Involvement of the scalloped gene in morphogenesis of the wing margin via regulating cell growth in a hemimetabolous insect Gryllus bimaculatus.

The acquisition of wings was a key event in insect evolution. As hemimetabolous insects were the first group to acquire functional wings, establishing the mechanisms of wing formation in this group could provide useful insights into their evolution. In this study, we aimed to elucidate the expression and function of the gene scalloped (sd), which is involved in wing formation in Drosophila melanogaster, and in Gryllus bimaculatus mainly during postembryonic development. Expression analysis showed that sd is expressed in the tergal edge, legs, antennae, labrum, and cerci during embryogenesis and in the distal margin of the wing pads from at least the sixth instar in the mid to late stages. Because sd knockout caused early lethality, nymphal RNA interference experiments were performed. Malformations were observed in the wings, ovipositor, and antennae. By analyzing the effects on wing morphology, it was revealed that sd is mainly involved in the formation of the margin, possibly through the regulation of cell proliferation. In conclusion, sd might regulate the local growth of wing pads and influence wing margin morphology in Gryllus.

Striking parallels between dorsoventral patterning in Drosophila and Gryllus reveal a complex evolutionary history behind a model gene regulatory network.

Dorsoventral pattering relies on Toll and BMP signalling in all insects studied so far, with variations in the relative contributions of both pathways. Drosophila and the beetle Tribolium share extensive dependence on Toll, while representatives of more distantly related lineages like the wasp Nasonia and bug Oncopeltus rely more strongly on BMP signalling. Here, we show that in the cricket Gryllus bimaculatus, an evolutionarily distant outgroup, Toll has, like in Drosophila, a direct patterning role for the ventral half of the embryo. In addition, Toll polarises BMP signalling, although this does not involve the conserved BMP inhibitor Sog/Chordin. Finally, Toll activation relies on ovarian patterning mechanisms with striking similarity to Drosophila. Our data suggest two surprising hypotheses: (1) that Toll's patterning function in Gryllus and Drosophila is the result of convergent evolution or (2) a Drosophila-like system arose early in insect evolution and was extensively altered in multiple independent lineages.

The Cricket Gryllus bimaculatus: Techniques for Quantitative and Functional Genetic Analyses of Cricket Biology.

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 animals have 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.

Cell cycle regulator, small silencing RNA, and segmentation patterning gene expression in relation to embryonic diapause in the band-legged ground cricket.

Insects enter diapause to synchronize their life cycle with biotic and abiotic conditions favorable for their development, reproduction, and survival. Adult females of the band-legged ground cricket Dianemobius nigrofasciatus (Orthoptera, Glyllidae) respond to environmental factors in autumn and lay diapause-destined eggs. The eggs arrest their development and enter diapause at a very early embryonic stage, specifically the cellular blastoderm. To elucidate the physiological mechanisms underlying this very early stage programmed developmental arrest, we investigated the cell division cycle as well as the expression of cell cycle regulators, small silencing RNAs, and segment patterning genes. The diapause embryo arrests its cell cycle predominantly at the G0/G1 phase. The proportion of cells in the S phase of the cell cycle abruptly decreased at the time of developmental arrest, but further changes of the G0/G1 and G2/M were later observed. Thus, cell cycle arrest in the diapause embryo is not an immediate event, but it takes longer to reach the steady state. We further elucidated molecular events possibly involved in diapause preparation and entry. Downregulation of Proliferating cellular antigen (PCNA; a cell cycle regulator), caudal and pumilio (cad and pum; early segmentation genes) as well as P-element induced wimpy testis (piwi) (a small silencing RNA) prior to the onset of developmental arrest was notable. The downregulation of PCNA, cad and pum continued even after entry into developmental arrest. In contrast to upregulation in non-diapause eggs, Cyclin D (another cell cycle regulator) and hunchback, Krüppel, and runt (gap and pair-rule genes) were downregulated in diapause eggs. These molecular events may contribute to embryonic diapause of D. nigrofasciatus.

Embryonic development of the cricket Gryllus bimaculatus.

