Insect Heads Research Articles

Competition among Flavescence Dorée Phytoplasma Strains in the Experimental Insect Vector Euscelidius variegatus.

Phytoplasmas are plant pathogenic wall-less bacteria transmitted in a persistent propagative manner by hemipteran insects, mainly belonging to the suborder Auchenorrhyncha (Fulgoromorpha and Cicadomorpha). Flavescence dorée (FD) is a quarantine disease of grapevine, causing great damage to European viticulture and associated with phytoplasmas belonging to 16SrV-C (FD-C) and -D (FD-D) subgroups. FD-C and FD-D strains share similar pathogenicity, but mixed infections are rare in nature. To investigate the competition among FDp strains, specimens of the laboratory vector Euscelidius variegatus (Hemiptera: Cicadellidae) were forced to acquire both phytoplasma haplotypes upon feeding on FD-C- and FD-D-infected plants or after the injection of both strains. The pathogen colonization of insect bodies and heads was monitored with multiplex qPCR, and the efficiencies of phytoplasma transmission were estimated. Single infection, irrespective of strain type, was more frequent than expected, indicating that competition among FD strains occurs. Hypotheses of competition for resources and/or host active sites or the direct antibiosis of one strain against the other are discussed, based on the genetic complexity of FDp populations and on the high genome variability of the FD-D strain. As FD management still mainly relies on insecticides against vectors, the characterization of FDp haplotypes and the description of their epidemiology also have practical implications.

An antennal electric signal detection system based on template matching

As the most efficient perception system in nature, the perception mechanism of the insect (such as honeybee) antennae is the key to imitating the high-performance sensor technology. An automated experimental device suitable for collecting electrical signals (including antenna reaction time information) of antennae was developed, in response to the problems of the non-standardized experimental process, interference of manual operation, and low efficiency in the study of antenna perception mechanism. Firstly, aiming at the automatic identification and location of insect heads in experiments, the image templates of insect head contour features were established. Insect heads were template-matched based on the Hausdorff method. Then, for the angle deviation of the insect heads relative to the standard detection position, a method that calculates the angle of the insect head mid-axis based on the minimum external rectangle of the long axis was proposed. Eventually, the electrical signals generated by the antennae in contact with the reagents were collected by the electrical signal acquisition device. Honeybees were used as the research object in this study. The experimental results showed that the accuracy of template matching could reach 95.3% to locate the bee head quickly, and the deviation angle of the bee head was less than 1°. The distance between antennae and experimental reagents could meet the requirements of antennae perception experiments. The parameters, such as the contact reaction time of honeybee antennae to sucrose solution, were consistent with the results of the manual experiment. The system collects effectively antenna contact signals in an undisturbed state and realizes the standardization of experiments on antenna perception mechanisms, which provides an experimental method and device for studying and analyzing the reaction time of the antenna involved in biological antenna perception mechanisms.

Protocol for preparation of heterogeneous biological samples for 3D electron microscopy: a case study for insects

Modern morphological and structural studies are coming to a new level by incorporating the latest methods of three-dimensional electron microscopy (3D-EM). One of the key problems for the wide usage of these methods is posed by difficulties with sample preparation, since the methods work poorly with heterogeneous (consisting of tissues different in structure and in chemical composition) samples and require expensive equipment and usually much time. We have developed a simple protocol allows preparing heterogeneous biological samples suitable for 3D-EM in a laboratory that has a standard supply of equipment and reagents for electron microscopy. This protocol, combined with focused ion-beam scanning electron microscopy, makes it possible to study 3D ultrastructure of complex biological samples, e.g., whole insect heads, over their entire volume at the cellular and subcellular levels. The protocol provides new opportunities for many areas of study, including connectomics.

