What does not kill it does not always make it stronger: High temperatures in pyriproxyfen treatments produce Aedes aegypti adults with reduced longevity and smaller females

Aedes aegypti control in Brazil comprises integrated actions, in which larvicide application is a supplementary measure. Despite the importance of analyzing the effects of temperature on the efficiency of larvicides to control mosquito populations, there is still a lack of information regarding the sublethal effect of larvicides. We hypothesized that mosquitoes which survived pyriproxyfen exposure, during their immature development, have small body sizes and live less than mosquitoes that were not exposed to this larvicide. We investigated the sublethal effects of five different pyriproxyfen concentrations (0.0001; 0.001; 0.01; 0.1 and 1 mg.L⁻¹), under three different temperatures (20, 25 and 30 °C). As we increased the larvicide concentration, less larvae survived and developed into adults. However, at 30 °C the survival was higher than at 25 °C and 20 °C comparing the concentrations of 0.001 mg.L⁻¹ and 0.01 mg.L⁻¹ (10% and 16% high, respectively). Mosquito survivors to pyriproxyfen exposure, in all thermal conditions, had shorter adult life spans than mosquitoes not exposed to pyriproxyfen during the larval stage. The females exposed at 30 °C showed smaller wings than females from experiments at 20 °C. These findings provide evidence that the biological parameters of the adult lifespan and wing centroid size are impaired due to larval exposure to pyriproxyfen, even at high temperatures. Reduced longevity and small wing size are fitness costs to survive this larvicide exposure. These findings provide support to assess resistance development to larvicides through future generations and contribute to the discussion on improving the rational application of larvicides.     

Disruption of insect isoprenoid biosynthesis with pyridinium bisphosphonates

Farnesyl diphosphate synthase (FPPS) catalyzes the condensation of the non-allylic diphosphate, isopentenyl diphosphate (IPP; C₅), with the allylic diphosphate primer dimethylallyl diphosphate (DMAPP; C₅) to generate the C₁₅ prenyl chain (FPP) used for protein prenylation as well as sterol and terpene biosynthesis. Here, we designed and prepared a series of pyridinium bisphosphonate (PyrBP) compounds, with the aim of selectively inhibiting FPPS of the lepidopteran insect order. FPPSs of Drosophila melanogaster and the spruce budworm, Choristoneura fumiferana, were inhibited by several PyrBPs, and as hypothesized, larger bisphosphonates were more selective for the lepidopteran protein and completely inactive towards dipteran and vertebrate FPPSs. Cell growth of a D. melanogaster cell line was adversely affected by exposure to PyrPBs that were strongly inhibitory to insect FPPS, although their effect was less pronounced than that observed upon exposure to the electron transport disrupter, chlorfenapyr. To assess the impact of PyrBPs on lepidopteran insect growth and development, we performed feeding and topical studies, using the tobacco hornworm, Manduca sexta, as our insect model. The free acid form of a PyrBP and a known bisphosphonate inhibitor of vertebrate FPPS, alendronate, had little to no effect on larval M. sexta; however, the topical application of more lipophilic ester PyrBPs caused decreased growth, incomplete larval molting, cuticle darkening at the site of application, and for those insects that survived, the formation of larval–pupal hybrids. To gain a better understanding of the structural differences that produce selective lepidopteran FPPS inhibition, homology models of C. fumiferana and D. melanogaster FPPS (CfFPPS2, and DmFPPS) were prepared. Docking of substrates and PyrBPs demonstrates that differences at the −3 and −4 positions relative to the first aspartate rich motif (FARM) are important factors in the ability of the lepidopteran enzyme to produce homologous isoprenoid structure and to be selectively inhibited by larger PyrBPs.     

The Insect Growth Regulator Pyriproxyfen Terminates Egg Diapause in the Asian Tiger Mosquito,Aedes albopictus

