Insect Embryos Research Articles

Hemocyanin contributes to embryonic adaptation to hypoxia in the migratory locust.

Ambient hypoxia can pose a major threat to the survival of metazoan organisms, especially insect embryos. Hemocyanin exhibits dominant expression in insect embryos, but its specific roles in hypoxia adaptation remain unexplored. Soil-dwelling locust eggs may frequently experience hypoxia during development. A comprehensive analysis of physiological and biochemical characters of hemocyanin was conducted in the embryos of migratory locust Locusta migratoria. Our results demonstrated that the revolution process was the critical hypoxia-sensitive event during locust embryogenesis. Hemocyanin presented a prominent expression in the revolution stage and exhibited strong responses to hypoxia. The relative duration of revolution was correlated negatively with the expression of hemocyanin subunit 2 (HC2), suggesting that HC2 might be closely associated with hypoxia adaptation of locust embryos. Furthermore, a HC2 mutant locust strain was established using the CRISPR/Cas9 technology, and higher hypoxia sensitivity was found for HC2-deficient locust embryos. Knockdown of HC2 increased anaerobic metabolism and oxidative stress while reducing oxidative metabolism. Overall, these findings clearly demonstrated the pivotal roles of hemocyanin in hypoxia adaptation of insect embryos.

Early embryonic development of the German cockroach Blattella germanica

BackgroundEarly embryogenesis is characterized by dramatic cell proliferation and movement. In most insects, early embryogenesis includes a phase called the uniform blastoderm, during which cells evenly cover the entirety of the egg. However, the embryo of the German cockroach, Blattella germanica, like those of many insects within the super order Polyneoptera, does not have a uniform blastoderm; instead, its first cells condense rapidly at the site of a future germband. We investigated early development in this species in order to understand how early gene expression is or is not conserved in these insect embryos with distinct early cell behaviors.ResultsWe present a detailed time series of nuclear division and distribution from fertilization through germband formation and report patterns of expression for the early patterning genes hunchback, caudal, and twist in order to understand early polarization and mesoderm formation. We show a detailed time course of the spatial expression of two genes involved in the segmentation cascade, hedgehog and even-skipped, and demonstrate two distinct dynamics of the segmentation process.ConclusionsDespite dramatic differences in cell distribution between the blastoderms of many Polyneopteran insects and those of more well-studied developmental models, expression patterns of early patterning genes are mostly similar. Genes associated with axis determination in other insects are activated relatively late and are probably not maternally deposited. The two phases of segmentation—simultaneous and sequential—might indicate a broadly conserved mode of morphological differentiation. The developmental time course we present here should be of value for further investigation into the causes of this distinct blastoderm type.

Permeability and Toxicity of Cryoprotective Agents in Silkworm Embryos: Impact on Cryopreservation.

The permeation of cryoprotectants into insect embryos is critical for successful cryopreservation. However, the permeability of silkworm embryos to cryoprotectants and the effects of cryopreservation remain poorly studied. In this study, we evaluated the permeability and toxicity of four cryoprotective agents (CPAs) as well as the vitrification effect on the viability of silkworm embryos. Among the four CPAs, propylene glycol (PG) showed the best permeability. Ethylene glycol (EG) and PG were the least toxic CPAs, but glycerol (GLY) and dimethyl sulfoxide (DMSO) were more toxic. Moreover, we examined several factors including the kind and the concentration of CPAs, exposure time, embryonic stage, and silkworm strains. Embryos at the earlier phases of stage 25 were more tolerant to vitrification using EG. We found that over 21% of embryos treated with EG at the early 2 phase of stage 25: 163 h after egg laying (AEL) developed and progressed to serosa ingestion after vitrification and rewarming. The result was the same in other strains as well. Our results are valuable for the development of new cryopreservation protocols of silkworm embryos.

Wet Beveling of Microinjection Needles Utilizing Constant Air Pressure for Feedback on Needle Opening.

