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[Ultrastructure and morphology of the mitochondriome of cardiomyocytes from invertebrates. I. The mitochondriome of cardiomyocytes from insects].

The cardiomyocyte mitochondrial ultrastructure of two insect species (the American cockroach Periplaneta americana, and a dragonfly Aeschna sp.) has been studied. Mitochondria in cardiomyocytes of these insects are connected by intermitochondrial contacts, similar in morphology to vertebrate intermitochondrial contacts. The number of intermitochondrial contacts differs in cardiomyocytes of the studied insects, numbering 12 and 18 per 100 mitochondria in cardiomyocytes of the cockroach and dragonfly, respectively, which is due presumably to differences in activity of these insects. Cardiomyocytes of both species have several features in common. It was shown that cross-striated myofibrils oriented in different directions occupy 50-58% of the cytoplasmic volume, while mitochondria cover only 16-18%. The pattern of mitochondrial localization differs in cardiomyocytes of the two studied insects. In the cockroach, cardiomyocyte mitochondria are seen both in the center of the cell and on its periphery, in protrusions; whereas in the dragonfly, mitochondria of cardiomyocytes are confined to the protrusions of the abluminal cell side. Mitochondrial profiles are small, their packing is not dense. Mitochondria in cardiomyocytes of these insects have few plastic cristae and dense matrix.

Animals↗

Agonist-induced phosphorylation and desensitization of human m2 muscarinic cholinergic receptors in Sf9 insect cells.

The human m1 (hm1) and m2 (hm2) muscarinic cholinergic receptors (mAChR) expressed in Sf9 insect cells using recombinant baculovirus were tested for their ability to undergo agonist-dependent phosphorylation and desensitization. The muscarinic agonist carbachol induced phosphorylation of the hm2 mAChR in the Sf9 cells incubated with 32P(i) to an extent of 4-5 mol of phosphate/mol of receptor. In contrast, no phosphorylation of the hm1 mAChR was observed. The hm2 mAChR stimulated [35S]GTP gamma S binding to, and GTPase activity of, the insect cell G-proteins. These receptor-mediated activities were reduced by 50% in membranes prepared from agonist-treated cells compared to control, suggesting that the agonist-induced phosphorylation of the hm2 mAChR resulted in desensitization of the receptors. No role for protein kinase C or cyclic nucleotide-dependent kinases in receptor phosphorylation and desensitization was suggested from studies using agents known to modulate the activity of these enzymes. However, pertussis toxin was found to completely eliminate the interaction of the hm2 receptors with the insect cell G-proteins, but did not perturb the ability of carbachol to induce agonist-dependent phosphorylation of the receptors. These results suggested that G-proteins and/or G-protein-activated signalling were not necessary for the agonist-induced phosphorylation of the receptors. Overall, the data indicated that the human m2 (but not the human m1) mAChR expressed in Sf9 insect cells undergo phosphorylation and desensitization in an agonist-dependent, G-protein-independent fashion by an endogenous insect cell kinase. The results demonstrated that a human G-protein-linked receptor is regulated in insect cells in a manner that is similar to that involving members of the G-protein receptor-kinase family.

Animals↗

[Inheritance and segregation of transformants in cotton with two types of insect-resistant genes].

A plant expression vector containing a chemeric Bt29K gene coding for the active Cry1Ac protein and the arrowhead proteinase inhibition gene API-B was introduced into an elite cotton cultivar Jihe 321 by Agrobactertium tumefaciens. Some insect-resistant cotton lines were developed. Segregation and stabilization of insect-resistant genes in six transformation lines were studied. Based on the results of kanamycin resistant test and insect bioassay using Heliethis armigera, PCR detection and Southern-blot, we found that the inheritance and segregation of Bt gene were complicated, some transformants were in accordance with Mendelian patterns of inheritance in the ratio of insect-resistant plants to non-resistant plants in Ti progeny, yet others were non-Mendelian patterns. But the inheritance and segregation of Bt gene in homozygous transformation lines were one or two pairs of major dominant genes through crossing of insect resistant homozygous lines with non-transformation cotton variety. That the insect resistance phenotype was conditioned by one or two pairs of dominant genes was ascertained in this study. There were two copies of Bt genes in two transformation lines DR248 and DR193, which was reported for the first time. The results were confirmed by Southern-blot. Through observation of segregation population of transgenic plants at different generations, we found that the exogenous Bt gene in cotton genome showed unstable in inheritance in early generations, but the gene could be stabilized through resistance screening generation by generation. The unstability of Bt gene may mean that it need time for the gene to compatibilize cotton genome.