Polyphenolic composition, antibacterial, modulator and neuroprotective activity of Tarenaya spinosa (Jacq.) Raf. (Cleomaceae)

Objective: To evaluate the antibacterial activity and neuroprotective capacity of the ethanolic and aqueous extracts of Tarenaya spinosa (T. spinosa) as well as to determine and quantify some of its polyphenols by high performance liquid chromatography with diode-array detection (HPLC-DAD). Methods: The bacterial Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa strains, grown in Heart Agar Infusion, were tested. The drugs gentamicin, norfloxacin and imipenem were used to evaluate the modulating or antagonistic capacity of the T. spinosa extracts. The extract was analysed by HPLC-DAD to determine the main phenolic compounds. For the cell viability tests, individual heads of the Nauphoeta cinerea arthropod model were removed, homogenized in Trifluoromethyl ketone and centrifuged afterwards. Subsequently, 20 μL of NaNO2 were added to the biological material, except in the control group, to evaluate the protection capacity of the extracts. The homogenate of the insect heads was incubated for 2 h in tubes containing tetrazolium bromide. Results: HPLC-DAD demonstrated that the ethanolic extract of T. spinosa presented caffeic acid as the major compound. The ethanolic extract also showed neuroprotective effects at concentrations ≥ 10 μg/mL, while aqueous extract was shown to have a protective effect only at the concentration of 100 μg/mL. The aqueous extract demonstrated a clinically relevant antibacterial activity against the Staphylococcus aureus multidrug resistant strain - MDR, with MIC 512 μg/mL. However, when the extracts were associated with gentamicin and imipenem, a synergism was detected against Staphylococcus aureus and Escherichia coli MDR strains. Conclusions: Although it does not present an antibacterial action, the extracts of T. spinosa can be used in the pharmaceutical industries since its extracts show modulating action of drugs. Besides, these natural products have neuroprotective capacity.

Identification of the genes in tea leafhopper, Empoasca onukii (Hemiptera: Cicadellidae), that encode odorant-binding proteins and chemosensory proteins using transcriptome analyses of insect heads

Empoasca onukii Matsuda is a serious pest affecting tea production throughout China. Chemosensory behaviors are important in the life cycle of E. onukii, especially for detecting plant odorants during host localizations. In this process, odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) mediate the initial biochemical recognition steps. We used a transcriptomics-based approach to identify 40 putative E. onukii OBP genes and 11 CSP genes. The encoded OBPs comprised 19 classic OBPs and 21 plus-C OBPs. A phylogenetic analysis revealed that the plus-C OBPs formed monophyletic groups, and most of the classic OBPs and CSPs were distributed among other orthologous groups. Additionally, there were significant differences between the sexes regarding the expression of five OBP genes and two CSP genes. Moreover, 36 OBP genes and six CSP genes were most highly expressed in the heads. Two CSP genes were most highly expressed in the thoraxes, while four OBP genes and three CSP genes exhibited high expression levels in the heads and thoraxes. Our data may represent a valuable resource for future functional characterizations of the E. onukii OBPs and CSPs.

Molecular cloning, characterization, genomic structure and functional analysis of catalase in Chilo suppressalis

Catalase (CAT), one of the key enzymes involved in protecting organisms from reactive oxygen species (ROS), degrades hydrogen peroxide to oxygen and water. In this study, we isolated the catalase gene (CsCAT) from the striped stem borer, Chilo suppressalis, a serious pest of rice in Asia. Sequence analysis of the genomic DNA indicated that CsCAT contains a 1023bp intron and a 1524bp coding sequence. The predicted CsCAT protein contains 507 amino acids and includes the proximal active site (FXRERIPERVVHAKGXGA) and heme-ligand sequence (RLFSYNDTX) that are conserved in catalase enzymes. Phylogenetic analysis indicated that CAT from Lepidopteran moths could be assigned to one well-supported cluster. qRT-PCR indicated that CsCAT was expressed in insect heads, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, and hemocytes. Expression of CsCAT was highest in the fat body and hemocytes of diapausing larvae, which suggests a potential important role in insect physiology during diapause period. Expression of CsCAT (measured by qRT-PCR) and enzymatic activity of CsCAT were not significantly altered by temperature stress, which indicates that CsCAT may insensitive to temperature.

