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PubMed2002

Neuropeptides in the nervous system of Drosophila and other insects: multiple roles as neuromodulators and neurohormones.

Neuropeptides in insects act as neuromodulators in the central and peripheral nervous system and as regulatory hormones released into the circulation. The functional roles of insect neuropeptides encompass regulation of homeostasis, organization of behaviors, initiation and coordination of developmental processes and modulation of neuronal and muscular activity. With the completion of the sequencing of the Drosophila genome we have obtained a fairly good estimate of the total number of genes encoding neuropeptide precursors and thus the total number of neuropeptides in an insect. At present there are 23 identified genes that encode predicted neuropeptides and an additional seven encoding insulin-like peptides in Drosophila. Since the number of G-protein-coupled neuropeptide receptors in Drosophila is estimated to be around 40, the total number of neuropeptide genes in this insect will probably not exceed three dozen. The neuropeptides can be grouped into families, and it is suggested here that related peptides encoded on a Drosophila gene constitute a family and that peptides from related genes (orthologs) in other species belong to the same family. Some peptides are encoded as multiple related isoforms on a precursor and it is possible that many of these isoforms are functionally redundant. The distribution and possible functions of members of the 23 neuropeptide families and the insulin-like peptides are discussed. It is clear that each of the distinct neuropeptides are present in specific small sets of neurons and/or neurosecretory cells and in some cases in cells of the intestine or certain peripheral sites. The distribution patterns vary extensively between types of neuropeptides. Another feature emerging for many insect neuropeptides is that they appear to be multifunctional. One and the same peptide may act both in the CNS and as a circulating hormone and play different functional roles at different central and peripheral targets. A neuropeptide can, for instance, act as a coreleased signal that modulates the action of a classical transmitter and the peptide action depends on the cotransmitter and the specific circuit where it is released. Some peptides, however, may work as molecular switches and trigger specific global responses at a given time. Drosophila, in spite of its small size, is now emerging as a very favorable organism for the studies of neuropeptide function due to the arsenal of molecular genetics methods available.

Amino Acid Sequence↗
PubMed2001

Characterization and cloning of a Tenebrio molitor hemolymph protein with sequence similarity to insect odorant-binding proteins.

The yellow mealworm beetle, Tenebrio molitor, produces a number of moderately abundant low molecular weight hemolymph proteins ( approximately 12 kDa) which behave in a similar manner during purification and share antigenic epitopes. The cDNA sequence of the major component (THP12) was determined and the deduced protein sequence was found to be similar to those of insect odorant-binding proteins. Southern blot analysis suggests that at least some of the diversity in this family of proteins is encoded at the gene level. Both northern and western blot analysis indicate that THP12 is present in a variety of developmental stages and both sexes. THP12 was originally classified as an antifreeze protein, but the lack of antifreeze activity in the recombinant protein, as well as the clear separation of the antifreeze activity from THP12 following HPLC purification, has ruled out this function. The abundance of THP12, the similarity of THP12 to insect odorant-binding proteins, and the presence of hydrophobic cavities inside the protein (Rothemund et al., A new class of hexahelical insect proteins revealed as putative carriers of small hydrophobic ligands. Structure, 7 (1999) 1325-1332.) suggest that THP12 may function to carry non-water soluble compounds in the hemolymph. THP12 is also similar, particularly in structurally important regions, to other insect proteins from non-sensory tissues, suggesting the existence of a large family of carrier proteins which may perform diverse functions throughout the insect.

Amino Acid Sequence↗
PubMed2002

Expression and functional characterization of a nucleotide sugar transporter from Drosophila melanogaster: relevance to protein glycosylation in insect cell expression systems.

Insect cells are used routinely to express recombinant mammalian glycoproteins. However, insect protein glycosylation pathways are not well understood and appear to differ from those of mammalian cells. One way to more clearly evaluate the protein glycosylation potential of insect cells is to use the Drosophila melanogaster genome to identify genes that might encode relevant functions. These genes can then be expressed and the functions of the gene products directly evaluated by biochemical assays. In this study, we used this approach to determine the function of a putative Drosophila nucleotide sugar transporter gene. The results showed that this gene encodes a protein that can transport UDP-galactose, but not CMP-sialic acid. Thus, Drosophila encodes at least some of the infrastructure needed to produce glycoproteins with complex glycans, but this particular gene product does not directly support glycoprotein sialylation. These findings are relevant to insect cell biology and to an informed consideration of insect cell expression systems as tools for recombinant glycoprotein production.

Amino Acid Sequence↗
PubMed2003

Insect adipokinetic hormones: release and integration of flight energy metabolism.

