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Museum specimens reveal the genomic consequences of long-term population decline in an insect pollinator.

Global insect pollinator populations are under threat, with reported declines attributed to increasing habitat loss, pesticide use, and disease. Tracking how genetic diversity has changed over time could reveal the rate and extent of these declines, and the adaptive capacity of affected species-providing an important complement to habitat-based conservation efforts. However, few studies have been able to reconstruct suitable historical baselines to link genomic changes with population change. Here, we use whole genome data from 101 museum specimens of the declining moss carder bumblebee (Bombus muscorum) collected across Britain and Ireland between 1894 and 2019 to reveal a dramatic drop in genetic diversity over the last century. We find a substantial (∼24.6%) reduction in genome-wide heterozygosity across Britain during this period. In England and Wales, where habitat fragmentation is most pronounced, we observe a 2.86-fold increase in runs of homozygosity, commensurate with population fragmentation and isolation. Our results reveal the extent to which human-induced environmental change can lead to severe decadal-scale genomic erosion in a functionally important insect. Identified using DNA from historic museum collections, our approach has widespread applicability for insect conservation and understanding the evolutionary consequences of environmental change.

Animals↗

Molecular characterization and phylogenetic relationships of a protein with potential oxygen-binding capabilities in the grasshopper embryo. A hemocyanin in insects?

Arthropodan hemocyanins, prophenoloxidases (PPOs), and insect hexamerins form a superfamily of hemolymph proteins that we propose to call the AHPH superfamily. The evolutionary and functional relationships of these proteins are illuminated by a new embryonic hemolymph protein (EHP) that is expressed during early stages of development in the grasshopper embryo. EHP is a 78-kDa soluble protein present initially in the yolk sac content, and later in the embryonic hemolymph. Protein purification and peptide sequencing were used to identify an embryonic cDNA clone coding for EHP. In situ hybridization identifies hemocytes as EHP-expressing cells. As deduced from the cDNA clone, EHP is a secreted protein with two potential glycosylation sites. Sequence analysis defines EHP as a member of the AHPH superfamily. Phylogenetic analyses with all the currently available AHPH proteins, including EHP, were performed to ascertain the evolutionary history of this protein superfamily. We used both the entire protein sequence and each of the three domains present in the AHPH proteins. The phylogenies inferred for each of the domains suggest a mosaic evolution of these protein modules. Phylogenetic and multivariate analyses consistently group EHP with crustacean hemocyanins and, less closely, with insect hexamerins, relative to cheliceratan hemocyanins and PPOs. The grasshopper protein rigorously preserves the residues involved in oxygen binding, oligomerization, and allosteric regulation of the oxygen transport proteins. Although insects were thought not to have hemocyanins, we propose that EHP functions as an oxygen transport or storage protein during embryonic development.

Amino Acid Sequence↗

Molecular docking of substrates and inhibitors in the catalytic site of CYP6B1, an insect cytochrome p450 monooxygenase.

Furanocoumarins represent plant toxins that are used in the treatment of a variety of skin diseases and are metabolized by cytochrome p450 monooxygenases (p450s) existing in insects such as Papilio polyxenes (the black swallowtail). To elucidate the active site in the CYP6B1 protein that is the principal p450 existing in this species, we have constructed a homology model of it based on sequence and structure alignments with the bacterial CYP102 protein whose crystal structure has been defined and with the insect CYP6B4 protein that also metabolizes furanocoumarins. In the derived CYP6B1 model, Phe116 and His117 in SRS1, Phe371 in SRS5 and Phe484 in SRS6 contribute to the formation of a resonant network that stabilizes the p450's catalytic site and allows for interactions with its furanocoumarin substrates. The first two of these residues are absolutely conserved in all members of the insect CYP6B subfamily and the last two are variable in different members of the CYP6B subfamily. A combination of theoretical and experimental docking analyses of two substrates (xanthotoxin and bergapten) and two inhibitors (coumarin and pilocarpine) of this p450 provide significant information on the positioning of furanocoumarins within this catalytic pocket. Molecular replacement models based on the results of variations at two of these critical amino acids provide support for our furanocoumarin-docked model and begin to rationalize the altered substrate reactivities observed in experimental analyses.

5-Methoxypsoralen↗

Stinging insect hypersensitivity in children.