Animals↗

Selecting the right statistical model for analysis of insect count data by using information theoretic measures.

Researchers and regulatory agencies often make statistical inferences from insect count data using modelling approaches that assume homogeneous variance. Such models do not allow for formal appraisal of variability which in its different forms is the subject of interest in ecology. Therefore, the objectives of this paper were to (i) compare models suitable for handling variance heterogeneity and (ii) select optimal models to ensure valid statistical inferences from insect count data. The log-normal, standard Poisson, Poisson corrected for overdispersion, zero-inflated Poisson, the negative binomial distribution and zero-inflated negative binomial models were compared using six count datasets on foliage-dwelling insects and five families of soil-dwelling insects. Akaike's and Schwarz Bayesian information criteria were used for comparing the various models. Over 50% of the counts were zeros even in locally abundant species such as Ootheca bennigseni Weise, Mesoplatys ochroptera Stål and Diaecoderus spp. The Poisson model after correction for overdispersion and the standard negative binomial distribution model provided better description of the probability distribution of seven out of the 11 insects than the log-normal, standard Poisson, zero-inflated Poisson or zero-inflated negative binomial models. It is concluded that excess zeros and variance heterogeneity are common data phenomena in insect counts. If not properly modelled, these properties can invalidate the normal distribution assumptions resulting in biased estimation of ecological effects and jeopardizing the integrity of the scientific inferences. Therefore, it is recommended that statistical models appropriate for handling these data properties be selected using objective criteria to ensure efficient statistical inference.

Animals↗

Unsteady aerodynamics of insect flight.

Over the past decade, the importance of unsteady aerodynamic mechanisms for flapping insect flight has become widely recognised. Even at the fastest flight speeds, the old quasi-steady aerodynamic interpretation seems inadequate to explain the extra lift produced by the wings. Recent experiments on rigid model wings have confirmed the effectiveness of several postulated high-lift mechanisms. Delayed stall can produce extra lift for several chords of travel during the translational phases of the wingbeat. Lift can also be enhanced by circulation created during pronation and supination by rotational mechanisms: the fling/peel, the near fling/peel and isolated rotation. These studies have revealed large leading-edge vortices which contribute to the circulation around the wing, augmenting the lift. The mechanisms show distinctive patterns of vortex shedding from leading and trailing edges. The results of flow visualization experiments on tethered insects are reviewed in an attempt to identify the high-lift mechanisms actually employed. The fling/peel mechanism is clearly used by some insects. The near fling/peel is the wing motion most commonly observed, but evidence for the production of high lift remains indirect. For many insects, lift on the upstroke probably results from delayed stall instead of the flex mechanism of isolated rotation. The large leading-edge vortices from experiments on rigid model wings are greatly reduced or missing around the real insect wings, often making the identification of aerodynamic mechanisms inconclusive. A substantial spanwise flow component has been detected over the aerodynamic upper wing surface, which should transport leading-edge vorticity towards the wingtip before it has much time to roll up. This spanwise transport, arising from centrifugal acceleration, is probably a general phenomenon for flapping insect flight. It should reduce and stabilise any leading-edge vortices that are present, which is essential for preventing stall and maintaining the circulation of high-lift mechanisms during translation.

Animals↗

Polydnaviruses of hymenopteran endoparasitoids knock out the host insect immune response.

The insect immune system reacts against invading microorganisms and parasites with the recruitment of haemocytes and with humoral response. Cellular immune reactions involve phagocytosis, nodule formation and encapsulation by different types of haemocytes whereas insect cell-free antibacterial immunity depends on the production of a number of peptides and proteins, among which lysozyme, cecropins and attacins represent the major group of immune proteins. Polydnaviruses from certain hymenopterous parasitoids interfere with both host immunity and host development. These immunosuppressive viruses exhibit an intimate genetic relationship with the parasitoid since viral sequences are integrated within the parasitoid chromosomal DNA. The viral genes expression in parasitized host induces immunosuppression and alters development of the host insect. The parasitoids developing in the host body cavity knock out the insect immune system, inducing a decline in cellular and humoral components of the immune system so that parasitoid eggs are not recognized as foreign and thereby are not encapsulated. Polydnaviruses carrying parasitoids escape the host immune response and may develop within the insect host whereas other invaders are normally destroyed by defense factors of insect haemolymph.

Animals↗

Diseases of insects of medical importance in Europe.

