Highly efficient hyPBase-mediated transgenesis facilitates gain-of-function analysis in the cricket Gryllus bimaculatus.

oskar Predates the Evolution of Germ Plasm in Insects

A conserved role for arrow in posterior axis patterning across Arthropoda

Divergent and conserved roles of extradenticle in body segmentation and appendage formation, respectively, in the cricket Gryllus bimaculatus

All extant species are an outcome of nature's "experiments" during evolution, and hence multiple species need to be studied and compared to gain a thorough understanding of evolutionary processes. The field of evolutionary developmental biology (evo-devo) aspires to expand the number of species studied, because most functional genetic studies in animalshave been limited to a small number of "traditional" model organisms, many of which belong to the same phylum (Chordata). The phylum Arthropoda, and particularly its component class Insecta, possesses many important characteristics that are considered favorable and attractive for evo-devo research, including an astonishing diversity of extant species and a wide disparity in body plans. The development of the most thoroughly investigated insect genetic model system to date, the fruit fly Drosophila melanogaster (a holometabolous insect), appears highly derived with respect to other insects and indeed with respect to most arthropods. In comparison, crickets (a basally branching hemimetabolous insect lineage compared to the Holometabola) are thought to embody many developmental features that make them more representative of insects. Here we focus on crickets as emerging models to study problems in a wide range of biological areas and summarize the currently available molecular, genomic, forward and reverse genetic, imaging and computational tool kit that has been established or adapted for cricket research. With an emphasis on the cricket species Gryllus bimaculatus, we highlight recent efforts made by the scientific community in establishing this species as a laboratory model for cellular biology and developmental genetics. This broad toolkit has the potential to accelerate many traditional areas of cricket research, including studies of adaptation, evolution, neuroethology, physiology, endocrinology, regeneration, and reproductive behavior. It may also help to establish newer areas, for example, the use of crickets as animal infection model systems and human food sources.

Localization of octopaminergic neurones in insects

Contents of Journal of Insect Physiology

BackgroundAnimals exhibit circadian rhythms with a period of approximately 24 h in various physiological functions, including locomotor activity. This rhythm is controlled by an endogenous oscillatory mechanism, or circadian clock, which consists of cyclically expressed clock genes and their product proteins. cryptochrome (cry) genes are thought to be involved in the clock mechanism, and their functions have been examined extensively in holometabolous insects, but in hemimetabolous insects their role is less well understood.ResultsIn the present study, the role of cry genes was investigated using RNAi technology in a hemimetabolous insect, the cricket Gryllus bimaculatus. Using a molecular cloning approach, we obtained cDNAs for two cry genes: Drosophila-type cry1 (Gb’cry1) and mammalian-type cry2 (Gb’cry2). Gb’cry2 has six splicing variants, most of which showed rhythmic mRNA expression. Gb’cry1RNAi treatment had only a limited effect at the behavioral and molecular levels, while Gb’cry2RNAi had a significant effect on behavioral rhythms and molecular oscillatory machinery, alone or in combination with Gb’cry1RNAi. In Gb’cry1/Gb’cry2 double-RNAi crickets, most clock genes showed arrhythmic expression, except for timeless, which retained clear rhythmic expression. Molecular analysis revealed that some combination of Gb’cry1 and Gb’cry2 variants suppressed CLK/CYC transcriptional activity in cultured cells.ConclusionBased on these results, we propose a new model of the cricket’s circadian clock, including a molecular oscillatory loop for Gb’cry2, which can operate independent of the Gb’per/Gb’tim loop.

Novel genes have the potential to drive the evolution of new biological mechanisms, or to integrate into preexisting regulatory circuits and contribute to the regulation of older, conserved biological functions. One such gene, the novel insect-specific gene oskar, was first identified based on its role in establishing the Drosophila melanogaster germ line. We previously showed that this gene likely arose through an unusual domain transfer event involving bacterial endosymbionts and played a somatic role before evolving its well-known germ line function. Here, we provide empirical support for this hypothesis in the form of evidence for a neural role for oskar. We show that oskar is expressed in the adult neural stem cells of a hemimetabolous insect, the cricket Gryllus bimaculatus. In these stem cells, called neuroblasts, oskar is required together with the ancient animal transcription factor Creb to regulate long-term (but not short-term) olfactory memory. We provide evidence that oskar positively regulates Creb, which plays a conserved role in long-term memory across animals, and that oskar in turn may be a direct target of Creb. Together with previous reports of a role for oskar in nervous system development and function in crickets and flies, our results are consistent with the hypothesis that oskar's original somatic role may have been in the insect nervous system. Moreover, its colocalization and functional cooperation with the conserved pluripotency gene piwi in the nervous system may have facilitated oskar's later co-option to the germ line in holometabolous insects.

