Model Insect Species Research Articles

Responses to a warming world: Integrating life history, immune investment, and pathogen resistance in a model insect species.

Environmental temperature has important effects on the physiology and life history of ectothermic animals, including investment in the immune system and the infectious capacity of pathogens. Numerous studies have examined individual components of these complex systems, but little is known about how they integrate when animals are exposed to different temperatures. Here, we use the Indian meal moth (Plodia interpunctella) to understand how immune investment and disease resistance react and potentially trade‐off with other life‐history traits. We recorded life‐history (development time, survival, fecundity, and body size) and immunity (hemocyte counts, phenoloxidase activity) measures and tested resistance to bacterial (E. coli) and viral (Plodia interpunctella granulosis virus) infection at five temperatures (20–30°C). While development time, lifespan, and size decreased with temperature as expected, moths exhibited different reproductive strategies in response to small changes in temperature. At cooler temperatures, oviposition rates were low but tended to increase toward the end of life, whereas warmer temperatures promoted initially high oviposition rates that rapidly declined after the first few days of adult life. Although warmer temperatures were associated with strong investment in early reproduction, there was no evidence of an associated trade‐off with immune investment. Phenoloxidase activity increased most at cooler temperatures before plateauing, while hemocyte counts increased linearly with temperature. Resistance to bacterial challenge displayed a complex pattern, whereas survival after a viral challenge increased with rearing temperature. These results demonstrate that different immune system components and different pathogens can respond in distinct ways to changes in temperature. Overall, these data highlight the scope for significant changes in immunity, disease resistance, and host–parasite population dynamics to arise from small, biologically relevant changes to environmental temperature. In light of global warming, understanding these complex interactions is vital for predicting the potential impact of insect disease vectors and crop pests on public health and food security.

Two distinctive β subunits are separately involved in two binding sites of imidacloprid with different affinities in Locusta migratoria manilensis

Due to great diversity of nicotinic acetylcholine receptor (nAChR) subtypes in insects, one β subunit may be contained in numerous nAChR subtypes. In the locust Locusta migratoria, a model insect species with agricultural importance, the third β subunits (Locβ3) was identified in this study, which reveals at least three β subunits in this insect species. Imidacloprid was found to bind nAChRs in L. migratoria central nervous system at two sites with different affinities, with Kd values of 0.16 and 10.31nM. The specific antisera (L1-1, L2-1 and L3-1) were raised against fusion proteins at the large cytoplasmic loop of Locβ1, Locβ2 and Locβ3 respectively. Specific immunodepletion of Locβ1 with antiserum L1-1 resulted in the selective loss of the low affinity binding site for imidacloprid, whereas the immunodepletion of Locβ3 with L3-1 caused the selective loss of the high affinity site. Dual immunodepletion with L1-1 and L3-1 could completely abolish imidacloprid binding. In contrast, the immunodepletion of Locβ2 had no significant effect on the specific [3H]imidacloprid binding. Taken together, these data indicated that Locβ1 and Locβ3 were respectively contained in the low- and high-affinity binding sites for imidacloprid in L. migratoria, which is different to the previous finding in Nilaparvata lugens that Nlβ1 was in two binding sites for imidacloprid. The involvement of two β subunits separately in two binding sites may decrease the risk of imidacloprid resistance due to putative point mutations in β subunits in L. migratoria.

Alternative splicing in nicotinic acetylcholine receptor subunits from Locusta migratoria and its influence on acetylcholine potencies