Extensive research into Drosophila melanogaster embryogenesis has improved our understanding of insect developmental mechanisms. However, Drosophila development is thought to be highly divergent from that of the ancestral insect and arthropod in many respects. We therefore need alternative models for arthopod development that are likely to be more representative of basally-branching clades. The cricket Gryllus bimaculatus is such a model, and currently has the most sophisticated functional genetic toolkit of any hemimetabolous insect. The existing cricket embryonic staging system is fragmentary, and it is based on morphological landmarks that are not easily visible on a live, undissected egg. To address this problem, here we present a complementary set of "egg stages" that serve as a guide for identifying the developmental progress of a cricket embryo from fertilization to hatching, based solely on the external appearance of the egg. These stages were characterized using a combination of brightfield timelapse microscopy, timed brightfield micrographs, confocal microscopy, and measurements of egg dimensions. These egg stages are particularly useful in experiments that involve egg injection (including RNA interference, targeted genome modification, and transgenesis), as injection can alter the speed of development, even in control treatments. We also use 3D reconstructions of fixed embryo preparations to provide a comprehensive description of the morphogenesis and anatomy of the cricket embryo during embryonic rudiment assembly, germ band formation, elongation, segmentation, and appendage formation. Finally, we aggregate and schematize a variety of published developmental gene expression patterns. This work will facilitate further studies on G. bimaculatus development, and serve as a useful point of reference for other studies of wild type and experimentally manipulated insect development in fields from evo-devo to disease vector and pest management.

Effects of temperature and moisture on Mormon cricket reproduction with implications for responses to climate change.

During the last decade, populations of flightless Mormon crickets Anabrus simplex (Orthoptera: Tettigoniidae) increased suddenly over vast areas of the Western United States, suggesting that climate is an important factor driving outbreaks. Moreover summer temperatures are predicted to increase and precipitation is expected to decrease in most areas of the U.S. Great Basin, but little is known of the response of Mormon crickets to changes in temperature and soil moisture. In a laboratory study, we varied ambient temperature and lighting and measured the propensity of mating pairs to mate, and the proportion of eggs that developed into embryos. We found that reproduction was optimal when ambient temperature reached 30°C and the insects were beneath broad-spectrum lights such that maternal body and soil temperatures reached 35°C. Fewer eggs that developed fully were laid when maternal body and soil temperatures reached 30°C or 37-39°C. We also varied initial soil moisture from 0% to 100% saturated and found that more eggs reached embryonic diapause when initial soil moisture was 25% or 50% of saturated volume. However more of the developed eggs hatched when treated in summer soils with 0-25% of saturated moisture. We conclude that small changes in temperature had large effects on reproduction, whereas large changes in moisture had very small effects on reproduction. This is the first report of Mormon crickets mating in a laboratory setting and laying eggs that hatched, facilitating further research on the role of maternal and embryonic environments in changes in population size.

Developmental gene discovery in a hemimetabolous insect: de novo assembly and annotation of a transcriptome for the cricket Gryllus bimaculatus.

Most genomic resources available for insects represent the Holometabola, which are insects that undergo complete metamorphosis like beetles and flies. In contrast, the Hemimetabola (direct developing insects), representing the basal branches of the insect tree, have very few genomic resources. We have therefore created a large and publicly available transcriptome for the hemimetabolous insect Gryllus bimaculatus (cricket), a well-developed laboratory model organism whose potential for functional genetic experiments is currently limited by the absence of genomic resources. cDNA was prepared using mRNA obtained from adult ovaries containing all stages of oogenesis, and from embryo samples on each day of embryogenesis. Using 454 Titanium pyrosequencing, we sequenced over four million raw reads, and assembled them into 21,512 isotigs (predicted transcripts) and 120,805 singletons with an average coverage per base pair of 51.3. We annotated the transcriptome manually for over 400 conserved genes involved in embryonic patterning, gametogenesis, and signaling pathways. BLAST comparison of the transcriptome against the NCBI non-redundant protein database (nr) identified significant similarity to nr sequences for 55.5% of transcriptome sequences, and suggested that the transcriptome may contain 19,874 unique transcripts. For predicted transcripts without significant similarity to known sequences, we assessed their similarity to other orthopteran sequences, and determined that these transcripts contain recognizable protein domains, largely of unknown function. We created a searchable, web-based database to allow public access to all raw, assembled and annotated data. This database is to our knowledge the largest de novo assembled and annotated transcriptome resource available for any hemimetabolous insect. We therefore anticipate that these data will contribute significantly to more effective and higher-throughput deployment of molecular analysis tools in Gryllus.