The Asian tiger mosquito, Aedes albopictus, is a highly invasive mosquito species that transmits chikungunya and dengue. This species overwinters as diapausing eggs in temperate climates. Early diapause termination may be a beneficial strategy for winter mosquito control; however, a mechanism to terminate the diapause process using chemicals is not known. We tested the hypothesis that a hormonal imbalance caused by the administration of juvenile hormone analog would terminate egg diapause in A. albopictus. We tested the insect growth regulator pyriproxyfen on all developmental stages to identify a susceptible stage for diapause termination. We found that pyriproxyfen treatment of mosquito eggs terminated embryonic diapause. The highest rates of diapause termination were recorded in newly deposited (78.9%) and fully embryonated (74.7%) eggs at 0.1 and 1 ppm, respectively. Hatching was completed earlier in newly deposited eggs (25–30 days) compared to fully embryonated eggs (71–80 days). The combined mortality from premature diapause termination and ovicidal activity was 98.2% in newly deposited and >98.9% in fully embryonated eggs at 1 ppm. The control diapause eggs did not hatch under diapausing conditions. Pyriproxyfen exposure to larvae, pupae and adults did not prevent the females from ovipositing diapausing eggs. There was no effect of pyriproxyfen on diapausing egg embryonic developmental time. We also observed mortality in diapausing eggs laid by females exposed to pyriproxyfen immediately after blood feeding. There was no mortality in eggs laid by females that survived larval and pupal exposures. In conclusion, diapausing eggs were the more susceptible to pyriproxyfen diapause termination compared to other life stages. This is the first report of diapause termination in A. albopictus with a juvenile hormone analog. We believe our findings will be useful in developing a new control strategy against overwintering mosquito populations.     

Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth,Manduca sexta

Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects.     

Egg exposure to pyriproxyfen in the tomato leaf miner Tuta absoluta: ovicidal activity or behavioural‐modulated hatching mortality?

Insecticide susceptibility varies with the insect life stage and although the egg stage is sometimes perceived as the most vulnerable, it is a difficult target for insecticide application and little studied as such. Egg susceptibility to insecticides is generally considered important for insect growth regulators and insect pests of reduced mobility like leaf miners because of their placement on the host plant part targeted by the insecticidal application. The egg exposure to the juvenile hormone mimic pyriproxyfen was studied here in the tomato leaf miner Tuta absoluta (Lepidoptera: Gelechiidae), a key tomato insect pest, aiming to assess if mortality is achieved due to insecticide ovicidal activity or due to behavioural impairment of the larvae preventing egg hatching. Survival analysis of treated tomato leaves and/or eggs indicated the importance of egg exposure to pyriproxyfen leading to a significant decrease in survival with a nearly 50% reduction in survival time, which peaked at the first instar. Ovicidal activity of pyriproxyfen in the tomato leaf miner was negligible, but image recording and behaviour analysis indicated behavioural impairment of larval activity compromising hatching and leaf mining by early instar larvae leading to high mortality at this stage (ca 50%). The likely causes and management implications of such findings are discussed.     

Composition of the Spruce Budworm (Choristoneura fumiferana) Midgut Microbiota as Affected by Rearing Conditions

The eastern spruce budworm (Choristoneura fumiferana) is one of the most destructive forest insect pests in Canada. Little is known about its intestinal microbiota, which could play a role in digestion, immune protection, communication and/or development. The present study was designed to provide a first characterization of the effects of rearing conditions on the taxonomic diversity and structure of the C. fumiferana midgut microbiota, using a culture-independent approach. Three diets and insect sources were examined: larvae from a laboratory colony reared on a synthetic diet and field-collected larvae reared on balsam fir or black spruce foliage. Bacterial DNA from the larval midguts was extracted to amplify and sequence the V6-V8 region of the 16S rRNA gene, using the Roche 454 GS-FLX technology. Our results showed a dominance of Proteobacteria, mainly Pseudomonas spp., in the spruce budworm midgut, irrespective of treatment group. Taxonomic diversity of the midgut microbiota was greater for larvae reared on synthetic diet than for those collected and reared on host plants, a difference that is likely accounted for by several factors. A greater proportion of bacteria from the phylum Bacteroidetes in insects fed artificial diet constituted the main difference between this group and those reared on foliage; within the phylum Proteobacteria, the presence of the genus Bradyrhizobium was also unique to insects reared on artificial diet. Strikingly, a Bray-Curtis analysis showed important differences in microbial diversity among the treatment groups, pointing to the importance of diet and environment in defining the spruce budworm midgut microbiota.     

