Identifying genes related to choriogenesis in insect panoistic ovaries by Suppression Subtractive Hybridization

<p>Abstract</p> <p>Background</p> <p>Insect ovarioles are classified into two categories: panoistic and meroistic, the later having apparently evolved from an ancestral panoistic type. Molecular data on oogenesis is practically restricted to meroistic ovaries. If we aim at studying the evolutionary transition from panoistic to meroistic, data on panoistic ovaries should be gathered. To this end, we planned the construction of a Suppression Subtractive Hybridization (SSH) library to identify genes involved in panoistic choriogenesis, using the cockroach <it>Blattella germanica </it>as model.</p> <p>Results</p> <p>We constructed a post-vitellogenic ovary library by SSH to isolate genes involved in choriogenesis in <it>B. germanica</it>. The tester library was prepared with an ovary pool from 6- to 7-day-old females, whereas the driver library was prepared with an ovary pool from 3- to 4-day-old females. From the SSH library, we obtained 258 high quality sequences which clustered into 34 unique sequences grouped in 19 contigs and 15 singlets. The sequences were compared against non-redundant NCBI databases using BLAST. We found that 44% of the unique sequences had homologous sequences in known genes of other organisms, whereas 56% had no significant similarity to any of the databases entries. A Gene Ontology analysis was carried out, classifying the 34 sequences into different functional categories. Seven of these gene sequences, representative of different categories and processes, were chosen to perform expression studies during the first gonadotrophic cycle by real-time PCR. Results showed that they were mainly expressed during post-vitellogenesis, which validates the SSH technique. In two of them corresponding to novel genes, we demonstrated that they are specifically expressed in the cytoplasm of follicular cells in basal oocytes at the time of choriogenesis.</p> <p>Conclusion</p> <p>The SSH approach has proven to be useful in identifying ovarian genes expressed after vitellogenesis in <it>B. germanica</it>. For most of the genes, functions related to choriogenesis are postulated. The relatively high percentage of novel genes obtained and the practical absence of chorion genes typical of meroistic ovaries suggest that mechanisms regulating chorion formation in panoistic ovaries are significantly different from those of meroistic ones.</p

Brownie, a Gene Involved in Building Complex Respiratory Devices in Insect Eggshells

Background: Insect eggshells must combine protection for the yolk and embryo with provisions for respiration and for the entry of sperm, which are ensured by aeropyles and micropyles, respectively. Insects which oviposit the eggs in an egg-case have a double problem of respiration as gas exchange then involves two barriers. An example of this situation is found in the cockroach Blattella germanica, where the aeropyle and the micropyle are combined in a complex structure called the sponge-like body. The sponge-like body has been well described morphologically, but nothing is known about how it is built up. Methodology/Principal Findings: In a library designed to find genes expressed during late chorion formation in B. germanica, we isolated the novel sequence Bg30009 (now called Brownie), which was outstanding due to its high copy number. In the present work, we show that Brownie is expressed in the follicle cells localized in the anterior pole of the oocyte in late choriogenesis. RNA interference (RNAi) of Brownie impaired correct formation of the sponge-like body and, as a result, the egg-case was also ill-formed and the eggs were not viable. Conclusions/Significance: Results indicate that the novel gene Brownie plays a pivotal role in building up the sponge-lik

Different Bla-g T cell antigens dominate responses in asthma versus rhinitis subjects

Dillon, M. B. C. et al.[Background and objective] The allergenicity of several German cockroach (Bla-g) antigens at the level of IgE responses is well established. However, less is known about the specificity of CD4+ TH responses, and whether differences exist in associated magnitude or cytokine profiles as a function of disease severity.[Methods] Proteomic and transcriptomic techniques were used to identify novel antigens recognized by allergen-specific T cells. To characterize different TH functionalities of allergen-specific T cells, ELISPOT assays with sets of overlapping peptides covering the sequences of known allergens and novel antigens were employed to measure release of IL-5, IFNγ, IL-10, IL-17 and IL-21.[Results] Using these techniques, we characterized TH responses in a cohort of adult Bla-g-sensitized subjects, either with (n = 55) or without (n = 17) asthma, and nonsensitized controls (n = 20). T cell responses were detected for ten known Bla-g allergens and an additional ten novel Bla-g antigens, representing in total a 5-fold increase in the number of antigens demonstrated to be targeted by allergen-specific T cells. Responses of sensitized individuals regardless of asthma status were predominantly TH2, but higher in patients with diagnosed asthma. In asthmatic subjects, Bla-g 5, 9 and 11 were immunodominant, while, in contrast, nonasthmatic-sensitized subjects responded mostly to Bla-g 5 and 4 and the novel antigen NBGA5.[Conclusions] Asthmatic and nonasthmatic cockroach-sensitized individuals exhibit similar TH2-polarized responses. Compared with nonasthmatics, however, asthmatic individuals have responses of higher magnitude and different allergen specificity.This project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract numbers HHSN272200900052C and HHSN272201000052I and grant numbers 1UM1AI114271-01 and U19AI100275.Peer reviewe