Insect flight involves mobilization, transport and utilization of endogenous energy reserves at extremely high rates. Peptide adipokinetic hormones (AKHs), synthesized and stored in neuroendocrine cells, integrate flight energy metabolism. The complex multifactorial control mechanism for AKH release in the locust includes both stimulatory and inhibitory factors. The AKHs are synthesized continuously, resulting in an accumulation of AKH-containing secretory granules. Additionally, secretory material is stored in large intracisternal granules. Although only a limited part of these large reserves appears to be readily releasable, this strategy allows the adipokinetic cells to comply with large variations in secretory demands; changes in secretory activity do not affect the rate of hormone biosynthesis. AKH-induced lipid release from fat body target cells has revealed a novel concept for lipid transport during exercise. Similar to sustained locomotion of mammals, insect flight activity is powered by oxidation of free fatty acids derived from endogenous reserves of triacylglycerol. However, the transport form of the lipid in the circulatory system is diacylglycerol (DAG) that is delivered to the flight muscles associated with lipoproteins. While DAG is loaded onto the multifunctional insect lipoprotein, high-density lipophorin (HDLp) and multiple copies of the exchangeable apolipoprotein III (apoLp-III) associate reversibly with the expanding particle. The resulting low-density lipophorin (LDLp) specifically shuttles DAG to the working muscles. Following DAG hydrolysis by a lipophorin lipase, apoLp-III dissociates from the particle, regenerating HDLp that is re-utilized for lipid uptake at the fat body cells, thus functioning as an efficient lipid shuttle mechanism. Many structural elements of the lipoprotein system of insects appear to be similar to their counterparts in mammals; however, the functioning of the insect lipoprotein in energy transport during flight activity is intriguingly different.

Animals↗
PubMed2006

Polymerase chain reaction identification of a hymenopteran insect in the cornea: a case report.

The type of corneal injuries associated with insect encounters is related to the composition of the foreign body. However, previous reports on corneal foreign bodies as insects were rarely based on scientific evidence. Here, we report on a 49-year-old male who was stung in his left eye by an unknown insect. Emergent keratotomy was performed to remove the embedded corneal foreign body. The removed foreign body was observed under light microscopy, and a fragment of insect was suspected. The sample was sent for molecular analysis. The polymerase chain reaction product was sequenced, subjected to a BLAST search, and identified as an ichneumonoid member of the insect order Hymenoptera.

Animals↗
PubMed2005

ACE inhibitory activity in enzymatic hydrolysates of insect protein.

In this paper, ACE inhibitory activity in insect protein hydrolyzed by various enzymes (gastrointestinal proteases, alcalase, and thermolysin) is reported for the first time. Four insects of different insect orders were tested: Spodoptera littoralis (Lepidoptera), Bombyx mori (Lepidoptera), Schistocerca gregaria (Orthoptera), and Bombus terrestris (Hymenoptera). ACE inhibitory activity was measured by two different methods: a spectrophotometric method using FAPGG (2-furanacryloyl-phenylalanyl-glycyl-glycine) as substrate, and an HPLC method using dansyltriglycine (DTG) as substrate. Hydrolysis of the insect protein resulted in an increased ACE inhibitory activity. Overall, the highest ACE inhibitory activity was obtained after gastrointestinal digestion. These results suggest a role for insect protein as antihypertensive component in functional foods and nutraceuticals. Furthermore, the ACE inhibitory activity differed according to the method used. As a consequence, there is a need to standardize methodologies to evaluate ACE inhibitory activity.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2004

Field-testing of methyl salicylate for recruitment and retention of beneficial insects in grapes and hops.

Evidence for recruitment and retention of beneficial insects in grapes and hops using controlled-release dispensers of methyl salicylate (MeSA), a component of herbivore-induced volatile blends, is presented. In a replicated experiment conducted in a juice grape vineyard, sticky cards in blocks baited with MeSA captured significantly greater numbers of five species of predatory insects (Chrysopa nigricornis, Hemerobius sp., Deraeocoris brevis, Stethorus punctum picipes, Orius tristicolor) than unbaited blocks. Four insect families (Syrphidae, Braconidae, Empididae, Sarcophagidae) were also significantly more abundant in the MeSA-baited blocks, as indicated by sticky card captures. Canopy shake samples and sticky card monitoring conducted in a MeSA-baited, unsprayed hop yard indicated development and maintenance of a beneficial arthropod population that was nearly four times greater than that present in an unbaited reference yard. Four times as many S. punctum picipes and six times as many O. tristicolor were sampled in the MeSA yard. Similar contrasts in abundance of these predators and others were apparent when compared with levels recorded in the yard in previous years. The large population of predatory insects in the MeSA-baited hop yard was associated with a dramatic reduction in spider mite numbers, the major arthropod pest of hops, in late June, and subeconomic populations were maintained for the rest of the season. The evidence presented here is highly suggestive that the use of controlled-release MeSA in a crop could increase recruitment and residency of populations of certain beneficial insects. This strategy may have the potential to enhance the efficacy and reliability of conservation biological control in crop pest management.