Germ Cell Specification Requires Zygotic Mechanisms Rather Than Germ Plasm in a Basally Branching Insect

BackgroundOpsins are key proteins in animal photoreception. Together with a light-sensitive group, the chromophore, they form visual pigments which initiate the visual transduction cascade when photoactivated. The spectral absorption properties of visual pigments are mainly determined by their opsins, and thus opsins are crucial for understanding the adaptations of animal eyes. Studies on the phylogeny and expression pattern of opsins have received considerable attention, but our knowledge about insect visual opsins is still limited. Up to now, researchers have focused on holometabolous insects, while general conclusions require sampling from a broader range of taxa. We have therefore investigated visual opsins in the ocelli and compound eyes of the two-spotted cricket Gryllus bimaculatus, a hemimetabolous insect.ResultsPhylogenetic analyses place all identified cricket sequences within the three main visual opsin clades of insects. We assign three of these opsins to visual pigments found in the compound eyes with peak absorbances in the green (515 nm), blue (445 nm) and UV (332 nm) spectral range. Their expression pattern divides the retina into distinct regions: (1) the polarization-sensitive dorsal rim area with blue- and UV-opsin, (2) a newly-discovered ventral band of ommatidia with blue- and green-opsin and (3) the remainder of the compound eye with UV- and green-opsin. In addition, we provide evidence for two ocellar photopigments with peak absorbances in the green (511 nm) and UV (350 nm) spectral range, and with opsins that differ from those expressed in the compound eyes.ConclusionsOur data show that cricket eyes are spectrally more specialized than has previously been assumed, suggesting that similar adaptations in other insect species might have been overlooked. The arrangement of spectral receptor types within some ommatidia of the cricket compound eyes differs from the generally accepted pattern found in holometabolous insect taxa and awaits a functional explanation. From the opsin phylogeny, we conclude that gene duplications, which permitted differential opsin expression in insect ocelli and compound eyes, occurred independently in several insect lineages and are recent compared to the origin of the eyes themselves.

Insects have evolved a diversity of regulatory mechanisms to determine their sex. Understanding the molecular regulation mechanisms of insect sex determination is of great significance in revealing the general law of insect sex determination and providing potential routes for the genetic manipulation of pest species. Although the sex determination cascade and doublesex (dsx) gene functions have been well described in some holometabolous insects, little is known about this cascade in hemimetabolous insects. In this study, we identified the dsx homolog in Gryllus bimaculatus, which belongs to the Orthoptera order and is an important model for developmental and evolutionary biology. We found that Gbdsx has two alternative splicing isoforms (male-specific GbdsxM and non-sex-specific GbdsxC). Using RNAi-mediated knock-down of GbdsxM in 6th-instar nymphs resulted in adult male forewings showing feminized vein development and abnormal external genitalia. CRISPR/Cas9 knockout of Gbdsx in embryos resulted in adult males becoming pseudofemales, with feminized forewings and abnormal external and internal genitalia. Additionally, the pseudofemales created by Gbdsx knockout demonstrated normal courtship trends and aggressive behavior but no actual mating behavior. However, the knockout and knock-down of Gbdsx in female crickets does not affect their sexual traits or fertility. Our results suggest that GbdsxM plays a critical role in the development of male cricket sexual traits, courtship and mating behavior, which furthers our understanding of sex determination in hemimetabolous insects.

The metamorphosis of insects and their regulation

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic pest management strategies. The most used transposable element in insect transformation is piggyBac, and piggyBac-based germline transformation has been successfully conducted in model insects. However, it is still challenging to employ this technology in non-model insects that include agricultural pests. This paper reports on germline transformation of a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda, using the hyperactive piggyBac transposase (hyPBase). In this work, the hyPBase mRNA was produced and used in place of helper plasmid in embryo microinjections. This change led to the successful generation of transgenic FAW. Furthermore, the methods of screening transgenic animals, PCR-based rapid detection of transgene insertion, and thermal asymmetric interlaced PCR (TAIL-PCR)-based determination of the integration site, are also described. Thus, this paper presents a protocol to produce transgenic FAW, which will facilitate piggyBac-based transgenesis in FAW and other lepidopteran insects.

