Inhibition of cuticular lipid synthesis and its effect on insect survival

A new approach to insect control—using sodium trichloroacetate (NaTCA) to inhibit synthesis of the hydrophobic cuticular lipids that protect insects from dehydration—was tested on Triatoma infestans. In vivo and in vitro studies of incorporation of radioactive precursors showed diminished cuticular hydrocarbon synthesis after NaTCA treatment. Thin layer chromatography and scanning electron microscopy showed disruption of the cuticular lipid layer of NaTCA-treated insects, which also have increased mortality and altered molting cycles. NaTCA treatment enhanced the penetration and increased the lethality of a contact insecticide. © 1994 Wiley-Liss, Inc.     

Hydrocarbons of Rhodnius prolixus, a Chagas disease vector

The surface hydrocarbons of the blood-sucking insect, Rhodnius prolixus, a major Chagas disease vector in Venezuela, Colombia and Central America, were characterized by capillary gas chromatography coupled to mass spectrometry (CGC-MS). A total of 54 single or multicomponent peaks of saturated, straight-chain and methyl-branched hydrocarbons were identified. Major n-alkanes were n-C27, n-C29, n-C31 and n-C33 hydrocarbons. In the branched fraction, methyl groups were at positions 3, 5, 7, 11, 13, 15 and 17- for monomethyl isomers, and separated by three or five methylene groups for the trimethyl or tetramethyl derivatives. For the higher molecular weight components of 37, 39 and 41 atoms in the carbon skeleton, the di-, tri- and tetramethyl branches were usually separated by three or five, and sometimes 7, 11 or 13, methylene groups. The internal hydrocarbon pool contained larger amounts of the higher molecular weight methyl-branched components. Qualitative differences among epicuticular and internal hydrocarbon compositions were detected, both in adult and nymphal stages. No significant sexual dimorphism was detected, but a significant shift in the major n-alkane components was evident from the nymphal to the adult stage, differing also in the relative amounts of the higher molecular weight methyl-branched chains. Comparison of the hydrocarbon components to that of other Chagas disease vectors is discussed.     

The cuticular hydrocarbons of the Triatoma sordida species subcomplex (Hemiptera: Reduviidae)

The cuticular hydrocarbons of the Triatoma sordida subcomplex (Hemiptera: Reduviidae: Triatominae) were ana-lysed by gas chromatography and their structures identified by mass spectrometry. They comprised mostly n-alkanes and methyl-branched alkanes with one-four methyl substitutions. n-alkanes consisted of a homologous series from C21-C33 and represented 33-45% of the hydrocarbon fraction; n-C29 was the major component. Methyl-branched alkanes showed alkyl chains from C24-C43. High molecular weight dimethyl and trimethylalkanes (from C35-C39) represented most of the methyl-branched fraction. A few tetramethylalkanes were also detected, comprising mostly even-numbered chains. Several components such as odd-numbered 3-methylalkanes, dimethylalkanes and trimethylalkanes of C37 and C39 showed patterns of variation that allowed the differentiation of the species and populations studied. Triatoma guasayana and Triatoma patagonica showed the most distinct hydrocarbon patterns within the subcomplex. The T. sordida populations from Brazil and Argentina showed significantly different hydrocarbon profiles that posed concerns regarding the homogeneity of the species. Triatoma garciabesi had a more complex hydrocarbon pattern, but it shared some similarity with T. sordida. The quantitative and qualitative variations in the cuticular hydrocarbons may help to elucidate the relationships between species and populations of this insect group.     

Is the social parasite Vespa dybowskii using chemical transparency to get her eggs accepted?

Both avian and insect cuckoos must trick their hosts into accepting foreign eggs. In birds this is achieved through egg mimicry. Within the hornets the only known social parasite is the rare Vespa dybowskii. We investigated how the V. dybowskii queen induces tens or hundreds of host workers to accept her eggs and offspring. Since hydrocarbons function as recognition cues in social insects, we investigated these compounds from the surface of eggs and workers of V. dybowskii, both host species (V. simillima and V. crabro) and an additional four non-host species. We found that chemical mimicry of the hosts’ colony odour and their eggs normally associated with wasps was not being employed by V. dybowskii. Chemical insignificance is also unlikely as the amounts of hydrocarbons extracted from parasite, host and non-host eggs were similar. Eggs of V. dybowskii may survive in part due to being chemically transparent, as methyl-branched compounds only represent a tiny proportion (<1%) of the parasites hydrocarbon profile but a large proportion (26–41%) in both host species. However, the functions of various hydrocarbon groups need to be investigated in the hornets before this new acceptance mechanism of parasite eggs and adults is understood.     