Animals↗
PubMed2002

A transgene-based, embryo-specific lethality system for insect pest management.

Biological approaches to insect pest management offer alternatives to pesticidal control. In area-wide control programs that cover entire regions, the sterile insect technique (SIT) can be used to successfully suppress economically important pest species by the mass release of sterilized pest organisms. However, conventional sterilization by ionizing radiation reduces insect fitness, which can result in reduced competitiveness of the sterilized insects. Here we report a transgene-based, dominant embryonic lethality system that allows for generation of large quantities of competitive but sterile insects without the need of irradiation. The system involves the ectopic expression of a hyperactive pro-apoptotic gene that causes embryo-specific lethality when driven by the tetracycline-controlled transactivator (tTA) under the regulation of a cellularization gene enhancer-promoter. We have successfully tested this system in Drosophila melanogaster. The embryonic lethality can be suppressed maternally, which will allow it to be combined with transgenic female-specific lethality systems to raise only vigorous but sterile males.

Animals↗
PubMed2003

Insect immunity and its implication in mosquito-malaria interactions.

Insects' resistance to infectious agents is essential for their own survival and also for the health of the plant, animal and human populations with which they closely interact. Several of the major human diseases are spread by insects and are rapidly expanding as a result of the development of insecticide resistance in vectors and drug resistance in parasites. A vector insects' permissiveness to a pathogen, and hence the spread of the disease, will largely depend on the compatibility of the molecular interactions between the two species and the capability of the insect immune system to recognize and kill the pathogen. The innate immune system comprises a variety of components and mechanisms that can discriminate between different microorganisms and mount specific responses to control pathogenic infections. An impressive body of knowledge on the insects' innate immunity has been generated from studies in the model organism Drosophila. These studies are now guiding the exploration of the immune system in the vector mosquito of human malaria, Anopheles, and its implication in the elimination of parasites. Anopheles immune responses have been linked to parasite losses and some refractory mosquitoes can kill all parasites through specific defence mechanisms. The recently sequenced Drosophila and Anopheles genomes provide a detailed and comparative view on their immune gene repertoires that in combination with post-genomic analyses is used to further dissect the complex mechanisms of Plasmodium killing in the mosquito.

Animals↗
PubMed2005

Inhibitor kappaB-like proteins from a polydnavirus inhibit NF-kappaB activation and suppress the insect immune response.

Complex signaling pathways regulate the innate immune system of insects, with NF-kappaB transcription factors playing a central role in the activation of antimicrobial peptides and other immune genes. Although numerous studies have characterized the immune responses of insects to pathogens, comparatively little is known about the counter-strategies pathogens have evolved to circumvent host defenses. Among the most potent immunosuppressive pathogens of insects are polydnaviruses that are symbiotically associated with parasitoid wasps. Here, we report that the Microplitis demolitor bracovirus encodes a family of genes with homology to inhibitor kappaB (IkappaB) proteins from insects and mammals. Functional analysis of two of these genes, H4 and N5, were conducted in Drosophila S2 cells. Recombinant H4 and N5 greatly reduced the expression of drosomycin and attacin reporter constructs, which are under NF-kappaB regulation through the Toll and Imd pathways. Coimmunoprecipitation experiments indicated that H4 and N5 bound to the Rel proteins Dif and Relish, and N5 also weakly bound to Dorsal. H4 and N5 also inhibited binding of Dif and Relish to kappaB sites in the promoters of the drosomycin and cecropin A1 genes. Collectively, these results indicate that H4 and N5 function as IkappaBs and, circumstantially, suggest that other IkappaB-like gene family members are involved in the suppression of the insect immune system.

Amino Acid Sequence↗
PubMed1989

Complementation of recombinant baculoviruses by coinfection with wild-type virus facilitates production in insect larvae of antigenic proteins of hepatitis B virus and influenza virus.