BioTechniquesVol. 56, No. 3 BioSpotlightOpen AccessBioSpotlightNathan BlowNathan BlowSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000114140AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Bridging the gap in chromatin analysisDetermining the structure and organization of chromatin within cells is important for understanding how gene expression is regulated and how genomes evolve. While a number of techniques, including fluorescence in situ hybridization (FISH) and various chromatin conformation capture analyses (e.g., 3C and Hi-C), have been developed to study chromatin organization and interactions, each of these approaches has drawbacks. While FISH allows single-cell studies of chromatin interactions, the resolution of this technique can be limited to the diffraction limit of light, ∼250 nm. Super resolution microscopy techniques have improved upon this limitation, but still not to sufficient resolution to discern specific chromatin features or enable three-dimensional reconstruction of chromatin interactions. Chromatin conformation capture techniques enable much higher resolution studies of chromatin interactions but also require large numbers of cells, resulting in an inability to assess the frequencies of specific interactions within individual cells. In an effort to bridge the gap between the high resolution of chromatin conformation analysis and the single cell perspective provided by FISH, Chen et al. (Karolinska Institute, Stockholm, Sweden) present a new technique, chromatin in situ proximity (ChrISP), in the current issue of BioTechniques. The methodological basis of ChrISP lies in the well-developed in situ proximity ligation assay (ISPLA), wherein two probes create a fluorescent amplification signal when in close proximity to one another. Two forms of ChrISP were described by the authors: In one, rolling circle amplification (RCA) is used to amplify the signal between the two probes, while the second avoids RCA, instead relying on labeled “splinter” and “backbone” oligos to create a signal between the two chromatin probes. The authors demonstrate that by using the “Green-Splinter ChrISP” (splinter plus backbone oligos with no RCA) they were able to obtain resolutions greater than 17 nm in all 3 dimensions, a 30-fold improvement in the Z-plane in comparison to the other approaches. In the end, given the versatility and simplicity of the ChrISP technique, Chen et al. predict that the method will prove extremely useful when examining chromatin structure alone as well as the alignment of chromatin with other cell landmarks, such as the nuclear membrane and nucleoli.See “Chromatin in situ proximity (ChrISP): Single-cell analysis of chromatin proximities at high resolution”Needle-ing butterfliesAfter years of development and optimization, it is easy for Drosophila researchers to take germline transformation for granted. While it is a demanding technique that quite often requires a large number of injections for success, a transformant can usually be obtained. For other species though, the tools for germline transformation are less well developed, presenting a challenge for scientists wanting to assess genotype/phenotype relationships. But in this issue of BioTechniques, a team of researchers led by Jeffrey Marcus (University of Manitoba, Winnipeg, Canada) report on an improved injection needle design that significantly increases transformation efficiencies and experimental outcomes when working with the buckeye butterfly, Junonia coenia. In pilot injections using a non-optimized needle design previously used for germline transformation in another butterfly species, the authors observed 100% mortality of injected Junonia ova. In an effort to find a needle that would work for Junonia transformation, the group took inspiration from standard Drosophila needles, which have a steep taper and large orifice. Employing these “new” needle designs while injecting Junonia ova resulted in a significantly improved outcome: 21.7% of injected ova hatched with a transformation rate of 3%. Marcus and his team next checked whether or not their new design also showed increased transformation efficiency with other butterfly and moth species beyond Junonia—finding that these needles were indeed more efficient than previous designs. The new needles should be a welcome advance for the large number of researchers working on lepidopteran species where higher transformation efficiency is especially important since fewer ova are available, making genetic analysis a more realistic possibility.See “Improved injection needles facilitate germline transformation of the buckeye butterfly Junonia coenia”A new needle design (Needle type 2) for ova injection during germline transformation experiments of butterfly and moth species significantly improved transformation efficiencies.FiguresReferencesRelatedDetails Vol. 56, No. 3 Follow us on social media for the latest updates Metrics Downloaded 122 times History Published online 3 April 2018 Published in print March 2014 Information© 2014 Author(s)PDF download

The cricket Gryllus bimaculatus is an emerging model insect of the order Orthoptera that is used in a wide variety of biological research themes. This hemimetabolous species appears highly complementary to Drosophila and other well-established holometabolous models. To improve transgenesis applications in G. bimaculatus, we have designed a transformation marker gene inspired from the widespread Drosophila mini-white+. Using CRISPR/Cas9, we first generated a loss-of-function mutant allele of the Gb-white gene (Gb-w), which exhibits a white eye coloration at all developmental stages. We then demonstrate that transgenic insertions of a piggyBac vector containing a 3xP3-Gb-w+ cassette rescue eye pigmentation. As an application, we used this vector to generate G. bimaculatus lines expressing a centromeric histone H3 variant (CenH3.1) fused to EGFP and validated EGFP-CenH3.1 detection at cricket centromeres. Finally, we demonstrate that Minos-based germline transformation and site-specific plasmid insertion with the ΦC31 integrase system function in G. bimaculatus.