Discovery of novel trimethylalkanes in the internal hydrocarbons of developing pupae of Heliothis virescens and Helicoverpa zea

Novel trimethyl-branched alkanes which eluted with the monomethylalkanes were identified in the internal lipids of Helicoverpa zea but were not present in Heliothis virescens. Their structures were unique in that the first methyl branch occurred on carbon 2 and the 2nd and 3rd methyl branch points were separated by a single methylene. Novel trimethylalkanes identified from their chemical ionization and electron impact mass spectra were 2,18,20-trimethyltetratriacontane, 2,18,20-trimethylhexatriacontane, and 2,24,26-trimethyldotetracontane. Previous reports did not find these trimethylalkanes in the cuticular surface lipids of larvae, pupae or adults of either species. The internal pupal hydrocarbons of H. virescens and H. zea amounted to 123 μg and 304 μg per pupa, respectively. They consisted of n-alkanes (8 and 4%, respectively) and methyl-branched alkanes (88 and 94%, respectively). The n-alkanes ranged in chain length from approximately 21 to 35 carbons and the methyl-branched alkanes from approximately 26 to 55 carbons vs. methyl-branched alkanes from 28 to 37 carbons previously reported for hydrocarbons from the pupal cuticular surface. The major n-alkane was heptacosane (3.3 and 1.2%, respectively, in H. virescens and H. zea). The major methyl-branched alkanes in H. virescens were methylhentriacontane (15%), methyltritriacontane (12%) and dimethyltritriacontane (10%), and in H. zea were methylnonacosane (17%), dimethylnonacosane (9%) and methylhentriacontane (20%). Except for the novel trimethylalkanes, the methylalkane branch points were predominantly on odd-numbered carbons as has been reported for these and other species.     

Influence of target plant density on herbivorous insect oviposition choice: Ambrosia artemisiifolia L. (Asteraceae) and Zygogramma suturalis F. (Coleoptera: Chrysomelidae)

Zygogramma suturalis F. has been introduced to Russia for the biological control of the common ragweed Ambrosia artemisiifolia L. The functional response of Z. suturalis to the density of the target plant was studied at two levels: between plants and between 0.1 m² sampling plots. The number of Z. suturalis eggs per unit of plant weight was almost independent of plant weight, indicating that a functional response between plants is absent. However, tall and intermediate height plants received more eggs per phytomass unit than short plants. Between plots, correlation analysis showed that absolute Z. suturalis egg density is positively dependent on the ragweed density. Experiments showed that the locomotor activity of ovipositing females decreased when on the ragweed, suggesting orthokinesis. The number of leaf beetle eggs per unit of ragweed weight was negatively correlated with host plant density in May‐June, when the majority of eggs are laid, but was positively correlated in July and loosely, or not significantly, correlated in August. Seasonal changes in the functional response of a herbivorous insect to host plant population density have not been reported previously. A negative functional response of a herbivorous insect to target plant density can be a serious obstacle to the biological control of weeds.     

Sex- and age-related changes in the biophysical properties of cuticular lipids of the housefly,Musca domestica

We examined the biophysical properties of cuticular lipids isolated from the housefly, Musca domestica. Melting temperatures (T_m) of surface lipids isolated from female houseflies decreased from 39.3 °C to 35.3 °C as the females attained sexual maturity and produced sex pheromone, whereas those prepared from males did not change with age. Lipids melted over a 10–25 °C temperature range, and their physical properties were a complex function of the properties of the component lipids. The T_m of total cuticular lipids was slightly below that of cuticular hydrocarbons (HC), the predominant lipid fraction. Hydrocarbons were further fractionated into saturated, unsaturated, and methyl-branched components. The order of decreasing T_m was total alkanes > total HCs > methyl-branched alkanes > alkenes. For 1-day-old flies, measured T_ms of hydrocarbons were 1.3–5.5 °C lower than T_ms calculated from a weighted average of T_ms for saturated and unsaturated components. For 4-day-old flies, calculated T_ms underestimated T_m by 11–14 °C. © 1995 Wiley-Liss, Inc.     