Microinjection needles are a critical tool in the delivery of genome modification reagents, CRISPR components (guide RNAs, Cas9 protein, and donor template), and transposon system components (plasmids and transposase mRNA) into developing insect embryos. Sharp microinjection needles are particularly important during the delivery of these modifying agents since they help minimize damage to the embryo being injected, thereby increasing the survival of these embryos as compared to injection with non-beveled needles. Further, the beveling of needles produces needles that are more consistent from needle to needle as compared to needles opened by randomly breaking the needle tip by brushing the tip against an object (side of a coverslip, the surface of the embryo to be injected, etc.). The process of wet beveling of microinjection needles with constant pressure air delivered to the needle allows the person beveling the needle to know when the needle is open (presence of bubbles) and also gives a relative indication of how large a needle opening has been created. The relative opening size in the needle can be determined by adjusting the air pressure delivered to the needle until an equilibrium is reached and bubbles stop flowing from the tip of the needle. The lower the pressure at which the equilibrium is reached, the larger the needle size; and conversely, the higher the pressure, the smaller the needle size.

Notch Signaling in Insect Development: A Simple Pathway with Diverse Functions.

Notch signaling is an evolutionarily conserved pathway which functions between adjacent cells to establish their distinct identities. Despite operating in a simple mechanism, Notch signaling plays remarkably diverse roles in development to regulate cell fate determination, organ growth and tissue patterning. While initially discovered and characterized in the model insect Drosophila melanogaster, recent studies across various insect species have revealed the broad involvement of Notch signaling in shaping insect tissues. This review focuses on providing a comprehensive picture regarding the roles of the Notch pathway in insect development. The roles of Notch in the formation and patterning of the insect embryo, wing, leg, ovary and several specific structures, as well as in physiological responses, are summarized. These results are discussed within the developmental context, aiming to deepen our understanding of the diversified functions of the Notch signaling pathway in different insect species.

Long non-coding RNAs regulate heavy metal-induced apoptosis in embryo-derived cells

Heavy metal pollution has been a worldwide prevalent problem, and particularly a threat to ecosystem integrity and animals’ health. Previous studies on the mechanisms of heavy metal toxicity have focused on protein-coding genes, whereas most genomic transcripts are long non-coding RNAs (lncRNAs). Although lncRNAs are known to play important regulatory roles in biological processes, their role in heavy metal stress regulation is still not fully understood. We here developed an insect embryo cell model for studying metal toxicity and the underlying regulatory mechanisms. We performed genome-wide screening and functional characterization of lncRNAs induced by two essential and two non-essential heavy metals in Drosophila embryo-derived S2 cells. We identified 4894 lncRNAs, of which 1410 were novel. Forty-one lncRNAs, together with 328 mRNAs, were induced by all the four heavy metals. LncRNA–mRNA co-expression network and pathway enrichment analysis showed that detoxification metabolism, circadian rhythm, and apoptosis regulation pathways were activated in response to heavy metal stress. LncRNA CR44138 was remarkably upregulated in cells exposed to the four heavy metals and was associated with the apoptosis pathway. Expression interference confirmed that CR44138 aggravated cytotoxicity-induced apoptosis in cells under heavy metals stress. This study highlights the important role of lncRNAs in regulating the cellular response to heavy metals. This study also lays the foundation for discovering the novel regulatory mechanisms and developing diagnostic biomarkers of the toxic effects of heavy metal pollutants on organisms.

A timer gene network is spatially regulated by the terminal system in the Drosophila embryo.