In the past two decades, many advances have been made in the treatment of patients with insect venom sensitivity. An effective treatment (venom immunotherapy) has been developed and indications for therapy refined. In the past year, reports concerning fatal toxic reactions to multiple stings, Africanized ("killer") bees, bumblebee venom allergy and treatment, and reactions to fire ant stings have been published. Papers have also appeared on the use of sting challenges in the diagnostic evaluation of insect sting allergy, laboratory investigations of the mechanism of action of venom immunotherapy, and the outcome of discontinuing venom treatment after 5 years in insect-allergic patients.

Animals↗

Abiotic conditions can modify the penetrance of transgene-based lethality systems for insect population control.

Modern genetic biocontrol techniques for insect pest management, when compared to chemical insecticide spraying, offer high species specificity and reduced environmental impact, and some of these methods require the environmental release of genetically modified (GM) insects. Because organisms exposed to different environments often show variability in phenotype and gene expression, it is likely that GM insects will also experience environmentally mediated variation, potentially compromising pest control efficiency. This study examines the impact of temperature and nutrition on the early embryonic Tet-off conditional lethality system in Drosophila melanogaster. By independently manipulating parental and offspring environments, we assessed how exposure to variable environments influenced the probability of larval hatching and the transcript abundance of the transgenic system. Our findings revealed that: (i) transgene performance distinctly responds to temperature and nutrition; (ii) thermal stress has a greater impact when embryos, rather than parents, are exposed; and (iii) extreme nutritional conditions can markedly reduce the penetrance of transgenic lethality. Although changes in transgene transcript abundance were observed across environments, these changes did not fully explain the phenotypic variation, suggesting that factors downstream of transcription probably drive variation in transgenic lethality.

Animals↗

Synthesis of immunogenic, but non-infectious, poliovirus particles in insect cells by a baculovirus expression vector.

A baculovirus expression vector (AcLeon) derived from Autographa californica nuclear polyhedrosis virus (AcNPV) was prepared containing the complete 6.6 kb coding region of the P3/Leon/37 strain of poliovirus type 3 placed under the control of the AcNPV polyhedrin promoter. The recombinant virus was used to infect Spodoptera frugiperda insect cells. As demonstrated by use of the appropriate antibodies, infected insect cells made poliovirus proteins that included the structural proteins VP0, VP1 and VP3. Poliovirus particles were recovered from extracts of the infected cells and demonstrated to be free from detectable levels of RNA and to be non-infectious in tissue culture. After particle purification by CsCl gradient centrifugation and immunization of outbred mice, antibodies to the structural proteins, including neutralizing antibodies, were obtained. Other recombinant baculoviruses, containing the majority of the capsid coding region of P3/Leon/37 (e.g. AcCAP21, nucleotide residues 742 to 3318), made an unprocessed precursor to the poliovirus structural proteins. These data suggested that processing of the poliovirus gene product by the AcLeon construct was catalysed by the poliovirus-encoded proteases. The results demonstrated that antigenic and immunogenic poliovirus proteins and empty particles can be made in insect cells by recombinant baculoviruses.

Animals↗

Insect sting anaphylaxis in patients without detectable serum venom-specific IgE.

Following insect sting anaphylaxis, twenty-five patients of over 500 evaluated, did not have detectable serum venom-specific IgE. Twenty-two were evaluated within 1 year of the reaction, fifteen within 6 months. Anaphylaxis occurred in six patients after their first sting exposure. The clinical features of the sting reaction were typical of the reaction occurring in insect-allergic patients with serum venom-specific IgE. Eleven of the twenty-five patients had negative venom skin tests. These observations suggest that a non-IgE mechanism may be responsible for a minority of insect sting reactions.

Adolescent↗

Insect allergy. Predictive value of diagnostic tests: a three-year follow-up study.