Bikaverin and fusaric acid from Fusarium oxysporum show antioomycete activity against Phytophthora infestans

Aims: To isolate and identify antioomycete substances from Fusarium oxysporum EF119 against Phytophthora infestans and to investigate their antimicrobial activities against various plant pathogenic bacteria, oomycetes and true fungi. Methods and Results: Two antioomycete substances were isolated from liquid cultures of F. oxysporum EF119, which shows a potent disease control efficacy against tomato late blight caused by P. infestans. They were identified as bikaverin and fusaric acid by mass and nuclear magnetic resonance spectral analyses. They inhibited the mycelial growth of plant pathogenic oomycetes and fungi. Fusaric acid also effectively suppressed the cell growth of various plant pathogenic bacteria, but bikaverin was virtually inactive. Treatment with bikaverin at 300 μg ml^?1 suppressed the development of tomato late blight by 71%. Fusaric acid provided effective control against tomato late blight and wheat leaf rust over 67% at concentrations more than 100 μg ml^?1. Conclusions: Both bikaverin and fusaric acid showed in vitro and in vivo antioomycete activity against P. infestans. Significance and Impact of the Study: Fusarium oxysporum EF119 producing both bikaverin and fusaric acid may be used as a biocontrol agent against tomato late blight caused by P. infestans.     

Spectral sensitivity of the photonegative reaction of the blood-sucking bug Triatoma infestans (Heteroptera: Reduviidae)

We studied the spectral sensitivity of the visual system of the blood-sucking bug Triatoma infestans, one of the main vectors of Chagas Disease in South America. We quantified the photonegative reaction of this insect in a rectangular arena, half of which was kept dark and the other half illuminated with various intensities of different monochromatic lights (or broadband stimuli for λ>665 nm). As a behavioral parameter of the photonegative response, we measured the time each insect spent in the dark half of the arena. We found that low intensity levels (under 0.06 μW/cm²) of monochromatic lights of 397, 458, 499, and 555 nm evoked a statistically significant (i.e., different from that of control groups) photonegative reaction. Insects were less sensitive to monochromatic lights of 357 nm (UV) and 621 nm (dark orange), and to broadband stimuli in the red part of the spectrum (665–695 nm). These findings indicate that the visual system of T. infestans is sensitive to broader regions of the spectrum than those previously reported.     

Inoculation of Transgenic Resistant Potato by Phytophthora infestans Affects Host Plant Choice of a Generalist Moth

Pathogen attack and the plant’s response to this attack affect herbivore oviposition preference and larval performance. Introduction of major resistance genes against Phytophthora infestans (Rpi-genes), the cause of the devastating late blight disease, from wild Solanum species into potato changes the plant-pathogen interaction dynamics completely, but little is known about the effects on non-target organisms. Thus, we examined the effect of P. infestans itself and introduction of an Rpi-gene into the crop on host plant preference of the generalist insect herbivore, Spodoptera littoralis (Lepidoptera: Noctuidae). In two choice bioassays, S. littoralis preferred to oviposit on P. infestans-inoculated plants of both the susceptible potato (cv. Desiree) and an isogenic resistant clone (A01-22: cv. Desiree transformed with Rpi-blb1), when compared to uninoculated plants of the same genotype. Both cv. Desiree and clone A01-22 were equally preferred for oviposition by S. littoralis when uninoculated plants were used, while cv. Desiree received more eggs compared to the resistant clone when both were inoculated with the pathogen. No significant difference in larval and pupal weight was found between S. littoralis larvae reared on leaves of the susceptible potato plants inoculated or uninoculated with P. infestans. Thus, the herbivore’s host plant preference in this system was not directly associated with larval performance. The results indicate that the Rpi-blb1 based resistance in itself does not influence insect behavior, but that herbivore oviposition preference is affected by a change in the plant-microbe interaction.     

Gut tract microorganisms supply the precursors for methyl-branched hydrocarbon biosynthesis in the termite,Zootermopsis nevadensis