Due to the great abundance within insect central nervous system (CNS), nicotinic acetylcholine receptors (nAChRs) play key roles in insect CNS, which makes it to be the targets of several classes of insecticides, such as neonicotinoids. Insect nAChRs are pentameric complexes consisting of five subunits, and a dozen subunits in one insect species can theoretically comprise diverse nAChRs. The alternative splicing in insect nAChR subunits may increase the diversity of insect nAChRs. In the oriental migratory locust (Locusta migratoria manilensis Meyen), a model insect species with agricultural importance, the alternative splicing was found in six α subunits among nine α and two β subunits, such as missing conserved residues in Loop D from Locα1, Locα6 and Locα9, a 34-residue insertion in Locα8 cytoplasmic loop, and truncated transcripts for Locα4, Locα7 and Locα9. Hybrid nAChRs were successfully constructed in Xenopus oocytes through co-expression with rat β2 and one α subunit from L. migratoria, which included Locα1, Locα2, Locα3, Locα4, Locα5, Locα8 and Locα9. Influences of alternative splicing in Locα1, Locα8 and Locα9 on acetylcholine potency were tested on hybrid nAChRs. The alternative splicing in Locα1 and Locα9 could increase acetylcholine sensitivities on recombinant receptors, while the splicing in Locα8 showed significant influences on the current amplitudes of oocytes. The results revealed that the alternative splicing at or close to the ligand-binding sites, as well as at cytoplasmic regions away from the ligand-binding sites, in insect nAChR subunits would change the agonist potencies on the receptors, which consequently increased nAChR diversity in functional and pharmacological properties.

Combinatory annotation of cell membrane receptors and signalling pathways of Bombyx mori prothoracic glands.

The cells of prothoracic glands (PG) are the main site of synthesis and secretion of ecdysteroids, the biochemical products of cholesterol conversion to steroids that shape the morphogenic development of insects. Despite the availability of genome sequences from several insect species and the extensive knowledge of certain signalling pathways that underpin ecdysteroidogenesis, the spectrum of signalling molecules and ecdysteroidogenic cascades is still not fully comprehensive. To fill this gap and obtain the complete list of cell membrane receptors expressed in PG cells, we used combinatory bioinformatic, proteomic and transcriptomic analysis and quantitative PCR to annotate and determine the expression profiles of genes identified as putative cell membrane receptors of the model insect species, Bombyx mori, and subsequently enrich the repertoire of signalling pathways that are present in its PG cells. The genome annotation dataset we report here highlights modules and pathways that may be directly involved in ecdysteroidogenesis and aims to disseminate data and assist other researchers in the discovery of the role of such receptors and their ligands.

Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta

Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects.

Superoxide dismutase 2 knockdown leads to defects in locomotor activity, sensitivity to paraquat, and increased cuticle pigmentation in Tribolium castaneum.

Insects can rapidly adapt to environmental changes through physiological responses. The red flour beetle Tribolium castaneum is widely used as a model insect species. However, the stress–response system of this species remains unclear. Superoxide dismutase 2 (SOD2) is a crucial antioxidative enzyme that is found in mitochondria. T. castaneum SOD2 (TcSOD2) is composed of 215 amino acids, and has an iron/manganese superoxide dismutase domain. qRT-PCR experiments revealed that TcSOD2 was present through all developmental stages. To evaluate TcSOD2 function in T. castaneum, we performed RNAi and also assessed the phenotype and antioxidative tolerance of the knockdown of TcSOD2 by exposing larvae to paraquat. The administration of paraquat resulted in significantly higher 24-h mortality in TcSOD2 knockdown larval groups than in the control groups. The TcSOD2 knockdown adults moved significantly more slowly, had lower ATP content, and exhibited a different body color from the control groups. We found that TcSOD2 dsRNA treatment in larvae resulted in increased expression of tyrosinase and laccase2 mRNA after 10 days. This is the first report showing that TcSOD2 has an antioxidative function and demonstrates that T. castaneum may use an alternative antioxidative system when the SOD2-based system fails.

Real-time cell analysis and heat shock protein gene expression in the TcA Tribolium castaneum cell line in response to environmental stress conditions.