The Genetic,Morphological, and Physiological Characterization of a Dark Larval Cuticle Mutation in the Butterfly,Bicyclus anynana

Studies on insect melanism have greatly contributed to our understanding of natural selection and the ultimate factors influencing the evolution of darkly pigmented phenotypes. Research on several species of melanic lepidopteran larvae have found that low levels of circulating juvenile hormone (JH) titers are associated with a melanic phenotype, suggesting that genetic changes in the JH biosynthetic pathway give rise to increased deposition of melanin granules in the cuticle in this group. But does melanism arise through different molecular mechanisms in different species? The present study reports on a Bicyclus anynana (Lepidoptera: Nymphalidae) dark larvae single locus mutation, in which larvae exhibit a darker cuticle relative to wild type. Unlike other lepidopteran melanic larvae mutations, this one is autosomal recessive and does not appear to involve a deficiency in JH titers. Unlike JH deficiency mutants, dark larvae mutants display similar growth rates and sexual behaviors as wild type, and topical application of a JH analogue failed to rescue the wild type cuticular coloration. Finally, transmission electron microscopy showed that sclerotization or deposition of diffuse melanin, rather than deposition of melanin granules, produces the dark coloration found in the cuticle of this species. We conclude that different molecular mechanisms underlie larval melanism in different species of Lepidoptera.     

Transport and kinetics of diflubenzuron and pyriproxyfen in the beet armywormSpodoptera exigua and its predatorPodisus maculiventris

Transport and retention of the insect growth regulators (IGRs) diflubenzuron and pyriproxyfen in larvae of the beet armywormSpodoptera exigua (Hübner) and in nymphs of the predatory bugPodisus maculiventris (Say) were investigated. In a first experiment, the retention of orally administered [¹⁴C]radiolabeled isotopes of both compounds in fifth-instar larvae of the beet armyworm was studied. Rate of excretion of both IGRs inS. exigua caterpillars was high, with a 50% excretion time of approximately 6 h after intake. In a second experiment, the transport of the compounds from prey to predator and their retention inside the predator were studied. Fifthinstar nymphs ofP. maculiventris were allowed to feed on caterpillars that had been given contaminated food. For both diflubenzuron and pyriproxyfen, more than 80% of the amount of radiolabel applied was recovered in consumed prey. Low levels of radioactivity (c. 3% of the applied amount of radiolabel) were also found in the fluid regurgitated by the prey larvae when attacked by the predatory bugs. Relatively small amounts of radiolabel (c. 8 and 15% of the amount orally applied to the prey for diflubenzuron and pyriproxyfen, respectively) were ingested byP. maculiventris nymphs when feeding on beet armyworm caterpillars. The data suggest that the predators did not use gut content as food. The pattern of excretion in nymphs ofP. maculiventris differed between compounds. For diflubenzuron, there was a drastic decrease of radioactivity inside the predator body of around 40% within the first 6 h and then the level of retained radiolabel remained stable at 3–4% up to 72 h. For pyriproxyfen, a slow decrease of radioactivity inside the body was observed and at 72 h only 2% of the applied quantity was detected. Results of this study are discussed in relation to the findings from previous studies on the toxicity of both IGRs toP. maculiventris.     

A Comparison of Growth and Development of Three Major Agricultural Insect Pests Infected with Heliothis virescens ascovirus 3h (HvAV-3h)

Ascoviruses are double-stranded DNA viruses that are pathogenic to lepidopteran hosts, particularly noctuid larvae. Infection of a larva is characterized by retarded growth, reduced feeding and yellowish body color. In this paper, we reported the growth and development of three major agricultural noctuid insect pests, Helicoverpa armigera (Hübner), Spodoptera exigua (Hübner) and Spodoptera litura (Fabricius), infected with Heliothis virescens ascovirus 3h (HvAV-3h). Using 10-fold serial dilutions (0 to 7) of HvAV-3h-containing hemolymph to infect S. litura larvae, we found no significant difference in larval mortalities from 0 to 10³-fold dilutions; however, significant differences were observed at 10⁴-fold dilution and above. Using a 10-fold dilution of HvAV-3h-containing hemolymph to infect H. armigera, S. exigua and S. litura larvae, we found that the growth and development were significantly affected. All infected larvae could not pupate; the survival times of treated H. armigera, S. litura and S. exigua larvae were significantly longer than untreated control larvae. Body weight showed significant difference between treated and untreated control group from day 1 after inoculation in H. armigera and S. exigua, but day 2 in S. litura. Additionally, food intake also showed significant difference between treated and untreated control group from day 2 after inoculation in H. armigera and S. litura, but day 3 in S. exigua.     