In vivo and in vitro experiments were performed to examine the role of succinate and other potential precursors of the methylmalonyl-CoA used for methyl-branched hydrocarbon biosynthesis in the termite Zootermopsis nevadensis. The in vivo incorporation of [1,4-¹⁴C]succinate and [2,3-¹⁴C]succinate into hydrocarbon confirmed that succinate is a direct precursor to the methyl branch unit. The other likely precursors, the branched chain amino acids valine and isoleucine, were not efficiently incorporated into hydrocarbon. Carbon-13 NMR showed that one of the labeled carbons of [1,4-¹³C]succinate labeled position 6 of 5-methylalkanes and positions 6 and 18 of 5,17-dimethylalkanes, indicating that succinate, as a methylmalonyl-CoA unit, was incorporated as the third unit to form 5-methylheneicosane and as both the third and ninth units to form 5,17-dimethylheneicosane. Analysis of organic acids after the in vivo metabolism of [2,3-¹⁴C]succinate showed that succinate was converted to propionate and methylmalonate. Labeled succinate injected into the hemolymph was readily taken up by the gut tract. Isolated gut tissue efficiently converted succinate to acetate and propionate, both of which were released into the incubation media. Mitochondria from termite tissue (minus gut tract) converted succinate to methylmalonate and propionate only in the presence of malonic acid, an inhibitor of succinate dehydrogenase. The results of these studies show that while termite mitochondria are able to convert succinate to propionate and methylmalonate, most of the propionate used for methyl-branched hydrocarbon biosynthesis is produced by gut tract microorganisms. The propionate is then presumably transported through the hemolymph to epidermal cells for use in methyl-branched hydrocarbon biosynthesis.     

Inoculation of susceptible and resistant potato plants with the late blight pathogen Phytophthora infestans: effects on an aphid and its parasitoid

Plants are exposed to microbial pathogens as well as herbivorous insects and their natural enemies. Here, we examined the effects of inoculation of potato plants, Solanum tuberosum L. (Solanaceae), with the late blight pathogen Phytophthora infestans (Mont.) de Bary (Peronosporales: Pythiaceae) on an aphid species commonly infesting potato crops and one of the aphid's major parasitoids. We observed the peach‐potato aphid, Myzus persicae Sulzer (Hemiptera: Aphididae), and its natural enemy, the biocontrol agent Aphidius colemani Viereck (Hymenoptera: Braconidae), on potato either inoculated with water or P. infestans. Population growth of the aphid, parasitism rate of its natural enemy, and other insect life‐history traits were compared on several potato genotypes, the susceptible cultivar Désirée and genetically modified (GM) isogenic lines carrying genes conferring resistance to P. infestans. Effects of P. infestans inoculation on the intrinsic rate of aphid population increase and the performance of the parasitoid were only found on the susceptible cultivar. Insect traits were similar when comparing inoculated with non‐inoculated resistant GM genotypes. We also tested how GM‐plant characteristics such as location of gene insertion and number of R genes could influence non‐target insects by comparing insect performance among GM events. Different transformation events leading to different positions of R‐gene insertion in the genome influenced aphids either with or without P. infestans infection, whereas effects of position of R‐gene insertion on the parasitoid A. colemani were evident only in the presence of inoculation with P. infestans. We conclude that it is important to study different transformation events before continuing with further stages of risk assessment of this GM crop. This provides important information on the effects of plant resistance to a phytopathogen on non‐target insects at various trophic levels.     

Competition between vectors of Chagas disease, Triatoma infestans and T. sordida: effects on fecundity and mortality

Abstract. Interspecific competition between two species of triatomine bugs (Hemiptera: Reduviidae), vectors of Chagas disease, was assessed for 16 months through comparative fecundity and mortality of experimental populations in chicken nests, maintained indoors with ambient conditions. Triatoma sordida (Stål), the secondary vector in north‐eastern Argentina, was compared with Triatoma infestans (Klug) the more widespread domestic vector in the southern cone of South America. Both species populations originated from females collected in 1995 from the community of Empedrado, Corrientes, Argentina. Three population units were monitored: T. infestans alone, T. sordida alone and both species together in equal proportions. Each population started with six male and six female adults, 116 eggs, and nymphal instars I to V numbering 82, 48, 16, 11 and 19, respectively. Numbers and weight of individual bugs were recorded monthly (August 1995 to December 1996). The pure populations of T. infestans and T. sordida showed temporal changes in abundance, rising in summer and falling in winter, similar to the typical trends under normal field conditions. In the mixed population, however, T. sordida fell to extinction after 6 months, whereas T. infestans reached similar abundance to the pure (control) population. For each nymphal instar of T. sordida, the mean body weight was significantly less and mortality rate was higher in the mixed population compared to the pure population, but there were no significant differences of adult longevity or fecundity between the pure and mixed populations of T. sordida. The apparent competitive displacement of T. sordida by T. infestans was attributed to the latter species having better ability to obtain bloodmeals. This might explain the rarity of mixed populations where these two species occur in sympatry.     