The biological control of insects carrying human diseases was first attempted many years ago, but the results were disappointing owing to the insufficiency of information on the ecology and pathology of such insects at the time. In recent years, however, increased knowledge of insect pathology and ecology and the development of insecticide-resistance have led to a revival of interest in this method of vector control. Most of the recent published work on this subject relates to findings in North America; comparatively little information has hitherto been available in respect of Europe. This paper, which is based on a survey of the literature and on the author's own experience, presents the results of research on diseases of insects of medical importance in Europe. Infection with viruses, rickettsiae, bacteria, fungi, protozoa and nematodes are discussed and suggested lines for future European research on biological control are put forward.

Animals↗

Approaches to vector control: new and trusted. 3. Prospects for genetic manipulation of insect vectors.

Insect vector control has proved an effective method of reducing the transmission of disease-causing organisms to human populations in many tropical countries. A variety of methods has been employed for suppressing vector populations, including the application of biological control agents and the elimination of breeding sites, with a continuing and heavy reliance on the use of chemical insecticides. However, the development of insecticide resistance by vector insects, the cost of developing and registering new insecticidal compounds, and the increase in legislation to combat the detrimental effects of insecticidal residues on the environment, have emphasized the need to assess a variety of alternatives to vector control. What is required is a completely novel approach either to suppress vector populations, or to alter their ability to transmit disease-causing organisms in such a way as to have a profound and long-lasting effect on disease transmission. Genetic manipulation of insect vectors may provide just such an approach. The major requirements for being able to manipulate the genomes of insects are reviewed together with the progress which has been made to create transgenic vector insects. The potential applications of this technology are then explored, emphasizing that its most immediate use will be as an analytical tool. Finally, the feasibility of creating refractory vector strains by genetic manipulation and releasing them into the environment is assessed in relation to its future use as a disease control strategy.

Animals↗

Calcofluor disrupts the midgut defense system in insects.

The insect midgut is generally lined with a unique protective chitin/protein structure, the peritrophic membrane (PM). We demonstrated that in Trichoplusia ni larvae, the majority of PM proteins were assembled with chitin as a consequence of their chitin binding properties. These proteins could be dissociated from the PM in vitro by Calcofluor, a well-known chemical with chitin binding properties. The chitin binding characteristics of PM proteins were confirmed by their high affinity binding in vitro to regenerated chitin. In vivo assays demonstrated that Calcofluor could inhibit PM formation in five lepidopteran insects tested. The inhibition of T. ni PM formation by Calcofluor, was accompanied by increased larval susceptibility to baculovirus infection. Continuous inhibition of PM formation by Calcofluor resulted in retarded larval development and mortality. The destructive effect of Calcofluor on PM formation was demonstrated to be transient and reversible depending on the presence of Calcofluor within the midgut. In addition, degradation of the insect intestinal mucin was observed concurrently with the inhibition of PM formation by Calcofluor. Our studies revealed a potential novel approach to develop strategies for insect control by utilizing chitin binding molecules to specifically target PM formation in a broad range of insect pest species.

Animals↗

Identification of a novel class of insect glutathione S-transferases involved in resistance to DDT in the malaria vector Anopheles gambiae.

The sequence and cytological location of five Anopheles gambiae glutathione S-transferase (GST) genes are described. Three of these genes, aggst1-8, aggst1-9 and aggst1-10, belong to the insect class I family and are located on chromosome 2R, in close proximity to previously described members of this gene family. The remaining two genes, aggst3-1 and aggst3-2, have a low sequence similarity to either of the two previously recognized classes of insect GSTs and this prompted a re-evaluation of the classification of insect GST enzymes. We provide evidence for seven possible classes of insect protein with GST-like subunits. Four of these contain sequences with significant similarities to mammalian GSTs. The largest novel insect GST class, class III, contains functional GST enzymes including two of the A. gambiae GSTs described in this report and GSTs from Drosophila melanogaster, Musca domestica, Manduca sexta and Plutella xylostella. The genes encoding the class III GST of A. gambiae map to a region of the genome on chromosome 3R that contains a major DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] resistance gene, suggesting that this gene family is involved in GST-based resistance in this important malaria vector. In further support of their role in resistance, we show that the mRNA levels of aggst3-2 are approx. 5-fold higher in a DDT resistant strain than in the susceptible strain and demonstrate that recombinant AgGST3-2 has very high DDT dehydrochlorinase activity.

Amino Acid Sequence↗

Insect chitin synthase cDNA sequence, gene organization and expression.