In insect embryos, anteroposterior patterning is coordinated by the sequential expression of the 'timer' genes caudal, Dichaete, and odd-paired, whose expression dynamics correlate with the mode of segmentation. In Drosophila, the timer genes are expressed broadly across much of the blastoderm, which segments simultaneously, but their expression is delayed in a small 'tail' region, just anterior to the hindgut, which segments during germband extension. Specification of the tail and the hindgut depends on the terminal gap gene tailless, but beyond this the regulation of the timer genes is poorly understood. We used a combination of multiplexed imaging, mutant analysis, and gene network modelling to resolve the regulation of the timer genes, identifying 11 new regulatory interactions and clarifying the mechanism of posterior terminal patterning. We propose that a dynamic Tailless expression gradient modulates the intrinsic dynamics of a timer gene cross-regulatory module, delineating the tail region and delaying its developmental maturation.

Impacts of oxygen deficiency on embryo life-history traits of migratory locust Locusta migratoria from low and high altitudes.

Hypoxia challenges aerobic organisms in numerous environments, and hypoxic conditions may become more severe under future climate-change scenarios. The impact of hypoxia on the development of terrestrial insect embryos is not well understood. Here, to address this gap, embryonic life-history traits of migratory locust Locusta migratoria from low-altitude and high-altitude regions were compared under 2 oxygen levels: normoxia (i.e., 21 kPa oxygen partial pressure and mild hypoxia (i.e., 10 kPa oxygen partial pressure). Our results demonstrated that, whether reared under normoxia or mild hypoxia, L. migratoria from high-altitude populations had longer developmental times, reduced weight, and lower mean relative growth rate as compared with those from low-altitude populations. When transferred from normoxia to mild hypoxia, nearly all the tested life-history traits presented significant negative changes in the low-altitude populations, but not in the high-altitude populations. The factor 'strain' alone explained 18.26%-54.59% of the total variation for traits, suggesting that the phenotypic differences between L. migratoria populations from the 2 altitudes could be driven by genetic variation. Significant genetic correlations were found between life-history traits, and most of these showed differentiation between the 2 altitudinal gradients. G-matrix comparisons showed significant structural differences between L. migratoria from the 2 regions, as well as several negative covariances (i.e., trade-offs) between traits in the low-altitude populations. Overall, our study provides clear evidence that evolutionary divergence of embryonic traits between L. migratoria populations from different altitudes has occurred.

Noninvasive examination of the cardiac properties of insect embryos enabled by optical coherence tomography

Understanding the cardiac properties of insect embryos at different development stages is important, however, few works have been conducted probably due to the lack of effective tools. Using locust embryos as an example, here we show, for the first time, that optical coherence tomography (OCT) is capable of obtaining detailed information of embryos' heart activities and irregularities, such as the heart rate, cardiac cycle, diastolic and systolic diameters, hemolymph pumping rate and ejection fraction at different stages of embryonic development and at different temperatures. We develop algorithms and mathematical methods for extracting and analyzing cardiac behavior information of locust embryos. We discover that locust embryos experienced suspended development (quiescence) caused by cold storage have a heart rate 20% more than that of embryos without experiencing quiescence and that the hemolymph pumping rate of the two types of embryos behaves differently as the embryos grow. In addition, using OCT as an accurate cardiac activity examination tool, we show that the heart rates of locust embryos are effectively reduced due to nitric oxide synthase gene silencing by RNA interference, indicating potential application of using locust embryos as a good model organism to study cardiovascular diseases, including the congenital heart disease and arrhythmia. Finally, the capabilities offered by OCT in the studies of locust embryonic development may also prove helpful to promote locust reproduction for nutritions or restrain locust reproduction for pest control.

Astral microtubule cross-linking safeguards uniform nuclear distribution in the Drosophila syncytium.