One hundred and six adults with various reactions to yellow jacket (YJ), honey bee (HB) or unidentified insects (UI) were tested for allergy to insect venoms. For various reasons none received immunotherapy. Individuals completed questionnaires annually for three consecutive years and described sting reactions within the previous season. Ninety subjects completed all the questionnaires and 77 of these were re-tested at the end of the period. Nine out of 25 patients reacted with a systemic reaction when re-stung. High IgE and low IgG venom-specific antibody levels indicated an unfavourable prognosis, since eight of 11 individuals who initially presented venom-specific IgE greater than RAST class 2 and venom-specific IgG below detection limit had systemic reactions at re-sting. No such reactions occurred in subjects with no specific IgE and only one out of six with specific IgE as well as IgG reacted systematically. Skin prick tests (SPT) of less than 3mm with YJ venom 1,000 micrograms/ml excluded later systemic symptoms to stings, whereas larger skin reactions gave an equal chance of systemic or local reactions at re-sting. In individuals not stung by UI, YJ and/or HB the decline in venom-specific IgG and IgE was significant, median values ranging from 41% to 75% over the 3-year period. The decline was unaffected by the type of sting reaction prior to the initial test. SPT results did not change significantly. The findings are relevant when testing patients several years after their last insect sting and the results might indicate that the antibody decline is accompanied by a decrease in clinical sensitivity.

Adult↗

Provocation test with a living insect as a diagnostic tool in systemic reactions to bee and wasp venom: a prospective study with emphasis on the clinical aspects.

The use of the sting challenge (SC) in insect allergy has brought into doubt the widely held belief that clinical data, specific IgE and the skin test are reliable tools in diagnosis and decision making with regard to therapy. We subjected ninety patients, known to have a systemic reaction to SC, in the Intensive Care Unit. Of these patients, 28% once more showed a systemic reaction; 72% had only a local reaction. No statistically significant difference could be demonstrated between positive and negative SC patients with regard to age; sex; the presence of allergic disease(s)--other than allergy to insects--and the severity of the preceding systemic reaction; the sting site and the related time interval; or specific IgE, IgG1, IgG4, their ratios and the skin test. A negative skin test and/or specific IgE, however, does not exclude the possibility of a recurrent systemic reaction. The long-term value of the SC appeared to be good since the results of the revision SC remained unchanged. Skin test and specific IgE are useful as diagnostic tools in establishing the insect concerned. SC provides information on the likelihood of a recurrent systemic reaction.

Adolescent↗

Allergy to venom from bee or wasp: the relation between clinical and immunological reactions to insect stings.

Sixty-three patients, applying to the casualty ward after a bee or wasp sting, were tested. On the day of admission, and 4 weeks later, blood was sampled to measure specific IgE and IgG against bee and wasp venom and total IgE. Four weeks after the insect sting, all patients were examined with skin-prick test (1, 10 and 100 micrograms/ml), and intracutaneous test (0.05 ml of extract 1.0 microgram/ml). The amount of venom-specific IgE and IgG increased significantly during the 4-week period, venom-specific IgE was most pronounced in patients experiencing the most severe clinical reactions. However, no significant correlations were found between the results of the immunological tests carried out 4 weeks after the insect sting and the clinical reaction upon the insect sting.

Animals↗

Identification of insect cell lines by DNA amplification fingerprinting (DAF).

Fingerprint profiles were generated from twenty insect cell lines spanning the Orders, Lepidoptera, Diptera, Coleoptera and Homoptera employing DNA amplification fingerprinting (DAF) with arbitrarily selected primers. The fingerprint pattern is a stable characteristic of the cell line because high and low passages generated the same profile. In addition, insect hosts and homologous cell lines generated similar profiles. All cell lines could be distinguished from each other with the following exceptions: Plutella xylostella (BCIRL-PX2-HNU3) and Mamestra brassicae (IZD-MB-0503) produced identical patterns to Trichoplusia ni (TN-CL1). Also Spodoptera exigua (UCR-SE-1C) produced the same profile as Spodoptera frugiperda (SF9) and the parent cell line IPLB-SF21. All these cases of identical patterns are believed to be due to cross contamination or mislabelling of cultures. DAF will serve as an additional, valuable and reliable technique for the identification of insect cell lines.

Aedes↗

Genomics of hybrid poplar (Populus trichocarpax deltoides) interacting with forest tent caterpillars (Malacosoma disstria): normalized and full-length cDNA libraries, expressed sequence tags, and a cDNA microarray for the study of insect-induced defences in poplar.