Chitin is a major component of the cuticle of arthropods. However, the synthesis of chitin is poorly understood. Feeding larvae of the insect Lucilia cuprina on the fungal chitin synthase competitive inhibitor, nikkomycin Z resulted in strong concentration-dependent mortality of the larvae (LD50 = 280 nM). This result demonstrates that chitin is an essential component of this insect. The complete cDNA and deduced amino-acid sequences of the first arthropod chitin synthase-like protein, LcCS-1, from the larvae of the insect L. cuprina have been determined. The cDNA sequence is 5757 bp in length and codes for a large complex protein containing 1592 amino acids (Mr = 180 717). Analysis of the whole protein sequence reveals low, but significant, similarity to yeast chitin synthases with stronger areas of conservation centred on local regions implicated in the active sites of the yeast enzymes. Strikingly, LcCS-1 contains 15-18 potential transmembrane segments, indicating that the protein is an integral membrane protein. Two alternative topographical models of LcCS-1 are described, which involve its association with either the plasma membrane or the membrane of intracellular vesicles. LcCS-1 mRNA is produced in all life stages of the insect with expression in the larval stage limited to the integument and trachea. In a third instar larva the mRNA was localized to a single layer of epidermal cells immediately underlying the procuticle region of the integument. cDNA or genomic sequences that are highly related to fragments of LcCS-1 were demonstrated in three insect orders, one arachnid and Caenorhabditis elegans, thereby attesting to the importance of this enzyme in these chitin-producing organisms. Bioinformatics has been used to deduce the gene sequence and organization of the highly homologous Drosophila melanogaster orthologue of LcCS-1, DmCS-1.

Amino Acid Sequence↗

Polydnavirus infection inhibits synthesis of an insect plasma protein, arylphorin.

The wasp Campoletis sonorensis injects a segmented, double-stranded DNA polydnavirus (CsPDV) along with its egg during parasitization of Heliothis virescens larvae. After parasitization, CsPDV protects the wasp egg and larva by selectively disabling the host's cellular immune response. Other host physiological systems including growth and development are affected to the apparent benefit of the parasite. To begin the characterization of the biochemical effects and mode of action of CsPDV on host growth, the titre of a developmentally regulated insect storage protein, arylphorin, was studied. Parasitized or virus-infected insects had substantially less circulating arylphorin than control insects. Fat bodies from parasitized larvae also synthesized less arylphorin in vitro. However, Northern blots of total RNA from parasitized and non-parasitized, control insects showed that the arylphorin transcript level was unaffected by parasitization suggesting a biochemical block at the translational level. In vitro translation followed by immunoprecipitation of arylphorin indicated that the mRNA was present and translatable at equal levels in both parasitized and control insects. Injection of purified virus elicited the response observed in naturally parasitized larvae, demonstrating that the effect on arylphorin synthesis is mediated, either directly or indirectly, by polydnavirus gene product(s).

Animals↗

Insect vectors of phytoplasmas.

Plant diseases caused by, or associated with, phytoplasmas occur in hundreds of commercial and native plants, causing minor to extensive damage. Insect vectors, primarily leafhoppers, planthoppers, and psyllids, have been identified for relatively few phytoplasma diseases, limiting the capacity of managers to make informed decisions to protect crops and endangered indigenous plants. In the past two decades our knowledge of insect vector-phytoplasma interactions has increased dramatically, allowing researchers to make more accurate predictions about the nature and epidemiology of phytoplasma diseases. These better-characterized systems also may provide clues to the identity of insect vectors of other phytoplasma-associated diseases. We review the literature addressing the ecology of insect vectors, phytoplasma-insect ecological and molecular interactions, vector movement and dispersal, and possible management strategies with an emphasis on research from the past 20 years.

Animals↗

Antibacterial peptides isolated from insects.

Insects are amazingly resistant to bacterial infections. To combat pathogens, insects rely on cellular and humoral mechanisms, innate immunity being dominant in the latter category. Upon detection of bacteria, a complex genetic cascade is activated, which ultimately results in the synthesis of a battery of antibacterial peptides and their release into the haemolymph. The peptides are usually basic in character and are composed of 20-40 amino acid residues, although some smaller proteins are also included in the antimicrobial repertoire. While the proline-rich peptides and the glycine-rich peptides are predominantly active against Gram-negative strains, the defensins selectively kill Gram-positive bacteria and the cecropins are active against both types. The insect antibacterial peptides are very potent: their IC50 (50% of the bacterial growth inhibition) hovers in the submicromolar or low micromolar range. The majority of the peptides act through disintegrating the bacterial membrane or interfering with membrane assembly, with the exception of drosocin, apidaecin and pyrrhocoricin which appear to deactivate a bacterial protein in a stereospecific manner. In accordance with their biological function, the membrane-active peptides form ordered structures, e.g. alpha-helices or beta-pleated sheets and often cast permeable ion-pores. Their cytotoxic properties were exploited in in vivo studies targeting tumour progression. Although the native peptides degrade quickly in biological fluids other than insect haemolymph, structural modifications render the peptides resistant against proteases without sacrificing biological activity. Indeed, a pyrrhocoricin analogue shows lack of toxicity in vitro and in vivo and protects mice against experimental Escherichia coli infection. Careful selection of lead molecules based on the insect antibacterial peptides may extend their utility and produce viable alternatives to the conventional antimicrobial compounds for mammalian therapy.