The early insect embryo develops as a multinucleated cell distributing the genome uniformly to the cell cortex. Mechanistic insight for nuclear positioning beyond cytoskeletal requirements is missing. Contemporary hypotheses propose actomyosin-driven cytoplasmic movement transporting nuclei or repulsion of neighbor nuclei driven by microtubule motors. Here, we show that microtubule cross-linking by Feo and Klp3A is essential for nuclear distribution and internuclear distance maintenance in Drosophila. Germline knockdown causes irregular, less-dense nuclear delivery to the cell cortex and smaller distribution in ex vivo embryo explants. A minimal internuclear distance is maintained in explants from control embryos but not from Feo-inhibited embryos, following micromanipulation-assisted repositioning. A dimerization-deficient Feo abolishes nuclear separation in embryo explants, while the full-length protein rescues the genetic knockdown. We conclude that Feo and Klp3A cross-linking of antiparallel microtubule overlap generates a length-regulated mechanical link between neighboring microtubule asters. Enabled by a novel experimental approach, our study illuminates an essential process of embryonic multicellularity.

Effect of chemical dechorionation on silkworm embryo viability

The chorion covering/protecting insect egg, which has some effective functions such as providing mechanical strength, protecting eggs from external environments, and keeping moisture adjustment, is one of the principal barriers to manipulation, cryopreservation, and study of insect embryos. Here we evaluated the silkworm embryo viability after dechorionation using chemical reagents. We have developed an easy and effective method for chemical dechorionation that enables embryos to develop in culture, so that the larvae could normally grow. Eggs attached to a nylon net were treated with potassium hydroxide (KOH) and sodium hypochlorite (NaClO) to remove the chorion, washed with the Grace’s insect medium, and then cultured using a dry-moist method which we created. The most effective treatment with regard to embryonic development, hatching, and production of second instar larvae was 30% KOH for 7 min and 2% NaClO for 5 min at 27 °C. Embryos at later embryonic stages were more tolerant to chemical dechorionation and over 75% of embryos treated at 168 h-old (Stage 25, appearance of taenidium) survived to the second larval instar, moreover, the larvae derived from the dechorionated embryos have developed into the moths which can lay the fertilized eggs. Our method would contribute to the establishment of cryopreservation using embryos and analysis of silkworm embryogenesis and might also be applicable to other insect species.

Timing of increased temperature sensitivity coincides with nervous system development in winter moth embryos.

ABSTRACTClimate change is rapidly altering the environment and many species will need to genetically adapt their seasonal timing to keep up with these changes. Insect development rate is largely influenced by temperature, but we know little about the mechanisms underlying the temperature sensitivity of development. Here, we investigate seasonal timing of egg hatching in the winter moth, one of the few species which has been found to genetically adapt to climate change, likely through selection on temperature sensitivity of egg development rate. To study when during development winter moth embryos are most sensitive to changes in ambient temperature, we gave eggs an increase or decrease in temperature at different moments during their development. We measured their developmental progression and time of egg hatching, and used fluorescence microscopy to construct a timeline of embryonic development for the winter moth. We found that egg development rate responded more strongly to temperature once embryos were in the fully extended germband stage. This is the phylotypic stage at which all insect embryos have developed a rudimentary nervous system. Furthermore, at this stage, timing of ecdysone signaling determines developmental progression, which could act as an environment dependent gateway. Intriguingly, this may suggest that, from the phylotypic stage onward, insect embryos can start to integrate internal and environmental stimuli to actively regulate important developmental processes. As we found evidence that there is genetic variation for temperature sensitivity of egg development rate in our study population, such regulation could be a target of selection imposed by climate change.

Image Processing Method of an Insect Embryo Based on Optical Coherence Tomography

Image Processing Method of an Insect Embryo Based on Optical Coherence Tomography

Molecular basis of the silkworm mutant rel causing red egg color and embryonic death.

The coloration and hatchability of insect eggs can affect individual and population survival. However, few genetic loci have been documented to affect both traits, and the genes involved in regulating these two traits are unclear. The silkworm recessive mutant rel shows both red egg color and embryo mortality. We studied the molecular basis of the rel phenotype formation. Through genetic analysis, gene screening and sequencing, we found that two closely linked genes, BGIBMGA003497 (Bm-re) and BGIBMGA003697 (BmSema1a), control egg color and embryo mortality, respectively. Six base pairs of the Bm-re gene are deleted in its open reading frame, and BmSema1a is expressed at abnormally low levels in mutant rel . BmSema1a gene function verification was performed using RNA interference and clustered randomly interspersed palindromic repeats (CRISPR)/CRISPR-associate protein 9. Deficiency of the BmSema1a gene can cause the death of silkworm embryos. This study revealed the molecular basis of silkworm rel mutant formation and indicated that the Sema1a gene is essential for insect embryo development.