We describe the coinfection of insects with wild-type and recombinant baculoviruses in which the polyhedrin gene promoter is used to express hepatitis B virus envelope protein (hepatitis B virus surface antigen; HBsAg) or influenza A virus neuraminidase (NA). Viruses were administered per os to larvae of the cabbage looper, Trichoplusia ni, causing an infection that within 5 days resulted in the production of approximately 0.15 mg of HBsAg per insect, representing 1.5% of the total extracted protein, or approximately 2.8 mg of NA per insect, representing 28% of the total extractable protein. The HBsAg and NA produced by infected larvae were purified from insect lysates. These proteins were antigenic as determined by conformation-dependent immunoassays. The NA was enzymatically active with conventional substrates. The method of infection described allows genetic complementation by wild-type virus of recombinant viruses lacking the polyhedrin gene essential for infection per os and has implications for the high-yield production in insect larvae of other recombinant proteins of baculoviruses.

Animals↗
PubMed1990

Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae.

Human adenosine deaminase (EC 3.5.4.4), a key purine salvage enzyme essential for immune competence, has been overproduced in Spodoptera frugiperda cells and in Trichoplusia ni (cabbage looper) larvae infected with recombinant baculovirus. The coding sequence of human adenosine deaminase was recombined into a baculovirus immediately downstream from the strong polyhedrin gene promoter. Approximately 60 hr after infection of insect cells with the recombinant virus, maximal levels of intracellular adenosine deaminase mRNA, protein, and enzymatic activity were detected. The recombinant human adenosine deaminase represented 10% of the total cellular protein and exhibited a specific activity of 70 units/mg of protein in crude homogenate. This specific activity is 70-350 times greater than that exhibited by the enzyme in homogenates of the two most abundant natural sources of human adenosine deaminase, thymus and leukemic cells. When the recombinant virus was injected into insect larvae, the maximum recombinant enzyme was produced 4 days postinfection and represented about 2% of the total insect protein with a specific activity of 10-25 units/mg of protein. The recombinant human adenosine deaminase was purified to homogeneity from both insect cells and larvae and demonstrated to be identical to native adenosine deaminase purified from human cells with respect to molecular weight, interaction with polyclonal anti-adenosine deaminase antibody, and enzymatic properties. A pilot purification yielded 8-9 mg of homogeneous enzyme from 22 larvae. The production of large quantities of recombinant human adenosine deaminase in insect larvae is inexpensive and rapid and eliminates the need for specialized facilities for tissue culture. This method should be applicable to large-scale production of many recombinant proteins.

Adenosine Deaminase↗
PubMed1999

Homeotic genes and the arthropod head: expression patterns of the labial, proboscipedia, and Deformed genes in crustaceans and insects.

cDNA fragments of the homologues of the Drosophila head homeotic genes labial (lab), proboscipedia (pb), and Deformed (Dfd) have been isolated from the crustacean Porcellio scaber. Because the accumulation domains of the head homeotic complex (Hox) genes had not been previously reported for crustaceans, we studied the expression patterns of these genes in P. scaber embryos by using in situ hybridization. The P. scaber lab homologue is expressed in the developing second antennal segment and its appendages. This expression domain in crustaceans and in the homologous intercalary segment of insects suggests that the lab gene specified this metamere in the last common ancestor of these two groups. The expression domain of the P. scaber pb gene is in the posterior part of the second antennal segment. This domain, in contrast to that in insects, is colinear with the domains of other head genes in P. scaber, and it differs from the insect pb gene expression domain in the posterior mouthparts, suggesting that the insect and crustacean patterns evolved independently from a broader ancestral domain similar to that found in modern chelicerates. P. scaber Dfd is expressed in the mandibular segment and paragnaths (a pair of ventral mouthpart structures associated with the stomodeum) and differs from insects, where expression is in the mandibular and maxillary segments. Thus, like pb, Dfd shows a divergent Hox gene deployment. We conclude that homologous structures of the mandibulate head display striking differences in their underlying developmental programs related to Hox gene expression.

Amino Acid Sequence↗
PubMed2002

Outbreaks of forest defoliating insects in Japan, 1950-2000.

In Japan, several forest-defoliating insects reach outbreak levels and cause serious defoliation. Stand mortality sometimes occurs after severe defoliation. However, in general, tree mortality caused by insect defoliation is low because of the prevailing moist climate in Japan. Evergreen conifers are more susceptible to tree mortality as a result of insect defoliation whereas deciduous broad-leaved trees are seldom killed. Insect defoliation occurs more frequently in man-made environments such as among shade trees, orchards, and plantations than in natural habitats. Outbreaks of some defoliators tend to occur in stands of a particular age: e.g. outbreaks of the pine caterpillar, Dendrolimus spectabilis Butler (Lepidoptera: Lasiocampidae) occur more frequently in young pine plantations. In contrast, defoliation caused by outbreaks of lepidopterous and hymenopterous pests in larch plantations is more frequent with stand maturation. There is a relationship between outbreaks of some defoliators and altitude above sea level. Most outbreaks of forest defoliators were terminated by insect pathogens that operated in a density-dependent fashion. Since the 1970s, Japan has been prosperous and can afford to buy timber from abroad. More recently, there has been an increasing demand for timber in Japan, that coincides with a huge demand internationally, so that the country will need to produce more timber locally in the future. The increasing pressure on the forestry industry to meet this demand will require more sophisticated methods of pest control coupled with more sustainable methods of silviculture.