Animals↗

Insect-virus relationships: sifting by informatics.

Several groups of large DNA viruses successfully utilise the rich resource provided by insect hosts. Defining the mechanisms that enable these pathogens to optimise their relationships with their hosts is of considerable scientific and practical importance, but our understanding of the processes involved is, as yet, rudimentary. Here we describe an informatics-based approach that uses comparison of viral genomic sequences to identify candidate genes likely to be specifically involved in this process. We hypothesise that such genes should satisfy two essential criteria, namely, that they should be (i) present in those members of a virus family that infect insects, but absent from those that infect other hosts, and (ii) found in at least two unrelated taxa of insect viruses. These criteria currently identify six groups of viral genes, including one that encodes the fusolin/gp37 proteins. Demonstration that the fusolin/gp37 proteins can enhance oral infectivity of insect viruses provides a primary validation of this approach to the examination of insect-virus relationships.

Animals↗

Leads for insect neuropeptide mimetic development.

Insect neuropeptides mediate a number of physiological processes critical for insect survival. The numerous neuropeptide sequences that have been reported present an opportunity to decipher the chemical and conformational requirements for neuropeptide-receptor interactions. Chemical and conformational requirements for activity represent a "template" from which agonist/antagonist peptide mimetics, with the potential to disrupt critical insect processes, can be developed. Information on structural requirements is presented for three neuropeptide families: the sulfakinins, pyrokinins, and leucokinin/achetakinins, including active core size, important side chains, peptide superagonists, and new data on pseudopeptide modification of the N- and C-terminal regions. Members of these peptide families have been associated with a variety of physiological activities such as myotropism, pheromonotropism, diapause induction, and diuresis in a number of insects. Spectroscopic data coupled with computer molecular dynamics/graphics studies on conformationally restricted analogs of insect neuropeptides reveal information on the active conformation adopted at the receptor site. Routes to development of peptide-mimetics from neuropeptide templates are discussed.

Amino Acid Sequence↗

An active insect kinin analog with 4-aminopyroglutamate, a novel cis-peptide bond, type VI beta-turn motif.

The insect kinins are potent diuretic peptides that preferentially form a cis-Pro, type VI beta-turn. An insect kinin analog containing (2S,4S)-4-aminopyroglutamate, a novel cis-peptide bond, type VI beta-turn motif, demonstrates significant activity in the physiological range in a cricket diuretic assay. This is the first instance of a 4-aminopyroglutamate analog of a peptide with a preference for a type VI turn that demonstrates significant bioactivity. The results provide further confirmatory evidence for the active conformation of the insect kinins, and a new scaffold with which to design biostable, peptidomimetic analogs capable of disrupting critical insect kinin-regulated processes in insects.

Animals↗

Beta-amino acid analogs of an insect neuropeptide feature potent bioactivity and resistance to peptidase hydrolysis.

Insect neuropeptides of the insect kinin class share a common C-terminal pentapeptide sequence F(1)X(1)(2)X(2)(3)W(4)G(5)-NH(2) (X(2)(3) = P, S, A) and regulate such critical physiological processes as water balance and digestive enzyme release. Analogs of the insect kinin class, in which the critical residues of F(1), P(3), and W(4) were replaced with beta(3)-amino acid or their beta(2)-homo-amino acid variants, have been synthesized by the solid phase peptide strategy. The resulting single- and double-replacement analogs were evaluated in an insect diuretic assay and enzyme digestion trials. Analogs modified in the core P(3) position produce a potent and efficacious diuretic response that is not significantly different from that obtained with the endogenous achetakinin peptides. The analogs also demonstrate enhanced resistance to hydrolysis by ACE and NEP, endopeptidases that inactivate the natural insect neuropeptides. This paper describes the first instance of beta-amino acids analogs of an arthropod peptide that demonstrate significant bioactivity and resistance to peptidase degradation.

Amino Acid Sequence↗