Interruption of Onchocerca volvulus transmission in Northern Venezuela.

BackgroundOnchocerciasis is caused by Onchocerca volvulus and transmitted by Simulium species (black flies). In the Americas, the infection has been previously described in 13 discrete regional foci distributed among six countries (Brazil, Colombia, Ecuador, Guatemala, Mexico and Venezuela) where more than 370,000 people are currently considered at risk. Since 2001, disease control in Venezuela has relied on the mass drug administration to the at-risk communities. This report provides empirical evidence of interruption of Onchocerca volvulus transmission by Simulium metallicum in 510 endemic communities from two Northern foci of Venezuela, after 10–12 years of 6-monthly Mectizan® (ivermectin) treatment to all the eligible residents.MethodsIn-depth entomologic and epidemiologic surveys were serially conducted from 2001–2012 in selected (sentinel and extra-sentinel) communities from the North-central (NC) and North-east (NE) onchocerciasis foci of Venezuela in order to monitor the impact of ivermectin treatment.ResultsFrom 2007–2009, entomological indicators in both foci confirmed that 0 out of 112,637 S. metallicum females examined by PCR contained L3 infection in insect heads. The upper bound of the 95% confidence intervals of the infective rate of the vector reached values below 1% by 2009 (NC) and 2012 (NE). Additionally, after 14 (NC) and 22 (NE) rounds of treatment, the seasonal transmission potential (±UL CIs) of S. metallicum was under the critical threshold of 20 L3 per person per season. Serological analysis in school children < 15 years-old demonstrated that 0 out of 6,590 individuals were harboring antibodies to Ov-16. Finally, epidemiological surveys made during 2010 (NC) and 2012 (NE) showed no evidence of microfilariae in the skin and eyes of the population.ConclusionsThese results meet the WHO criteria for absence of parasite transmission and disease morbidity in these endemic areas which represent 91% of the population previously at-risk in the country. Consequently, the two Northern foci are currently under post-treatment onchocerciasis surveillance status in Venezuela.

The Identification of Concerted Convergence in Insect Heads Corroborates Palaeoptera

The relationships of the 3 major clades of winged insects-Ephemeroptera, Odonata, and Neoptera-are still unclear. Many morphologists favor a clade Metapterygota (Odonata +Neoptera), but Chiastomyaria (Ephemeroptera + Neoptera) or Palaeoptera (Ephemeroptera +Odonata) has also been supported in some older and more recent studies. A possible explanation for the difficulties in resolving these relationships is concerted convergence-the convergent evolution of entire character complexes under the same or similar selective pressures. In this study, we analyze possible instances of this phenomenon in the context of head structures of Ephemeroptera, Odonata, and Neoptera. We apply a recently introduced formal approach to detect the occurrence of concerted convergence. We found that characters of the tentorium and mandibles in particular, but also some other head structures, have apparently not evolved independently, and thus can cause artifacts in tree reconstruction. Our subsequent analyses, which exclude character sets that may be affected by concerted convergence, corroborate the Palaeoptera concept. We show that the analysis of homoplasy and its influence on tree inference can be formally improved with important consequences for the identification of incompatibilities between data sets. Our results suggest that modified weighting (or exclusion of characters) in cases of formally identified correlated cliques of characters may improve morphology-based tree reconstruction.

