick metabolites as the substrate was shown to be the most satisfactory for maintaining a laboratory colony of C. felis felis, followed by the one containing meat flour.

Description of an automatic copulation induction system used to establish a free-mating laboratory colony of Nyssorhynchus deaneorum from Brazil.

BACKGROUND Nyssorhynchus deaneorum is a potential malaria vector because it has been shown to be competent to transmit Plasmodium vivax and Plasmodium falciparum, and because it exhibits antropophilic and endophilic behaviors in some regions of the Amazon. This profile makes Ny. deaneorum a useful mosquito for experiments that model Plasmodium-vector interactions in the Amazon.OBJECTIVE Herein we describe how a free-mating colony of Ny. deaneorum has been established using an automated light stimulation system.METHODS Mosquitoes were captured in São Francisco do Guaporé, Rondônia. The F1 generation was reared until adult emergence at which point copulation was induced using an automatic copulation induction system (ACIS).FINDINGS After four generations, natural mating and oviposition began to occur without light stimulation. The number of pupae and adult mosquitoes increased from the F5 to F10 generations. The new Ny. deaneorum colony exhibited susceptibility to P. vivax. MAIN CONCLUSIONS Automated light stimulation is an effective method for establishing an Ny. deaneorum colony under laboratory conditions as it produces enough adults to create a stenogamic colony. The establishment of a stable, P. vivax-susceptible colony of Ny. deaneorum makes it possible to model parasite-vector interactions and to test novel drug therapies that target parasite development in mosquitoes.