Insecticide resistance monitoring in whitefly (Bemisia tabaci) (Hemiptera: Aleyrodidae) in Oman

The sweet potato whitefly, Bemisia tabaci Gennadius, is an important insect pest of many crops including vegetables through direct feeding damage and as a vector of several plant viruses. Intensive use of insecticides has led to the development of insecticide resistance in global B. tabaci populations. This study was conducted to establish susceptibility levels to deltamethrin, thiamethoxam and pyriproxyfen in seven geographically different populations of B. tabaci MEAM1 adults in Oman. All B. tabaci populations showed very low to low level of resistance (2.1–12.3 fold) to deltamethrin. All B. tabaci populations showed no resistance to very low level of resistance to thiamethoxam (2.2–6.2 fold) and pyriproxyfen (2.4–3.5 fold). A likelihood analysis showed the possibility for control failure in two populations (Barka and Salalah) to deltamethrin, however, no possible failure was detected in all populations for thiamethoxam and pyriproxyfen. An insecticide resistance dynamics study in one population (SQU-1) showed a loss in susceptibility to deltamethrin with increase in the LC₅₀ value from 25.1 mg L⁻¹ to 84.5 mg L⁻¹ between 2017 and 2019 resulting in 5.3 fold increase in RF. The study results determined that several B. tabaci populations are at the initial stages of resistance development to deltamethrin and cross-resistance with thiamethoxam and pyriproxyfen. Vegetable farmers in Oman, the Barka and Salalah regions in particular, should be cautious in the repeated use of one class of insecticide alone.     

Recovering More than Tree Cover: Herbivores and Herbivory in a Restored Tropical Dry Forest

Intense and chronic disturbance may arrest natural succession, reduce environmental quality and lead to ecological interaction losses. Where natural succession does not occur, ecological restoration aims to accelerate this process. While plant establishment and diversity is promoted by restoration, few studies have evaluated the effect of restoration activities on ecological processes and animal diversity. This study assessed herbivory and lepidopteran diversity associated with two pioneer tree species growing in 4-year-old experimental restoration plots in a tropical dry forest at Sierra de Huautla, in Morelos, Mexico. The study was carried out during the rainy season of 2010 (July-October) in eleven 50 x 50 m plots in three different habitats: cattle-excluded, cattle-excluded with restoration plantings, and cattle grazing plots. At the beginning of the rainy season, 10 juveniles of Heliocarpus pallidus (Malvaceae) and Ipomoea pauciflora (Convolvulaceae) were selected in each plot (N = 110 trees). Herbivory was measured in 10 leaves per plant at the end of the rainy season. To evaluate richness and abundance of lepidopteran larvae, all plants were surveyed monthly. Herbivory was similar among habitats and I. pauciflora showed a higher percentage of herbivory. A total of 868 lepidopteran larvae from 65 morphospecies were recorded. The family with the highest number of morphospecies (9 sp.) was Geometridae, while the most abundant family was Saturnidae, with 427 individuals. Lepidopteran richness and abundance were significantly higher in H. pallidus than in I. pauciflora. Lepidopteran richness was significantly higher in the cattle-excluded plots, while abundance was significantly higher in the non-excluded plots. After four years of cattle exclusion and the establishment of plantings, lepidopteran richness increased 20 –fold in the excluded plots compared to the disturbed areas, whereas herbivory levels were equally high in both restored and disturbed sites. Restoration with plantings and exclusion of cattle and plantings was shown to be a successful strategy for attracting lepidopterans and cattle exclusion was the main factor explaining lepidopteran diversity.     

Transgenic protein production in silkworm silk glands requires cathepsin and chitinase of Autographa californica multicapsid nucleopolyhedrovirus

The silkworm Bombyx mori represents an established in vivo system for the production of recombinant proteins. Baculoviruses have been extensively investigated and optimised for the expression of high protein levels inside the haemolymph of larvae and pupae of this lepidopteran insect. Current technology includes deletion of genes responsible for the activity of virus-borne proteases, which in wild-type viruses, cause liquefaction of the host insect and enhance horizontal transmission of newly synthesised virus particles. Besides the haemolymph, the silk gland of B. mori provides an additional expression system for recombinant proteins. In this paper, we investigated how silk gland can be efficiently infected by a Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). We demonstrated that the viral chitinase and the cysteine protease cathepsin are necessary to permit viral entry into the silk gland cells of intrahaemocoelically infected B. mori larvae. Moreover, for the first time, we showed AcMNPV crossing the basal lamina of silk glands in B. mori larvae, and we assessed a new path of infection of silk gland cells that can be exploited for protein production.     