Animals↗
PubMed1994

Genetic manipulation of insect vectors as a strategy for the control of vector-borne disease.

A variety of very effective methods have been employed for suppressing insect 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 alternative strategies for vector control. What is required is a completely novel approach to either 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 genome manipulation in insects and the progress which has been made to create transgenic vector insects are reviewed. The potential applications of this methodology are then explored in the context of its future use for the control of vector-borne diseases.

Animals↗
PubMed1997

Evaluation of commercial insect repellents on human skin against Aedes aegypti (Diptera: Culicidae).

An olfactometer was used to evaluate the efficacy of selected commercial insect repellent products against Aedes aegypti (L.). A comparison of 12 commercial repellent products was made on human skin. The products tested included 2 natural oil insect repellent formulations (Buzz Away and Green Ban) containing plant extracts, 2 proprietary products (Skin-So-Soft lotion and bath oil), and 8 commercial deet preparations in various concentrations and forms of spray, aerosol, stick, cream, and lotion. Behavioral responses and time to probe were determined in triplicate using 10 female mosquito challenges per replicate for each product. Generally, products with higher concentrations of deet were found to have longer repellence times. OFF spray and Muskol lotion offered the longest repellence times. However, there was no significant difference in time for mosquitoes to probe among the formulations. Skin-So-Soft lotion and bath oil were not as effective as deet in repelling Ae. aegypti. Natural oil insect repellent formulations offered essentially no repellency against Ae. aegypti. This is a simple and reproducible method to evaluate the efficacy of insect repellents and is recommended for preliminary screening of new insect repellents or formulations.

Aedes↗
PubMed2005

Filter paper for preservation, storage, and distribution of insect and pathogen DNA samples.

Proper DNA storage is critical for studies involving genetic analysis of insects and for molecular diagnostics of pathogens carried by them. Molecular surveillance of pathogens carried by insects can involve screening of thousands of insect DNA samples. Problems with storage and degradation of these samples can arise. In this study, a simple filter paper-based method for storage and preservation of insect DNA was evaluated using polymerase chain reaction (PCR). DNA was isolated from individual house fly, Musca domestica L., adults by using a cell lysis technique. From 50 house flies known to carry Campylobacter spp., a portion of the DNA sample was stored frozen and another portion was pipetted onto filter paper. At monthly intervals for 7 mo, PCR was conducted using 1 microl of the frozen DNA sample and a 2.0-mm disk from the filter paper samples as the PCR template. Two markers were used, a 450-bp region of the insect mitochondrial DNA (mtDNA) ND5 gene and a 857-bp region of the Campylobacter spp. mtDNA 16S rDNA gene. PCR amplification was successful for all of the samples regardless of the storage method. The filter paper method is a simple and economical way to store, preserve, and distribute DNA samples for PCR analysis.

Animals↗
PubMed2006

Emergency treatment and prevention of insect-sting anaphylaxis.

PURPOSE OF REVIEW: Hymenoptera stings are thought to cause systemic allergic reactions in 0.4-5% of individuals, and to account for 40-100 deaths annually in the USA. This review examines current research on insect-sting anaphylaxis in the emergency department (ED), and provides suggestions on how to improve ED treatment and prevention. RECENT FINDINGS: Few published studies since January 2005 examine the emergency management of insect-sting allergic reactions. Earlier studies found that few ED patients with this problem received health education, a prescription for self-injectable epinephrine or referral to an allergy specialist at ED discharge. A recent multicenter study by our group demonstrated that ED patients with insect-sting allergic reactions continue to receive care discordant with national guidelines for the emergency management of anaphylaxis. SUMMARY: Concordance with national guidelines for the treatment of anaphylaxis remains poor among insect-sting allergy patients. The development of a simple, clinical definition of anaphylaxis is urgently needed to facilitate identification of cases. Such a definition would help tremendously with dissemination of emergency management guidelines and the creation of systems for their implementation. All of these steps are essential for the improved treatment and prevention of insect-sting anaphylaxis in the ED.

Anaphylaxis↗