Molecular characterization of glutaredoxin 2 from Ostrinia furnacalis

Glutaredoxins (GRXs) play very important roles in maintaining intracellular redox homeostasis. In the present study, the full-length cDNA sequence encoding GRX2, named OfurGRX2 (GenBank accession no. GU393246), was obtained from Ostrinia furnacalis, using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. Sequence analysis revealed that the open reading frame of OfurGRX2 consists of 351 nucleotides encoding 116 amino acid residues with a predicted molecular weight of 12.6 kDa. Homolog research revealed that OfurGRX2 shares a common active site, CPYC/CPFC, with other insect counterparts. Expression profiles revealed that OfurGRX2 is a ubiquitous gene expressed in insect heads, fat bodies, epidermises, mid guts and muscles. The OfurGRX2 transcript peaked in 36-h larvae of 4th instars, and then suddenly declined in the molting stage. Hormone treatment experiments revealed that 20-hydroxyecodyson (20e) significantly induces the expression of the OfurGRX2 transcript, whereas juvenile hormone (JH) counteracts 20e effects. Adverse stress factors (including starvation, ultraviolet light, mechanical injury, Escherichia coli exposure, and high and low temperatures) dramatically induced OfurGRXGRX2 transcript expression, which confirmed for the first time that GRX2 play important roles in insecta during exposure to adverse environments.

Identification of Circulifer haematoceps (Hemiptera:Cicadellidae) as vector of Spiroplasma citri in the Kerman province of Iran

A study of leafhoppers collected in citrus-growing areas of Kerman province as vectors of Spiroplasma citri, the causal agent of citrus stubborn disease, was carried out by transmission tests to liquid medium and/or to healthy periwinkle plants. From seven field-collected leafhopper species only Orosius albicinctus and Circulifer haematoceps delivered S. citri into LD10 broth medium by feeding through parafilm membrane and C. haematoceps was the sole leafhopper species capable of transmitting S. citri to healthy periwinkle plants. C. haematoceps, O. albicinctus and Austroagallia sinuata collected from sesame fields in citrus- growing areas, were raised on garden stock and sesame plants in the greenhouse. For S. citri acquisition, each progeny leafhopper colony was caged on S. citri infected periwinkles. After the acquisition access period leafhoppers were transferred to healthy periwinkle plants. Detection of spiroplasmas in leafhoppers was done by PCR performed separately on insect bodies and heads. Spiroplasmas were detected in the bodies of all three leafhopper species but in the head of C. haematoceps only. Symptoms typical of S. citri infection were observed only on periwinkle plants fed on by C. haematoceps. Results indicate that among the leafhoppers species collected in Kerman province, only C. haematoceps was capable of transmitting S. citri, and the other two species while capable of acquiring S. citri, were unable to transmit it to healthy plants.

Imaging applications of synchrotron X‐ray phase‐contrast microtomography in biological morphology and biomaterials science. I. General aspects of the technique and its advantages in the analysis of millimetre‐sized arthropod structure

Synchrotron-generated X-rays provide scientists with a multitude of investigative techniques well suited for the analysis of the composition and structure of all types of materials and specimens. Here, we describe the properties of synchrotron-generated X-rays and the advantages that they provide for qualitative morphological research of millimetre-sized biological organisms and biomaterials. Case studies of the anatomy of insect heads, of whole microarthropods and of the three-dimensional reconstruction of the cuticular tendons of jumping beetles, all performed at the beamline ID19 of the European Synchrotron Radiation Facility (ESRF), are presented to illustrate the techniques of phase-contrast tomography available for anatomical and structural investigations. Various sample preparation techniques are described and compared and experimental settings that we have found to be particularly successful are given. On comparing the strengths and weaknesses of the technique with traditional histological thin sectioning, we conclude that synchrotron radiation microtomography has a great potential in biological microanatomy.

The Genotype and Heredity of Modified Acetylcholinesterase of the Green Rice Leafhopper (Nephotettix cincticeps Uhler)

Heredity of modified acetylcholinesterase (AChE) of the green rice leafhopper (Nephotettix cincticeps Uhler) was assessed by crossing experiments between different strains and by analyzing AChE activity of individual insect heads to both metolcarb and N-propyl metolcarb. The green rice leafhopper which shows resistance to metolcarb was found to possess different AChE isozymes, which show negatively correlated cross-resistance to N-propyl metolcarb. However, each insect possessed only one type of AChE isozyme. By crossing experiments between different strains of the green rice leafhopper, at least seven different AChE phenotypes were observed. The method for analyzing AChE of individual insect was found to be a useful tool for the genetic analysis of organophosphorus and carbamate insecticide resistance of the green rice leafhopper.