Oskar predates the evolution of germ plasm in insects.

oskar is the only gene in the animal kingdom necessary and sufficient for specifying functional germ cells. However, oskar has only been identified in holometabolous ("higher") insects that specify their germline using specialized cytoplasm called germ plasm. Here we show that oskar evolved before the divergence of higher insects and provide evidence that its germline role is a recent evolutionary innovation. We identify an oskar ortholog in a basally branching insect, the cricket Gryllus bimaculatus. In contrast to Drosophila oskar, Gb-oskar is not required for germ cell formation or axial patterning. Instead, Gb-oskar is expressed in neuroblasts of the brain and CNS and is required for neural development. Taken together with reports of a neural role for Drosophila oskar, our data demonstrate that oskar arose nearly 50 million years earlier in insect evolution than previously thought, where it may have played an ancestral neural role, and was co-opted to its well-known essential germline role in holometabolous insects.

Identification and expression analyses of a novel serotonin receptor gene, 5-HT2ß, in the field cricket, Gryllus bimaculatus.

Biogenic amine serotonin (5-HT) modulates various aspects of behaviors such as aggressive behavior and circadian behavior in the cricket. In our previous report, in order to elucidate the molecular basis of the cricket 5-HT system, we identified three genes involved in 5-HT biosynthesis, as well as four 5-HT receptor genes (5-HT1A, 5-HT1B, 5-HT2α, and 5-HT7) expressed in the brain of the field cricket Gryllus bimaculatus DeGeer [7]. In the present study, we identified Gryllus 5-HT2ß gene, an additional 5-HT receptor gene expressed in the cricket brain, and examined its tissue-specific distribution and embryonic stage-dependent expression. Gryllus 5-HT2ß gene was ubiquitously expressed in the all examined adult tissues, and was expressed during early embryonic development, as well as during later stages. This study suggests functional differences between two 5-HT2 receptors in the cricket.

Notch/Delta signalling is not required for segment generation in the basally branching insect Gryllus bimaculatus.

Arthropods and vertebrates display a segmental body organisation along all or part of the anterior-posterior axis. Whether this reflects a shared, ancestral developmental genetic mechanism for segmentation is uncertain. In vertebrates, segments are formed sequentially by a segmentation 'clock' of oscillating gene expression involving Notch pathway components. Recent studies in spiders and basal insects have suggested that segmentation in these arthropods also involves Notch-based signalling. These observations have been interpreted as evidence for a shared, ancestral gene network for insect, arthropod and bilaterian segmentation. However, because this pathway can play multiple roles in development, elucidating the specific requirements for Notch signalling is important for understanding the ancestry of segmentation. Here we show that Delta, a ligand of the Notch pathway, is not required for segment formation in the cricket Gryllus bimaculatus, which retains ancestral characteristics of arthropod embryogenesis. Segment patterning genes are expressed before Delta in abdominal segments, and Delta expression does not oscillate in the pre-segmental region or in formed segments. Instead, Delta is required for neuroectoderm and mesectoderm formation; embryos missing these tissues are developmentally delayed and show defects in segment morphology but normal segment number. Thus, what initially appear to be 'segmentation phenotypes' can in fact be due to developmental delays and cell specification errors. Our data do not support an essential or ancestral role of Notch signalling in segment generation across the arthropods, and show that the pleiotropy of the Notch pathway can confound speculation on possible segmentation mechanisms in the last common bilaterian ancestor.

Allocation of maternal- and ejaculate-derived proteins to reproduction in female crickets, Teleogryllus oceanicus.