The rust red flour beetle, Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae), is a pest of stored grain and one of the most studied insect model species. Some of the previous studies involved heat response studies in terms of survival and heat shock protein expression, which are regulated to protect other proteins against environmental stress conditions. In the present study, we characterize the impedance profile with the xCELLigence Real-Time Cell Analyzer and study the effect of increased temperature in cell growth and viability in the cell line BCIRL-TcA-CLG1 (TcA) of T. castaneum. This novel system measures cells behavior in real time and is applied for the first time to insect cells. Additionally, cells are exposed to heat shock, increased salinity, acidic pH and UV-A light with the aim of measuring the expression levels of Hsp27, Hsp68a, and Hsp83 genes. Results show a high thermotolerance of TcA in terms of cell growth and viability. This result is likely related to gene expression results in which a significant up-regulation of all studied Hsp genes is observed after 1 h of exposure to 40 °C and UV light. All 3 genes show similar expression patterns, but Hsp27 seems to be the most affected. The results of this study validate the RTCA method and reveal the utility of insect cell lines, real-time analysis and gene expression studies to better understand the physiological response of insect cells, with potential applications in different fields of biology such as conservation biology and pest management.

Sensory and immune genes identification and analysis in a widely used parasitoid wasp Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae).

Trichogramma dendrolimi Matsumura (Hymenoptera: Trichogrammatidae) is one of the preponderant egg parasitoids of Dendrolimus spp., which are important defoliators of coniferous forests. This parasitoid wasp has been widely released to control pine caterpillar and other lepidopteran pests, but its control efficiency needs to be improved. Sensory systems are crucial for T. dendrolimi to locate hosts, and immunity is probably involved after egg deposition in the host cavity; however, few reports have focused on the molecular mechanism of olfactory detection and survival of T. dendrolimi. It is necessary to identify these genes before further functional research is conducted. In this study, we assembled and analyzed the transcriptome of T. dendrolimi using next-generation sequencing technology. The sequencing and assembly resulted in 38565 contigs with N50 of 3422 bp. Sequence comparison indicate that T. dendrolimi sequences are very similar to those of another parasitoid Nasonia vitripennis. Then the olfactory, vision, and immune-related gene families were identified, and phylogenetic analyses were performed with these genes from T. dendrolimi and other model insect species. Furthermore, phylogenetic tree with odorant binding proteins of T. dendrolimi and their host Dendrolimus was constructed to determine whether convergent evolution exists. These genes can be valid targets for further gene function research. The present study may help us to understand host location and survival mechanisms of T. dendrolimi and to use them more efficiently for pest control in the future.

Down syndrome cell adhesion molecule 1: testing for a role in insect immunity, behaviour and reproduction.

Down syndrome cell adhesion molecule 1 (Dscam1) has wide-reaching and vital neuronal functions although the role it plays in insect and crustacean immunity is less well understood. In this study, we combine different approaches to understand the roles that Dscam1 plays in fitness-related contexts in two model insect species. Contrary to our expectations, we found no short-term modulation of Dscam1 gene expression after haemocoelic or oral bacterial exposure in Tribolium castaneum, or after haemocoelic bacterial exposure in Drosophila melanogaster. Furthermore, RNAi-mediated Dscam1 knockdown and subsequent bacterial exposure did not reduce T. castaneum survival. However, Dscam1 knockdown in larvae resulted in adult locomotion defects, as well as dramatically reduced fecundity in males and females. We suggest that Dscam1 does not always play a straightforward role in immunity, but strongly influences behaviour and fecundity. This study takes a step towards understanding more about the role of this intriguing gene from different phenotypic perspectives.

Antennal sensory organs of Scathophaga stercoraria (Linnaeus, 1758)(Diptera: Scathophagidae): ultramorphology and phylogenetic implications.

Scathophaga stercoraria (Linnaeus, 1758) is a well-established insect model species involved in numerous investigations on behavior, biology, phylogeny, genetics and evolution. The antennal sensilla of S. stercoraria are examined via scanning electron microscopy in order to emphasize their importance on taxonomy and phylogeny. On antennal scape and pedicel, both microtrichiae and several sharp-tipped mechanoreceptors are observed, while another two structures, setiferous plaques and pedicellar button, are also detected on antennal pedicel. One type of sensory pit and four types of antennal sensilla, including trichoid sensilla, basiconic sensilla, coeloconic sensilla and clavate sensilla, are observed on antennal funiculus. Similarity and disparity of setiferous plaques among different calyptrate groups are analyzed in terms of phylogeny. The phylogenetic results supported by morphology of setiferous plaques strongly accord with the cladistic relations based on known molecular tree, implying the potential taxonomic and phylogenetic implications of the plaques in Calyptratae.

