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The transcription factor Ftz-f1 has multiple functions in insect development in a spatial-temporal line. One of these roles is in the insect ovaries, specifically in the regulation of steroidogenic enzymes production. We studied the function of F in Blattella germanica oogenesis, as it shows two moments of high expression in ovaries: before the imaginal moult, and just before ovulation in the adult. Injecting dsftz-f1 into adult females, either just after the imaginal moult or just prior to choriogenesis, prevented oviposition, with differences between the two approaches. In 3-day-old adult females treated with dsftz-f1 just after the emergence, the expression of ftz-f1 was not modified, but the steroidogenic genes increased their expression. ftz-f1 transcript levels in the ovaries of 5-day-old dsftz-f1-treated females were significantly depleted, and the expression levels of the same steroidogenic genes began to decrease. These results suggest that Ftz-f1 regulates the expression of steroidogenic genes in B. germanica, with phm possibly being a key target. Ftz-f1 has a different temporal function in the cytoskeleton of follicular cells of the basal ovarian follicles. Early in the gonadotrophic cycle, Ftz-f1 promotes the expression of genes related to the cytoskeleton and muscle proteins, while at the end of the cycle it maintains the expression levels of these genes, thus ensuring correct ovulation.

Ecdysone regulates essential processes in insect life. Perhaps the most well-known of these are related to metamorphosis. However, ecdysone is also required to regulate the proliferation and differentiation of germ cells in the ovary. The role of ecdysone in insect oogenesis has been studied in depth in holometabolan species with meroistic ovaries, such as Drosophila melanogaster, while in hemimetabolan species with panoistic ovaries their functions are still poorly understood. In the present work, we studied the role of ecdysone in the ovary of the last nymphal instar of the cockroach Blattella germanica by using RNA interference to reduce the levels of the ecdysone receptor (EcR), and thereby deplete the expression of ecdysteroidogenic genes in the prothoracic gland. However, the expression of ecdysteroidogenic genes was upregulated in the ovary, resulting in cell overproliferation in the germarium, which appeared swollen. By analysing the expression of genes that respond to ecdysone, we found that when the source of 20E is the nymphal ovary, EcR appears to repress 20E-associated genes bypassing early genes signalling.

Cytokinesis is the physical process of cell separation in the cell division, in which the cytoplasm of a parental eukaryotic cell divides into two daughter cells. The cytoskeleton is crucial to carry out the cytokinesis. shrub encodes a filament-forming subunit of ESCRT-III (Endosomal Sorting Complex Required for Transport) essential in the cell abscission, the last step of cytokinesis. In female germline stem cells of Drosophila melanogaster, shrub controls the timing of cell division by recruiting ESCRT-III complex in the midbody before the abscission and its absence stops germinal stem cells division. The current knowledge of the function of shrub in insect oogenesis is limited to germinal stem cells, and its role in ovarian somatic cells is still unknown. Our objective is to unveil the function of shrub in the oogenesis of adult cockroaches of Blattella germanica using RNA interference (RNAi). Experiments were performed injecting freshly emerged adult females with double-strand RNA (dsRNA) against shrub and dissecting insects at day 5.

Current approaches for insect gene editing require microinjection of materials into early embryos. This severely limits the application of gene editing to a great number of insect species, especially to those whose reproduction systems preclude access to early embryos for injection. To overcome these limitations, we report a simple and accessible method for insect gene editing, termed “direct parental” CRISPR (DIPA-CRISPR). We show that injection of Cas9 ribonucleoproteins (RNPs) into the haemocoel of adult females efficiently introduces heritable mutations in developing oocytes. Importantly, commercially available standard Cas9 protein can be directly used for DIPA-CRISPR, which makes this approach highly practical and feasible. DIPA-CRISPR enables highly efficient gene editing in the cockroaches, on which conventional approaches cannot be applied, and in the model beetle Tribolium castaneum. Due to its simplicity and accessibility, DIPA-CRISPR will greatly extend the application of gene editing technology to a wide variety of insects.

[Background] RNA interference (RNAi) is a cellular mechanism used to fight various threats, including transposons, aberrant RNAs, and some types of viruses. This mechanism relies on the detection of dsRNA molecules, which through a pathway involving Dicer-2 (Dcr-2) and Argonaute 2 (AGO2), produces small interfering RNAs (siRNAs) that bind to the complementary RNAs triggering their degradation., [Methods] Using the cockroach Blattella germanica as a model, we examined AGO2 activity by depleting its mRNA using RNAi and analyzing the phenotypes produced., [Results] Depleting AGO2 expression had no remarkable effect on nymphal development or reproduction. dsRNA treatment triggered an immediate and transitory increase in AGO2 expression, independently of Dcr-2 action. In addition, we analyzed the siRNAs generated after injecting a heterologous dsRNA in control and AGO2-depleted animals. The results revealed that obtained siRNAs mapped non-uniformly along the dsRNA sequence. In AGO2-depleted animals, the proportion of 22 nucleotide reads was higher and accumulations of reads appeared in areas less well-represented in the controls. We also detected a preference for cytosine as the first nucleotide in controls that was significantly attenuated in AGO2-depleted individuals., [Conclusions/general significance] The siRNAs produced from a dsRNA mapped heterogeneously along the length of the dsRNA and this arrangement depends on the dsRNA sequence. AGO2 exerts its role as nuclease on the siRNA duplexes independently of its action on the corresponding mRNA. This study sheds light on an extremely useful process for reverse genetics in laboratories, in addition to the design of more effective, specific, and eco-friendly pest-control strategies.