Female fitness has traditionally been thought to be maximized with one or a few matings. More recent research suggests that polyandry, mating with two or more males, can generate an increase in the viability of offspring females produce. However, the mechanism(s) underlying enhanced offspring viability remain largely unknown. The Australian field cricket Teleogryllus oceanicus has proved a useful model for examining the evolutionary significance of polyandry. Embryo viability appears to be associated with a male's investment in accessory gland tissue, implicating a role for seminal fluid. Here, I used amino acids labelled with different radio isotopes to identify proteins manufactured by males and females before they engaged in reproduction. Males incorporated 95% of the radiolabel into the testes, accessory glands and the ejaculate that was transferred to the female at mating. Male ejaculate compounds were incorporated predominantly into the female's somatic tissue. Relatively more female compounds were incorporated into the ovaries and into laid eggs than ejaculate compounds, and relatively fewer female compounds were sequestered in the somatic tissue than ejaculate compounds. The patterns observed suggest that while ejaculate compounds may be incorporated directly into eggs, they are likely to have a larger effect on maternal allocation to offspring.

Imaging of transgenic cricket embryos reveals cell movements consistent with a syncytial patterning mechanism.

The mode of insect embryogenesis varies among species, reflecting adaptations to different life history strategies [1, 2]. In holometabolous insects, which include the model systems, such as the fruit fly and the red flour beetle, a large proportion of the blastoderm produces an embryo, whereas hemimetabolous embryos generally arise from a small region of the blastoderm [3]. Despite their importance in evolutionary studies, information of early developmental dynamics of hemimetabolous insects remains limited. Here, to clarify how maternal and gap gene products act in patterning the embryo of basal hemimetabolous insects, we analyzed the dynamic segmentation process in transgenic embryos of an intermediate-germ insect species, the cricket, Gryllus bimaculatus. Our data based on live imaging of fluorescently labeled embryonic cells and nuclei suggest that the positional specification of the cellular blastoderm may be established in the syncytium, where maternally derived gradients could act fundamentally in a way that is similar to that of Drosophila, namely throughout the egg. Then, the blastoderm cells move dynamically, retaining their positional information to form the posteriorly localized germ anlage. Furthermore, we find that the anterior head region of the cricket embryo is specified by orthodenticle in a cellular environment earlier than the gnathal and thoracic regions. Our findings imply that the syncytial mode of the early segmentation in long-germ insects evolved from a dynamic syncytial-to-cellular mode found in the present study, accompanied by a heterochronic shift of gap gene action.

Early embryonic development of the head region of Gryllus assimilis Fabricius, 1775 (Orthoptera, Insecta).

We report our investigations on the embryonic development of Gryllus assimilis, with particular attention to the head. Significant findings revealed with scanning electron microscopy (SEM) images include: (1) the pre-antennal lobes represent the anterior-most segment that does not bear any appendages; (2) each of the lobes consists of central and marginal regions; (3) the central region thereof develops into the protocerebrum and the optic lobes, whereas the marginal region thereof becomes the anterior portion of the head capsule; (4) the initial position of the antennal segment is posterior to the mouth region; (5) appendage anlagen are transitorily present in the intercalary segment, and they later vanish together with the segment itself; (6) a bulged sternum appears to develop from the ventral surface of the mandibular, maxillary and labial segments. Embryonic features are then compared across the Insecta and further extended to the embryos of a spider (Araneae, Chelicerata). Striking similarities shared by the anterior-most region of the insect and spider embryos lead the authors to conclude that such comparison should be further undertaken to cover the entire Euarthropoda. This will help us to understand the embryology and evolution of the arthropod head.

Decoupling development and energy flow during embryonic diapause in the cricket, Allonemobius socius.