Comparative genomic analysis and evolution of family-B G protein-coupled receptors from six model insect species

Family-B G protein-coupled receptors (GPCR-Bs) play vital roles in many biological processes, including growth, development and reproduction. However, the evolution and function of GPCR-Bs have been poorly understood in insects.We have identified 87 GPCR-Bs from six model insect species, 20 from Tribolium castaneum, 9 from Apis mellifera, 11 from Bombyx mori, 9 from Acyrthosiphon pisum, 14 from Anopheles gambiae and 24 from Drosophila melanogaster. 22 of them were reported in this study for the first time. Phylogenetic analysis revealed that there are three kinds of evolutionary patterns that occurred among GPCR-Bs during insect evolution: one-to-one orthologous relationships, species-specific expansion and episodic duplication or loss in certain insect lineages. A striking finding was the discovery of a parathyroid hormone receptor like gene (pthrl) in invertebrates, which was independently duplicated in vertebrates and invertebrates, whereas this gene was lost at least twice during insect evolution. These results indicate that PTHRL is possibly divergent in the functions between mammals and insects.The information of family-B GPCRs in nondrosophiline insects has been established, and will promote the further study on the function of these GPCRs and deorphanization of them. On the other hand, this study provides us with multiple function of GPCR-Bs in differential organisms, which will be also the potential attacking targets for new pesticides and drugs.

Plant surfaces with cuticular folds and their replicas: Influence of microstructuring and surface chemistry on the attachment of a leaf beetle

Plant surfaces covered either with epicuticular wax crystals or cuticular folds have been shown to strongly reduce the ability of insects to attach to them. However, the relative impact of surface structuring vs. surface chemistry on insect attachment remains unclear. To understand the mechanisms reducing adhesion of insects on plant surfaces in more detail, we performed traction experiments (i) on plant surfaces covered with cuticular folds of different dimensions, and on their (ii) untreated and (iii) hydrophobized replicas. As a reference, measurements were performed on replicas of smooth plant surfaces and of glass. Traction forces were measured with a highly sensitive force transducer, using tethered male Colorado potato beetles (Leptinotarsa decemlineata) as a model insect species. Contact angle measurements with water and diiodomethane were also performed to examine the physicochemical properties of the test surfaces. We found that surface structuring has a strong influence on the magnitude of the attachment force. In contrast, under the chosen experimental conditions, surface chemistry had no significant influence. Our results indicate that attachment of the beetles is reduced solely by the dimensions of the folds, with cuticular folds of about 0.5μm in both height and width being the most effective. Contrary to the attachment of beetles, the wettability of the surfaces was considerably influenced by both surface structuring and chemistry. These results contribute to a better understanding of plant–insect interactions and the function of microstructured surfaces, and may facilitate the development of biomimetic anti-adhesive surfaces.

Colour in insect thermoregulation: Empirical and theoretical tests in the colour-changing grasshopper, Kosciuscola tristis

Body colours can result in different internal body temperatures, but evidence for the biological significance of colour-induced temperature differences is inconsistent. We investigated the relationship between body colour and temperature in a model insect species that rapidly changes colour. We used an empirical approach and constructed a heat budget model to quantify whether a colour change from black to turquoise has a role in thermoregulation for the chameleon grasshopper (Kosciuscola tristis). Our study shows that colour change in K. tristis provides relatively small temperature differences that vary greatly with wind speed (0.55 °C at ms(-1) to 0.05 °C at 10 ms(-1)). The biological significance of this difference is unclear and we discuss the requirement for more studies that directly test hypotheses regarding the fitness effects of colour in manipulating body temperature.