Electrogenic K+-basic amino-acid cotransport in the midgut of lepidopteran larvae

Summary Experiments performed on isolated midgut demonstrate that the model proposed for the absorption of neutral amino acids in the K⁺-transporting intestinal epithelium of lepidopteran larvae applies also to the transport of the basic amino acids histidine and lysine. The characteristics of these K⁺-basic amino-acid cotransports have been studied in brush-border membrane vesicles. Histidine and lysine are transported by different transport agencies, which share, to a different degree, a high sensitivity to transmembrane electrical potential difference. Kinetic analysis showed thatK _m for histidine and lysine increased 10-fold and three-fold, respectively, whereasV _max was only slightly modified when the electrical potential difference was abolished. The relationship between potassium concentration and histidine uptake indicates a cooperative binding of more than one potassium to the transporter. Countertransport experiments with glutamine as elicitor show that histidine and glutamine are transported through the same system.     

Stages of Infection during the Tripartite Interaction between Xenorhabdus nematophila, Its Nematode Vector, and Insect Hosts

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.     

Identification of the bioactive core component of the insecticidal Vip3A toxin peptide of Bacillus thuringiensis

The potential of insecticidal Vip3Aa toxin peptide of Bacillus thuringiensis (Bt) as a resource for development of lepidopteran insect resistant transgenic crop plants has not yet been fully fathomed. The single mode of protection offered by the insecticidal Vip3Aa toxin against a broad spectrum of lepidopteran insect pests that invade crop field as secondary insect pests, carry definitive significance. However, lack of diversity amongst insecticidal Vip3A toxin towards toxicity for lepidopteran insects is often considered as disadvantage. In order to bring in improvement at this front, search for diversity and protein engineering of the toxin molecule for creation of diversity require to be undertaken in future. In that context, identification of the bioactive core component of Vip3BR toxin peptide of Bt an analogue of Vip3Aa toxin has been accomplished. The core component was found to contain enhanced potency of the insecticidal property 2–3 folds more than the native toxin against four major crop pests.     

Proteomics of larval hemolymph in Bombyx mori reveals various nutrient-storage and immunity-related proteins

The silkworm, Bombyx mori, is an important economic insect for its production of silk. The larvae of many lepidopteran insects are major agricultural pests and often silkworm is explored as a model organism for other lepidopteran pest species. The hemolymph of caterpillars contains a lot of nutrient and immune components. In this study, we applied liquid chromatography–tandem mass spectrometry to gain a better understanding of the larval hemolymph proteomics in B. mori. We identified 752 proteins in hemolymph collected from day-4 fourth instar and day-7 fifth instar. Nearly half the identified proteins (49 %) were predicted to function as binding proteins and 46 % were predicted to have catalytic activities. Apolipophorins, storage proteins, and 30K proteins constituted the most abundant groups of nutrient-storage proteins. Of them, 30K proteins showed large differences between fourth instar larvae and fifth instar larvae. Besides nutrient-storage proteins, protease inhibitors are also expressed very highly in hemolymph. The analysis also revealed lots of immunity-related proteins, including recognition, signaling, effectors and other proteins, comprising multiple immunity pathways in hemolymph. Our data provide an exhaustive research of nutrient-storage proteins and immunity-related proteins in larval hemolymph, and will pave the way for future physiological and pathological studies of caterpillars.     

In vitro metabolism of pyriproxyfen by microsomes from susceptible and resistant housefly larvae

Levels of cytochrome P450 and b₅ were investigated in microsomal enzymes of houseflies from the gut and fat body of the third instar larvae of a pyriproxyfen-resistant strain (YPPF) and two pyriproxyfen-susceptible strains (YS and SRS). In comparison to the YS and SRS strains, YPPF microsomes had higher levels of total cytochrome P450s in both the gut and fat body. Furthermore, microsomes from the gut and fat body of YPPF larvae were found to have a much greater ability to hydroxylate aniline than YS larvae. In vitro metabolism studies of pyriproxyfen indicated that the metabolic rates were much higher in both the gut and fat body of YPPF larvae than of YS and SRS larvae. The major metabolites of pyriproxyfen in houseflies were identified to be 4′-OH-pyriproxyfen and 5′-OH-pyriproxyfen. Cytochrome P450 inhibitors, piperonyl butoxide (PB) and 2-propynyl 2,3,6-trichlorophenyl ether (PTPE), decreased the metabolic rates significantly in all three strains. This study confirmed that microsomal cytochrome P450 monooxygenases play an important role in the pyriproxyfen resistance of the housefly. Furthermore, it suggests that the fat body must be as important as the gut for the metabolism of pyriproxyfen in resistant housefly larvae. Arch. Insect Biochem. Physiol. 37:215–224, 1998. © 1998 Wiley-Liss, Inc.