Respiration rate increases 6.3-fold during 15 days of post-oviposition development in embryos of the Southern ground cricket, Allonemobius socius. This ontogenetic increase in metabolism of non-diapause insects is blocked during diapause, such that metabolic rate is only 36% of the rate measured for 15 days developing embryos. Surprisingly, however, there is not an acute metabolic depression during diapause entry at the point when developmental ceases (4-5 days post-oviposition), as measured by blockage of morphological change and DNA proliferation. The results indicate a decoupling of developmental arrest from metabolism. Both non-diapause and diapause embryos have unusually high [AMP]:[ATP] ratios and low [ATP]:[ADP] ratios during early embryogenesis, which suggests embryos may have experienced hypoxia as a result of an insect chorion that limits water loss but may restrict oxygen diffusion. The similar adenylate profiles for these two developmental states indicate the atypical energy status is not a specific feature of diapause. In addition embryos at day 3 have high levels of lactate that decrease as development proceeds up to day 7. Calorimetric-respirometric (CR) ratios of -353 (day 3) to -333 (day 7) kJ mol(-1) O2 are consistent with embryos that are aerobically recovering from hypoxia, but are inconsistent with an ongoing anaerobic contribution to metabolism. Superfusing 3-day embryos with O2 enriched air (40% O2) forces these metabolic indicators toward a more aerobic poise, but only partially. Taken together these biochemical data indicate the metabolic poise of A. socius is only partly explained by hypoxia in early development, and that the atypical set points are also intrinsic features of this ontogenetic period in the life cycle.

Embryonic diapause highlighted by differential expression of mRNAs for ecdysteroidogenesis, transcription and lipid sparing in the cricket Allonemobius socius.

Embryos of the ground cricket, Allonemobius socius, enter diapause 4-5 days post-oviposition and overwinter in this dormant state that is characterized by developmental arrest. Suppressive subtractive hybridization and quantitative real-time PCR reveal eight candidate genes in pre-diapause embryos that show promise as regulators of diapause entry, when compared with embryos not destined for diapause. Identifications are based both on the magnitude/consistency of differential mRNA abundances and the predicted functions of their products when placed in context of the physiological and biochemical events of diapause characterized in our companion paper. The proteins CYP450, AKR and RACK1 (associated with ecdysteroid synthesis and signaling) are consistently upregulated in pre-diapause, followed by major downregulation later in diapause. The pattern suggests that elevated ecdysone may facilitate onset of diapause in A. socius. Upregulation seen for the transcription factors Reptin and TFDp2 may serve to depress transcription and cell cycle progression. Cathpesin B-like protease, ACLY and MSP are three downregulated genes associated with yolk mobilization and/or metabolism that we predict may promote lipid sparing. Finally, embryos that have been in diapause for 10 days show a substantially different pattern of mRNA expression compared with either pre-diapause or embryos not destined for diapause, with the majority of mRNAs examined being downregulated. These transcript levels in later diapause suggest that a number of upregulated genes in pre-diapause are transiently expressed and are less essential as diapause progresses.

Early embryonic development and diapause stage in the band-legged ground cricket Dianemobius nigrofasciatus.

The band-legged ground cricket Dianemobius nigrofasciatus enters diapause at an early embryonic stage when adults are reared under short-day conditions or the eggs are exposed to a low temperature. We examined the morphological features of the embryo during early development and determined the exact stage of entry into diapause. In non-diapause eggs, no periplasmic space was observed in the surface region and a small number of nuclei surrounded by cytoplasm (energids) were found among the yolk granules and lipid droplets 12 h after egg laying (AEL) at 25 degrees C. The energids sparsely but evenly populated the surface region at 40 h AEL, but there were some gaps between these energids. A continuous thin layer of nuclei with cytoplasm had completely covered the egg surface at 56 h AEL, suggesting that the blastoderm is formed between 40 and 56 h AEL. At 72 h AEL, we found a germ band at the posterior pole. Electron microscopy revealed clear cell membranes at 40 h AEL. Staining with rhodamine-dextran dye demonstrated that the cell membrane is formed when the nuclei appear on the egg surface at 12-24 h AEL. These results indicate that cellularization occurs before blastoderm formation. In diapause eggs, neither the embryonic rudiment nor germ band was formed, but a continuous layer of cells covered the egg surface. It is concluded that D. nigrofasciatus enters diapause at the cellular blastoderm.