As part of a genomics strategy to characterize inducible defences against insect herbivory in poplar, we developed a comprehensive suite of functional genomics resources including cDNA libraries, expressed sequence tags (ESTs) and a cDNA microarray platform. These resources are designed to complement the existing poplar genome sequence and poplar (Populus spp.) ESTs by focusing on herbivore- and elicitor-treated tissues and incorporating normalization methods to capture rare transcripts. From a set of 15 standard, normalized or full-length cDNA libraries, we generated 139,007 3'- or 5'-end sequenced ESTs, representing more than one-third of the c. 385,000 publicly available Populus ESTs. Clustering and assembly of 107,519 3'-end ESTs resulted in 14,451 contigs and 20,560 singletons, altogether representing 35,011 putative unique transcripts, or potentially more than three-quarters of the predicted c. 45,000 genes in the poplar genome. Using this EST resource, we developed a cDNA microarray containing 15,496 unique genes, which was utilized to monitor gene expression in poplar leaves in response to herbivory by forest tent caterpillars (Malacosoma disstria). After 24 h of feeding, 1191 genes were classified as up-regulated, compared to only 537 down-regulated. Functional classification of this induced gene set revealed genes with roles in plant defence (e.g. endochitinases, Kunitz protease inhibitors), octadecanoid and ethylene signalling (e.g. lipoxygenase, allene oxide synthase, 1-aminocyclopropane-1-carboxylate oxidase), transport (e.g. ABC proteins, calreticulin), secondary metabolism [e.g. polyphenol oxidase, isoflavone reductase, (-)-germacrene D synthase] and transcriptional regulation [e.g. leucine-rich repeat transmembrane kinase, several transcription factor classes (zinc finger C3H type, AP2/EREBP, WRKY, bHLH)]. This study provides the first genome-scale approach to characterize insect-induced defences in a woody perennial providing a solid platform for functional investigation of plant-insect interactions in poplar.

Animals↗

Insect allergy. A comparative study including case histories and immunological parameters.

One hundred and seventeen persons all stung by yellow jacket (YJ) and/or bee were examined by means of skin prick test with venom of these insects, skin prick test with 10 inhalant allergens and analyses of total IgE, specific IgE and IgG against honey bee and YJ venom. Eighty-seven persons had had a systemic reaction to YJ or bee sting, the rest had reacted normally or with a large local reaction. Positive correlations (P less than 0.05) were found between results of skin prick tests and specific IgE against venoms and, for YJ, between the severity of symptoms after sting and the size prick test with the venom. That some of the more severe symptoms could have been caused by non-immunological mechanisms could explain why a significant correlation was present only between the results of the prick test and specific IgE and not between these tests and the clinical symptoms. Specific IgE values against YJ and honey bee venom showed covariation, although no correlation could be demonstrated between the clinical symptoms after stings from these insects, or between skin prick test results using the two different extracts. The severity of the sting reactions was not correlated to age, atopic disposition, amount of total IgE, number of stings during life, or positive skin prick test to inhalant allergens. It is concluded that in insect allergy, specific IgE analysis and skin prick tests are supplementary.

Adolescent↗

Basophil histamine release in insect venom allergy.

The aim of the present study was to investigate venom-related and venom-non-related immunological reactions in patients stung by bee or wasp. Sixteen consecutive patients (7 with local and 9 with systemic reactions) were tested with skin tests, RAST and basophil histamine release (BHR) test immediately after the insect sting and 2, 4, and 16 weeks later. No test was useful immediately after the insect sting, the "anergic period". In agreement with earlier findings, the SPT was the only allergy test that showed statistically significant differences between patients with local and systemic reactions, although a great overlap was found. Release of histamine from basophils after incubation with anti-IgE also showed statistically significant differences between local and systemic reactions. Further studies are needed, especially measurement of BHR after incubation with anti-IgE before insect stings.

Adult↗

Immunotherapy with aluminum hydroxide adsorbed insect venom extracts (Alutard SQ): immunologic and clinical results of a prospective study over 3 years.

This prospective study over 3 years investigated the safety, immunogenicity, and effectiveness of immunotherapy (IT) with aluminum hydroxide adsorbed insect venom extracts (Alutard SQ, ALK Laboratories) in patients with previous, severe, systemic, IgE-mediated, anaphylactic reactions to Hymenoptera stings. Seventeen patients were treated with honeybee venom (BV), 13 with yellow-jacket venom (YJV), and 5 with both. No severe reactions to IT were noted. Only 3 BV-allergic patients experienced mild systemic reactions of grades I or II (1 per 139 injections) during the increase phase. As for the immunologic data, there was a significant decrease in specific IgE antibodies after 1 year, and a significant increase in specific IgG and IgG4 antibodies on reaching the maintenance dose, with a further rise after 1, 2, and 3 years. Moreover, a significant decrease in anti-IgE autoantibodies was observed in the BV group. Out of the 11 patients that were occasionally restung by the relevant insect (totaling 19 stings in all), only one patient developed mild systemic allergic symptoms after a third sting. In view of these results, we consider IT with aluminum hydroxide adsorbed insect venom extracts to be safe, immunogenic, and effective. The low rate of side-effects may be due to the slow release of the venom in the aluminum hydroxide adsorbed form.