Knickkopf protein protects and organizes chitin in the newly synthesized insect exoskeleton

During each molting cycle of insect development, synthesis of new cuticle occurs concurrently with the partial degradation of the overlying old exoskeleton. Protection of the newly synthesized cuticle from molting fluid enzymes has long been attributed to the presence of an impermeable envelope layer that was thought to serve as a physical barrier, preventing molting fluid enzymes from accessing the new cuticle and thereby ensuring selective degradation of only the old one. In this study, using the red flour beetle, Tribolium castaneum, as a model insect species, we show that an entirely different and unexpected mechanism accounts for the selective action of chitinases and possibly other molting enzymes. The molting fluid enzyme chitinase, which degrades the matrix polysaccharide chitin, is not excluded from the newly synthesized cuticle as previously assumed. Instead, the new cuticle is protected from chitinase action by the T. castaneum Knickkopf (TcKnk) protein. TcKnk colocalizes with chitin in the new cuticle and organizes it into laminae. Down-regulation of TcKnk results in chitinase-dependent loss of chitin, severe molting defects, and lethality at all developmental stages. The conservation of Knickkopf across insect, crustacean, and nematode taxa suggests that its critical roles in the laminar ordering and protection of exoskeletal chitin may be common to all chitinous invertebrates.

Effects of intensified forestry on the landscape-scale extinction risk of dead wood dependent species

In the future, a significant proportion of Northern forests may become intensively managed through the planting of monospecific stands of native or introduced trees, and the use of multiple silvicultural treatments such as forest fertilization. Such an intensification of management in selected parts of the landscape is suggested by different zoning models, for example the Triad approach, which is under evaluation in some regions of North America. In this study, based on Fennoscandian conditions, we predicted landscape-scale extinction risks of five hypothetical model insect species dependent on fresh dead wood from Norway spruce (Picea abies), by simulating colonizations and local extinctions in forest stands. Intensified forestry applied to 50% of the spruce stands led to strongly increased extinction risks of all species during the following 150 years. For one species—the sun-exposure specialist—there were strong effects already after 50 years. The negative effects of intensive plantation forestry could be compensated for by taking greater biodiversity conservation measures in other managed forests or by setting aside more forests. This is consistent with the Triad model, which is according to our analyzes an effective way to decrease extinction risks, especially for the short-dispersing species and the species associated with closed forest. A zoning of forest land into intensive forestry, conventional forestry, and set asides may be better at combining increased timber production and maintenance of biodiversity in comparison to landscapes where all production forests are managed in the same way.

Programmed cell death of the ovarian nurse cells during oogenesis of the ladybird beetle Adalia bipunctata (Coleoptera: Coccinellidae)

Programmed cell death (PCD) is an evolutionary conserved and genetically regulated form of cell death, in which the cell plays an active role in its own demise. It is widely recognized that PCD can be morphologically classified into three major types: type I, known as apoptosis, type II, called autophagy, and type III, specified as cytoplasmic cell death. So far, PCD has been morphologically analyzed in certain model insect species of the meroistic polytrophic ovary-type, but has never been examined before in insects carrying meroistic telotrophic ovaries. In the present study, we attempted to thoroughly describe the three different types (I, II and III) of PCD occurring during oogenesis in the meroistic telotrophic ovary of the Coleoptera species Adalia bipunctata, at different developmental ages of the adult female insects. We reveal that in the ladybird beetle A. bipunctata, the ovarian tropharia undergo age-dependent forms of apoptotic, autophagic and cytoplasmic (paraptotic-like) cell death, which seem to operate in a rather synergistic fashion, in accordance with previous observations in Diptera and Lepidoptera species. Furthermore, we herein demonstrate the occurrence of morphogenetically abnormal ovarioles in A. bipunctata female insects. These atretic ovarioles collapse and die through a PCD-mediated process that is characterized by the combined activation of all three types of PCD. Conclusively, the distinct cell death programs (I, II and III) specifically engaged during oogenesis of A. bipunctata provide strong evidence for the structural and functional conserved nature of PCD during insect evolution among meroistic telotrophic and meroistic polytrophic ovary-type insects.