Callosobruchus embryo struggle to guarantee progeny production

We conducted a series of experiments to test insect embryo capability to survive and increase reproductive investment during early development after short exposure to essential oils. We used Callosobruchus maculatus as a model insect and eucalyptus leaf and flower essential oils. Both essential oils exhibited toxicity against C. maculatus embryos and adults.However, flower essential oil was more toxic. A fetus exposed to essential oils tried to make the best of a bad situation and compensate essential oils harmful effects in the later life stages. Insect progeny production guarantee resulted in a trade-off between reproduction and female longevity. The insect also could alter fitness and reproductive behavior including, mating latency reduction, copulation duration increase, and copulation success rate raise in adulthood. Flower essential oil-exposed embryos were more successful in increasing copulation duration, and leaf essential oil-exposed embryos achieved more copulation success and less mating latency. These consequences persisted until F1 generation that was not directly exposed to essential oil. However, the F2 generation could concur with the harmful effects of essential oils. C. maculatus embryo might use epigenetic mechanisms to guarantee progeny production. Reproductive behavior changes and the trade-off can be evolutionary mechanisms to save species from possible extinction in deleterious situations.

CRISPR/Cas9-induced vitellogenin knockout lead to incomplete embryonic development in Plutella xylostella

Vitellogenin (Vg) is important for insect egg maturation and embryo development. In the present study, we characterized the molecular structure and expression profile of Vg gene, and analyzed its reproductive functions in diamondback moth, Plutella xylostella (L.), a destructive pest of cruciferous crops, using CRISPR/Cas9 system. The P. xylostella Vg (PxVg) included all conserved domains and motifs that were commonly found in most insect Vgs except for the polyserine tract. PxVg gene was highly expressed in female pupae and adults. PxVg protein was detected in eggs and female adults. PxVg was mainly expressed in the fat body and its protein was detected in most tissues, except in the midgut. CRISPR/Cas9-induced PxVg knockout successfully constructed a homozygous mutant strain with a 5-base pair nucleotide deletion. No PxVg protein was found in the mutant individuals and in their ovaries. There were no significant differences between wild (WT) and mutant (Mut-5) types of P. xylostella in terms of ovariole length and the number of fully developed oocytes in newly emerged females. No significant difference was observed in the number of eggs laid within two days, but there was a lower egg hatchability (84% for WT vs. 47% for Mut-5). This is the first study presenting the functions of Vg in ovary development, egg maturation, oviposition and embryonic development of P. xylostella. Our results suggest that the reproductive functions of Vg may be species-specific in insects. It is possible that Vg may not be the major egg yolk protein precursor in P. xylostella. Other “functional Vgs” closely involved in the yolk formation and oogenesis would need to be further explored in P. xylostella.

Scanning laser optical tomography resolves developmental neurotoxic effects on pioneer neurons

Developmental neurotoxic compounds impair the developing human nervous system at lower doses than those affecting adults. Standardized test methods for assessing developmental neurotoxicity (DNT) require the use of high numbers of laboratory animals. Here, we use a novel assay that is based on the development of an intact insect embryo in serum-free culture. Neural pathways in the leg of embryonic locusts are established by a pair of afferent pioneer neurons, extending axons along a well-defined pathway to the central nervous system. After exposure to test chemicals, we analyze pioneer neuron shape with conventional fluorescence microscopy and compare it to 3D images, obtained by scanning laser optical tomography (SLOT) and processed by a segmentation algorithm. The segmented SLOT images resolve the 3D structure of the pioneers, recognize pathfinding defects and are thus advantageous for detecting DNT-positive compounds. The defects in axon elongation and pathfinding of pioneer axons caused by two DNT-positive reference compounds (methylmercury chloride; sodium(meta)arsenite) are compared to the biochemically measured general viability of the embryo. Using conventional fluorescence microscopy to establish concentration-response curves of axon elongation, we show that this assay identifies methylmercury chloride and the pro-apoptotic compound staurosporine as developmental neurotoxicants.