Control of Pyrethroid-Resistant Chagas Disease Vectors with Entomopathogenic Fungi

Background

Triatoma infestans-mediated transmission of Tripanosoma cruzi, the causative agent of Chagas disease, remains as a major health issue in southern South America. Key factors of T. infestans prevalence in specific areas of the geographic Gran Chaco region—which extends through northern Argentina, Bolivia, and Paraguay—are both recurrent reinfestations after insecticide spraying and emerging pyrethroid-resistance over the past ten years. Among alternative control tools, the pathogenicity of entomopathogenic fungi against triatomines is already known; furthermore, these fungi have the ability to fully degrade hydrocarbons from T. infestans cuticle and to utilize them as fuel and for incorporation into cellular components.

Methodology and Findings

Here we provide evidence of resistance-related cuticle differences; capillary gas chromatography coupled to mass spectrometry analyses revealed that pyrethroid-resistant bugs have significantly larger amounts of surface hydrocarbons, peaking 56.2±6.4% higher than susceptible specimens. Also, a thicker cuticle was detected by scanning electron microscopy (32.1±5.9 µm and 17.8±5.4 µm for pyrethroid-resistant and pyrethroid-susceptible, respectively). In laboratory bioassays, we showed that the virulence of the entomopathogenic fungi Beauveria bassiana against T. infestans was significantly enhanced after fungal adaptation to grow on a medium containing insect-like hydrocarbons as the carbon source, regardless of bug susceptibility to pyrethroids. We designed an attraction-infection trap based on manipulating T. infestans behavior in order to facilitate close contact with B. bassiana. Field assays performed in rural village houses infested with pyrethroid-resistant insects showed 52.4% bug mortality. Using available mathematical models, we predicted that further fungal applications could eventually halt infection transmission.

Conclusions

This low cost, low tech, ecologically friendly methodology could help in controlling the spread of pyrethroid-resistant bugs.     

A microsomal fatty acid synthetase from the integument of Blattella germanica synthesizes methyl-branched fatty acids, precursors to hydrocarbon and contact sex pheromone.

Methyl-branched fatty acids present in the integument of the German cockroach, Blattella germanica, were identified by gas chromatography-mass spectrometry of their methyl esters and reduction products (alkanes) as n-3-, n-4-, n-5-, n-7-, n-8-, and n-9-monomethyl fatty acids and as n-5,9-, n-3,9-, and n-3,11-dimethyl fatty acids with 16 to 20 total carbons. These fatty acids have the same branching patterns as do the major hydrocarbons of this insect, including 3,11-dimethylnonacosane, the precursor to the major contact sex pheromone, and are presumed to be intermediates in hydrocarbon formation. A novel microsomal fatty acid synthetase (FAS) located in the integument of this insect incorporated [methyl-14C]methylmalonyl-CoA into methyl-branched fatty acids as demonstrated by radio-high-performance liquid chromatography. A cytosolic FAS is also present in the integument. Both the microsomal and the soluble FAS incorporated [methyl-14C]methylmalonyl-CoA into fatty acids, but only the microsomal FAS was able to efficiently use methylmalonyl-CoA as the sole elongating agent. This is the first report of the characterization of methyl-branched fatty acids from the integument of an insect and of an integumental microsomal FAS that incorporates methylmalonyl-CoA into branched fatty acids.     

Effect of formulated Metarhizium anisopliae on eggs and eclosing nymphs of Triatoma infestans

Little is known about the ovicidal effects of fungi that attack nymphs and adults of triatomine vectors. A combined formulation of Metarhizium anisopliae IP 46 conidia prepared with diatomaceous earth (DE) and vegetable oil was tested against eggs of Triatoma infestans. Eggs were highly susceptible to fungal infection at relative humidity close to saturation [>98% relative humidity (RH)] but not at 75% RH regardless of the formulation applied. Susceptibility of eggs decreased with longer post‐ovipositional embryonation periods before treatments. The eventual eclosion of nymphs was best suppressed by application of conidia prepared with DE + oil and at a >98% RH incubation. Moreover, nymphs were less affected by the fungus when exposed for only a 24‐h period after eclosion to a treated surface than individuals that were in constant contact with the conidia. These findings contribute to a better understanding of the potential of M. anisopliae as an agent against all developmental stages of T. infestans.     

Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS–PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K_i = 3.29 nM) and human cathepsin L (K_i = 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.