Onchocerca lienalis:comparison of techniques for the cryopreservation of microfilariae within skin-snips or free of host tissues

Onchocera lienalis microfilariae (mf) were cryopreserved in liquid nitrogen within skin-snips using methanol as a cryoprotectant and their viability evaluated and compared to mf cryopreserved free of host tissues using ethanediol as a cryoprotectant. Despite an initial delay in emergence, the methanol technique did not significantly affect the total numbers of mf emerging from skin-snips of various sizes (3.3-59.81 mg) compared to untreated controls over a 6 h period. Following thawing, the initial motility index (MI) scores of mf cryopreserved by either method were not significantly different from untreated controls; however, over a period of 15 days in culture the MI scores of both cryopreserved groups showed a small but significant overall decline, with the methanol technique producing the lowest scores. These changes in motility levels correlated with the numbers of mf which developed to the infective stage following intrathoracic injection into Simulium ornatum, although this ability to develop was a much more sensitive measure of parasite viability; compared to untreated control recoveries of 3rd-stage larvae, 63.9-71.7% (ethanediol technique) and 34.2-36.9% (methanol technique) of this number were recovered from cryopreserved groups. There were no significant differences in the lengths of infective larvae recovered from the insect heads from each treatment group, nevertheless there were higher numbers of 2nd-stage larvae recovered from the cryopreserved groups compared to the untreated controls. The methanol technique has the advantage of being easier to carry out under field conditions, while parasite viability is significantly better using the ethanediol technique.

Determination of Internal Insect Infestation of Oats: Collaborative Study

An improved method has been developed for determining internal insect infestation of oat kennels. The method involves alcohol defatting and acid hydrolysis of the cracked oats, wet sieving to remove the acid, transfer to a 2 L Wildman trap flask, deaeration by boiling, and treatment with Tween 80-Na4EDTA. Insects are extracted with light mineral oil. Reports from 6 collaborators showed that recoveries averaged 88.98% for adult insect heads and 97.22% for larvae. The method has been adopted official first action.

Microanalytical Quality of Wheat Flour

A survey was made to determine the sanitary quality of wheat flour. Samples were collected and analyzed by the USDA Federal Grain Inspection Service under the quality check program for contract government purchases. The Association of Official Analytical Chemists' method for light filth in flour was used to extract fragments of insects, feathers and rodent hairs; insect heads, larvae, mites and psocids. Insect fragments were most frequently encountered: 83% of the samples contained insect fragments, with counts ranging from 0 to 721 and a mean of 12.52. Ranges of other counts were rodent hair fragments, 0 to 75; feather barbules, 0 to 3; insect heads, 0 to 2; mites, 0 to 2; insect larvae, 0 to 3; psocids 0 to 1.

Determination of Internal Insect Infestation of Wheat: Collaborative Study

An improved method has been developed for determining internal insect infestation of wheat kernels. The method involves acid hydrolysis of cracked wheat, wet sieving to remove the acid, transferring to a 2 L Wildman trap flask, deaeration by boiling, and treatment with Tween 80-Na4EDTA. Insects are extracted with light mineral oil. Reports from 6 collaborators showed that recovery was 95.92% for adult insect heads and 97.22% for larvae by the proposed method as compared with 87.05% and 6.12%, respectively, by the official method. The method has been adopted official first action.

Comparison of Methods for Recovery of Insect Fragments From Date and Fig Paste

Abstract Comparison of two flotation methods for the recovery of insect heads and other insect fragments from both date and fig paste indicated no significant differences between the two methods, although the gasoline flotation method was found preferable. There was also no significant difference between the participating analysts or laboratories. Three trappings were found necessary for complete recovery, irrespective of the method. The use of insect heads was found to be a sufficiently reliable criterion of the number of whole insects.