Let's Move toward Genomics for Butterfly Ecology and Insect Conservation

Let's Move toward Genomics for Butterfly Ecology and Insect Conservation

The Organization of the Antennal Lobe Correlates Not Only with Phylogenetic Relationship, But Also Life History: A Basal Hymenopteran as Exemplar

The structure of the brain is a consequence of selective pressures and the ancestral brain structures modified by those pressures. The Hymenoptera are one of the most behaviorally complex insect orders, and the olfactory system of honeybees (one of the most derived members) has been extensively studied. To understand the context in which the olfactory system of the Hymenoptera evolved, we performed a variety of immunocytochemical and anatomical labeling techniques on the antennal lobes (ALs) of one of its most primitive members, the sawflies, to provide a comparison between the honeybee and other insect model species. The olfactory receptor neurons project from the antennae to fill the entire glomerular volume but do not form distinct tracts as in the honeybee. Labeling of projection neurons revealed 5 output tracts similar to those in moths and immunolabeling for several transmitters revealed distinct populations of local interneurons and centrifugal neurons that were also similar to moths. There were, however, no histaminergic or dopaminergic AL neurons. The similarities between sawflies and moths suggest that along with the great radiation and increased complexity of behavioral repertoire of the Hymenoptera, there were extensive modifications of AL structure.

Mdm2 and p53 are highly conserved from placozoans to man

The p53 protein is the most commonly mutated tumor suppressor gene in man. Understanding of its evolutionary origins have been enhanced by the recent discovery of p53 family genes in the sea anemone Nematostella vectensis. This amino acid sequence conservation has been reflected in biological activity since the early p53 proteins, like their human counterparts, are responsible for DNA damage-induced cellular apoptosis, albeit restricted to the germ cell compartment in model organisms such as the nematode and fruit fly. In vertebrates from zebrafish to man the function of p53 is tightly and absolutely constrained by a negative regulator Mdm2. However the Mdm2 gene has not been detected in the genome of the model nematode (C. elegans) and insect (D. melanogaster) species. We have found that the p53 gene and the Mdm2 gene are present in Placozoans, one of the simplest of all free living multi-cellular organisms, implying that both proteins arose much earlier in evolution than previously thought. Detailed sequence analysis shows the exceptional retention of key features of both proteins from man to placazoan implying that the p53-Mdm2 interaction and its regulation have been conserved from a basal eumetazoan since the pre-cambrian era over one billion years ago.

The development of PCR‐based markers for molecular sex identification in the model insect species Tribolium castaneum

AbstractThe red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), is a major pest of stored grain and cereal crops. It is also an important model species in genetic, ecological, and evolutionary research. The majority of its genome was recently sequenced and published. However, the genomic sequence of the small Y‐chromosome is still undetermined, which hinders the development of molecular sex identification methods. Traditional methods for sexing adult forms of Tribolium beetles are troublesome. Therefore, a method for molecular sex identification in the red flour beetle was developed. One sex‐linked randomly amplified polymorphic DNA marker was converted into a sequence‐characterized amplified region (SCAR). The SCAR was aligned with the T. castaneum reference whole‐genome sequence and fully matched a fragment of a single contig of unknown genomic location. The novelty of the method is that the fragment consists of shorter DNA fragments that are also present at other locations around the genome, but the particular order of these fragments within the sequenced region appeared to be Y‐specific and this property was utilized for marker development. A set of three primers for multiplex PCR reaction was designed resulting in amplification of different length Y‐specific and not‐Y‐specific (control) DNA fragments in a single PCR, which allows to distinguish males from females. The primers successfully sexed pre‐sexed pupae and adult beetles from six laboratory strains, showing that the order of the repeated fragments is conserved in the species and is not strain‐specific.