Eyes absent (Eya), is a protein structurally conserved from hydrozoans to humans, for which two basic roles have been reported: it can act as a transcription cofactor and as a protein tyrosine phosphatase. Eya was discovered in the fly Drosophila melanogaster in relation to its function in eye development, and the same function was later reported in other insects. Eya is also involved in insect oogenesis, although studies in this sense are limited to D. melanogaster, which has meroistic ovaries, and where eya mutations abolish gonad formation. In the present work we studied the function of eya in the panoistic ovary of the cockroach Blattella germanica. We show that eya is essential for correct development of panoistic ovaries. In B. germanica, eya acts at different level and in a distinct way in the germarium and the vitellarium. In the germarium, eya contributes to maintain the correct number of somatic and germinal cells by regulating the expression of steroidogenic genes in the ovary. In the vitellarium, eya facilitates follicle cells proliferation and contributes to regulate the cell program, in the context of basal ovarian follicle maturation. Thus, eya-depleted females of B. germanica arrest the growth and maturation of basal ovarian follicles and become sterile.

The chytrid fungus Batrachochytrium dendrobatidis has caused the most prominent loss of vertebrate diversity ever recorded, which peaked in the 1980s. Recent incursion by its sister species B. salamandrivorans in Europe raised the alarm for a new wave of declines and extinctions in western Palearctic urodeles. The European Commission has responded by restricting amphibian trade. However, private amphibian collections, the main end consumers, were exempted from the European legislation. Here, we report how invasion by a released, exotic newt coincided with B. salamandrivorans invasion at over 1000 km from the nearest natural outbreak site, causing mass mortality in indigenous marbled newts (Triturus marmoratus), and posing an acute threat to the survival of nearby populations of the most critically endangered European newt species (Montseny brook newt, Calotriton arnoldi). Disease management was initiated shortly after detection in a close collaboration between policy and science and included drastic on site measures and intensive disease surveillance. Despite these efforts, the disease is considered temporarily contained but not eradicated and continued efforts will be necessary to minimize the probability of further pathogen dispersal. This precedent demonstrates the importance of tackling wildlife diseases at an early stage using an integrated approach, involving all stakeholders and closing loopholes in existing regulations.

“One of the fundamental events during lifetime is founding the next generation” (Büning [1]). “…oogenesis is the necessary first step for development of the animal embryo since only in this unique cell does the nucleus exhibit its totipotent properties required for supporting normal embryogenesis.” (Mahowald [2]).

The Piwi-interacting RNA (piRNA) system is an evolutionarily conserved mechanism involved in the control of transposable elements and maintenance of genomic stability, especially in germ-line cells and in early embryo stages. However, relevant particularities, both in mechanism and function, exist across species among metazoans and even within the insect class. As a member of the scarcely studied hemimetabolan group, Blattella germanica can be a suitable reference model to study insect evolution. We present the results of a stringent process of identification and study of expressed piRNAs for B. germanica across 11 developmental stages, ranging from non-fertilized egg to nymphs and adult female. Our results confirm the dual origin of piRNA in this species, with a majority of them being generated from the primary pathway, and a smaller but highly expressed set of sequences participating in the secondary (“pingpong”) reamplification pathway. An intriguing partial complementarity in expression is observed between the piRNA of the two biogenesis pathways, with those generated in the secondary pathway being quite restricted to early embryo stages. In addition, many piRNAs are exclusively expressed in late embryo and nymphal stages. These observation point at piRNA functions beyond the role of transposon control in early embryogenesis. Our work supports the view of a more complex scenario, with different sets of piRNAs acting in different times and having a range of functions wider than previously thought.

The German cockroach, Blattella germanica, is a worldwide pest that infests buildings, including homes, restaurants, and hospitals, often living in unsanitary conditions. As a disease vector and producer of allergens, this species has major health and economic impacts on humans. Factors contributing to the success of the German cockroach include its resistance to a broad range of insecticides, immunity to many pathogens, and its ability, as an extreme generalist omnivore, to survive on most food sources. The recently published genome shows that B. germanica has an exceptionally high number of protein coding genes. In this study, we investigate the functions of the 93 significantly expanded gene families with the aim to better understand the success of B. germanica as a major pest despite such inhospitable conditions. We find major expansions in gene families with functions related to the detoxification of insecticides and allelochemicals, defense against pathogens, digestion, sensory perception, and gene regulation. These expansions might have allowed B. germanica to develop multiple resistance mechanisms to insecticides and pathogens, and enabled a broad, flexible diet, thus explaining its success in unsanitary conditions and under recurrent chemical control. The findings and resources presented here provide insights for better understanding molecular mechanisms that will facilitate more effective cockroach control.