Dynamic expression patterns of vasa during embryogenesis in the cricket Gryllus bimaculatus.

The specification of germ cells during embryogenesis is an important issue in the development of metazoans. In insects, the mode of germ cell specification appears to be highly variable among species and molecular data are not sufficient to provide an evolutionary perspective to this issue. Expression of vasa can be used as a germ line marker. Here, we report the isolation of a vasa-like gene in a hemimetabolous insect, the cricket Gryllus bimaculatus (Gb'vas), and its expression patterns during oogenesis and embryogenesis. Gb'vas is preferentially expressed in the germarium and the expression of Gb'vas is detectable throughout vitellogenesis including mature eggs subjected to oviposition, suggesting that Gb'vas is maternally contributed to the cricket eggs. The zygotic expression of Gb'vas appears to start at the mid blastoderm stage in the posterior region of the egg, expanding in a developing germ anlage. In early germbands, an intense expression of Gb'vas is restricted to the posterior end. In later embryos, Gb'vas expression extends over the whole body and then distinctly localized to the embryonic gonad at the stage immediately before hatching. These results suggest that, in the cricket, germ cells are specified early in development at the posterior end of an early germband, as proposed by Heymons (1895) based on cytological criteria.

EGFR signaling is required for re-establishing the proximodistal axis during distal leg regeneration in the cricket Gryllus bimaculatus nymph.

Nymphs of hemimetabolous insects, such as cockroaches and crickets, possess functional legs with a remarkable capacity for epimorphic regeneration. In this study, we have focused on the role of epidermal growth factor receptor (EGFR) signaling in regeneration of a nymphal leg in the cricket Gryllus bimaculatus. We performed loss-of-function analyses with a Gryllus Egfr homolog (Gb'Egfr) and nymphal RNA interference (RNAi). After injection of double-stranded RNA for Gb'Egfr in the body cavity of the third instar cricket nymph, amputation of the leg at the distal tibia resulted in defects of normal distal regeneration. The regenerated leg lacked the distal tarsus and pretarsus. This result indicates that EGFR signaling is required for distal leg patterning in regeneration during the nymphal stage of the cricket. Furthermore, we demonstrated that EGFR signaling acts downstream of the canonical Wnt/Wg signaling and regulates appendage proximodistal (PD) patterning genes aristaless and dachshund during regeneration. Our results suggest that EGFR signaling influences positional information along the PD axis in distal leg patterning of insects, regardless of the leg formation mode.

Antenna and all gnathal appendages are similarly transformed by homothorax knock-down in the cricket Gryllus bimaculatus.

Our understanding of the developmental mechanisms underlying the vast diversity of arthropod appendages largely rests on the peculiar case of the dipteran Drosophila melanogaster. In this insect, homothorax (hth) and extradenticle (exd) together play a pivotal role in appendage patterning and identity. We investigated the role of the hth homologue in the cricket Gryllus bimaculatus by parental RNA interference. This species has a more generalized morphology than Oncopeltus fasciatus, the one other insect besides Drosophila where homothorax function has been investigated. The Gryllus head appendages represent the morphologically primitive state including insect-typical mandibles, maxillae and labium, structures highly modified or missing in Oncopeltus and Drosophila. We depleted Gb'hth function through parental RNAi to investigate its requirement for proper regulation of other appendage genes (Gb'wingless, Gb'dachshund, Gb'aristaless and Gb'Distalless) and analyzed the terminal phenotype of Gryllus nymphs. Gb'hth RNAi nymphs display homeotic and segmentation defects similar to hth mutants or loss-of-function clones in Drosophila. Intriguingly, however, we find that in Gb'hth RNAi nymphs not only the antennae but also all gnathal appendages are homeotically transformed, such that all head appendages differentiate distally as legs and proximally as antennae. Hence, Gb'hth is not specifically required for antennal fate, but fulfills a similar role in the specification of all head appendages. This suggests that the role of hth in the insect antenna is not fundamentally different from its function as cofactor of segment-specific homeotic genes in more posterior segments.