Transcriptome of pleuropodia from locust embryos supports that these organs produce enzymes enabling the larva to hatch

BackgroundPleuropodia are limb-derived glandular organs that transiently appear on the first abdominal segment in embryos of insects from majority of “orders”. They are missing in the genetic model Drosophila and little is known about them. Experiments carried out on orthopteran insects 80 years ago indicated that the pleuropodia secrete a “hatching enzyme” that digests the serosal cuticle to enable the larva to hatch, but evidence by state-of-the-art molecular methods is missing.ResultsWe used high-throughput RNA-sequencing to identify the genes expressed in the pleuropodia of the locust Schistocerca gregaria (Orthoptera). First, using transmission electron microscopy we studied the development of the pleuropodia during 11 stages of the locust embryogenesis. We show that the glandular cells differentiate and start secreting just before the definitive dorsal closure of the embryo and the secretion granules outside the cells become more abundant prior to hatching. Next, we generated a comprehensive embryonic reference transcriptome for the locust and used it to study genome wide gene expression across ten morphologicaly defined stages of the pleuropodia. We show that when the pleuropodia have morphological markers of functional organs and produce secretion, they are primarily enriched in transcripts associated with transport functions. They express genes encoding enzymes capable of digesting cuticular protein and chitin. These include the potent cuticulo-lytic Chitinase 5, whose transcript rises just before hatching. Unexpected finding was the enrichment in transcripts for immunity-related enzymes. This indicates that the pleuropodia are equipped with epithelial immunity similarly as barrier epithelia in postembryonic stages.ConclusionsThese data provide transcriptomic support for the historic hypothesis that pleuropodia produce cuticle-degrading enzymes and function in hatching. They may also have other functions, such as facilitation of embryonic immune defense. By the genes that they express the pleuropodia are specialized embryonic organs and apparently an important though neglected part of insect physiology.

An intact insect embryo for developmental neurotoxicity testing of directed axonal elongation_suppl

An intact insect embryo for developmental neurotoxicity testing of directed axonal elongation_suppl

An intact insect embryo for developmental neurotoxicity testing of directed axonal elongation.

Developmental neurotoxicity (DNT) of chemicals poses a serious threat to human health worldwide. Current in vivo test methods for assessing DNT require the use of high numbers of laboratory animals. Most alternative in vitro testing methods monitor rather simple toxicological endpoints, whereas the formation of a functional brain requires precisely timed navigation of axons within a complex tissue environment. We address this complexity by monitoring defects in axonal navigation of pioneer axons of intact locust embryos after exposure to chemicals. Embryos develop in serum-free culture with test chemicals, followed by immunolabeling of pioneer neurons. Defects in axon elongation of pioneer axons are quantified in concentration-response curves and compared to the general viability of the embryo, as measured by a resazurin assay. We show that selected chemical compounds interfering with calcium signaling, the cytoskeletal organization, and the reference developmental neurotoxicant rotenone, can be classified as DNT positive. The pesticide rotenone inhibits pioneer neuron elongation with a lower IC50 than the viability assay. The rho kinase inhibitor Y27632 can partially rescue outgrowth inhibition, supporting the classification of rotenone as a specific DNT positive compound. Since mechanisms of axonal guidance, such as growth cone navigation along molecular semaphorin gradients are conserved between locust and mammalian nervous systems, we will further explore the potential of this invertebrate preparation as an assay for testing the DNT potential of chemicals in humans.