Adolescent↗

An evolutionarily conserved mechanism for sensitization of soluble guanylyl cyclase reveals extensive nitric oxide-mediated upregulation of cyclic GMP in insect brain.

Soluble guanylyl cyclase (SGC) is the main receptor for the gaseous signalling molecule nitric oxide (NO) in vertebrates and invertebrates. Recently, a novel class of drugs that regulate mammalian SGC by NO-independent allosteric mechanisms has been identified [e.g. 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole, YC-1]. To assess the evolutionary conservation and hence the potential physiological relevance of these mechanisms, we have tested YC-1 on the brains of two model insects, the cockroach Periplaneta americana and the locust Schistocerca gregaria. YC-1 strongly potentiated the NO-induced elevation of total cyclic 3',5'-guanosine monophosphate (cGMP) and amplified the intensity and consistency of NO-induced cGMP-immunoreactivity in the brain. Our data indicate that the effect of YC-1 was independent of phosphodiesterase inhibition and thus mediated by direct sensitization of SGC. Immunohistopharmacology and co-labelling with antibodies against the SGC alpha-subunit confirmed that cGMP induced by co-application of NO and YC-1 is predominantly attributable to SGC. The staggering number of NO-responsive neurons revealed by YC-1 suggests that previous studies may have considerably underestimated the number of cellular targets for NO in the insect brain. Moreover, a subset of these targets exhibited cGMP-immunoreactivity without application of exogenous NO, demonstrating that YC-1 can be exploited for visualization of physiological cGMP signals in response to endogenous NO production. In conclusion, our discovery that YC-1 is a potent sensitizer of insect SGC indicates that a NO-independent regulatory site is an evolutionarily conserved feature of SGC. Our findings add considerable momentum to the concept of an as yet unidentified endogenous ligand that regulates the gain of the NO-cGMP signalling pathway.

Amino Acid Sequence↗

Unique accumulation of neuropeptides in an insect: FMRFamide-related peptides in the cockroach, Periplaneta americana.

FMRFamides belong to the most extensively studied neuropeptides in invertebrates and exhibit diverse physiological effects on different target organs, such as muscles, intestine and the nervous system. This study on the American cockroach confirms for the first time that extended FMRFamides occur in non-dipteran insects. By means of tandem mass spectrometry, these neuropeptides were structurally elucidated, and sequence information was used for subsequent cloning of the cockroach FMRFamide gene. This precursor gene encodes for 24 putative peptides and shows sufficient similarity with the Drosophila FMRFamide gene. Of the 24 peptides, 23 were detected by mass spectrometric methods; it is the highest number of neuropeptide forms shown to be expressed from a single precursor in any insect. The expression was traced back to single neurons in the thoracic ganglia. The unique accumulation of these FMRFamide-related peptides in thoracic perisympathetic organs provides the definite evidence for a tagma-specific distribution of peptidergic neurohormones in neurohaemal release sites of the insect CNS. Excitatory effects of the cockroach FMRFamides were observed on antenna-heart preparations. In addition, the newly described FMRFamides reduce the spike frequency of dorsal-unpaired median neurons and reduce the intracellular calcium concentration, which may affect the peripheral release of the biogenic amine octopamine.

Action Potentials↗

A baculovirus blocks insect molting by producing ecdysteroid UDP-glucosyl transferase.

The predicted amino acid sequence of a newly identified gene of the insect baculovirus Autographa californica nuclear polyhedrosis virus was similar to several uridine 5'-diphosphate (UDP)-glucuronosyl transferases and at least one UDP-glucosyl transferase. Genetic and biochemical studies confirmed that this gene encodes an ecdysteroid UDP-glucosyl transferase (egt). This enzyme catalyzes the transfer of glucose from UDP-glucose to ecdysteroids, which are insect molting hormones. Expression of the egt gene allowed the virus to interfere with normal insect development so that molting was blocked in infected larvae of fall armyworm (Spodoptera frugiperda).

Amino Acid Sequence↗