Chorion formation in panoistic ovaries requires windei and trimethylation of histone 3 lysine 9

Epigenetic modifications play key roles in transcriptional regulation. Trimethylation of histone 3 lysine 9 (H3K9me3) is one of the most widely studied histone post-translational modifications, and has been linked to transcriptional repression. In Drosophila melanogaster, Windei is needed for H3K9me3 in female germ line cells. Here, we report the occurrence of a D. melanogaster Windei (Wde) ortholog in the ovary of the hemimetabolous insect Blattella germanica, which we named BgWde. Depletion of BgWde by RNAi reduced H3K9me3 in follicular cells, which triggered changes in transcriptional regulation that led to the prevention of chorion gene expression. In turn, this impaired oviposition (and the formation of the ootheca) and, therefore, prevented reproduction. Windei and H3K9me3 have already been reported in follicular cells of D. melanogaster, but this is the first time that the function of these modifications has been demonstrated in the said cells. This is also the first time that an epigenetic marker is reported as having a key role in choriogenesis.Support for this research was provided by the Spanish Ministry of Science and Innovation (Grant nos. BFU2011-22404 to MDP and CGL2008-03517/BOS to XB), by the CSIC (Grant no. 2010TW0019, Formosa program) and from FEDER funds to XB. Support from Generalitat de Catalunya (Grant no. 2005 SGR 00053) and LINC-Global is also gratefully acknowledged. AH received a pre-doctoral research grant (JAE-LINCG program) from the CSIC, and The European Social Fund (ESF)

Chorion formation in panoistic ovaries requires windei and trimethylation of histone 3 lysine 9

Epigenetic modifications play key roles in transcriptional regulation. Trimethylation of histone 3 lysine 9 (H3K9me3) is one of the most widely studied histone post-translational modifications, and has been linked to transcriptional repression. In Drosophila melanogaster, Windei is needed for H3K9me3 in female germ line cells. Here, we report the occurrence of a D. melanogaster Windei (Wde) ortholog in the ovary of the hemimetabolous insect Blattella germanica, which we named BgWde. Depletion of BgWde by RNAi reduced H3K9me3 in follicular cells, which triggered changes in transcriptional regulation that led to the prevention of chorion gene expression. In turn, this impaired oviposition (and the formation of the ootheca) and, therefore, prevented reproduction. Windei and H3K9me3 have already been reported in follicular cells of D. melanogaster, but this is the first time that the function of these modifications has been demonstrated in the said cells. This is also the first time that an epigenetic marker is reported as having a key role in choriogenesis.Support for this research was provided by the Spanish Ministry of Science and Innovation (Grant nos. BFU2011-22404 to MDP and CGL2008-03517/BOS to XB), by the CSIC (Grant no. 2010TW0019, Formosa program) and from FEDER funds to XB. Support from Generalitat de Catalunya (Grant no. 2005 SGR 00053) and LINC-Global is also gratefully acknowledged. AH received a pre-doctoral research grant (JAE-LINCG program) from the CSIC, and The European Social Fund (ESF)

Comparative analysis of miRNA expression during the development of insects of different metamorphosis modes and germband types

BACKGROUND: Do miRNAs contribute to specify the germ-band type and the body structure in the insect embryo? Our goal was to address that issue by studying the changes in miRNA expression along the ontogeny of the German cockroach Blattella germanica, which is a short germ-band and hemimetabolan species. RESULTS: We sequenced small RNA libraries representing 11 developmental stages of B. germanica ontogeny (with especial emphasis on embryogenesis) and the changes in miRNA expression were examined. Data were compared with equivalent data for two long germ-band holometabolan species Drosophila melanogaster and Drosophila virilis, and the short germ-band holometabolan species Tribolium castaneum. The identification of B. germanica embryo small RNA sequences unveiled miRNAs not detected in previous studies, such as those of the MIR-309 family and 54 novel miRNAs. Four main waves of miRNA expression were recognized (with most miRNA changes occurring during the embryonic stages): the first from day 0 to day 1 of embryogenesis, the second during mid-embryogenesis (days 0-6), the third (with an acute expression peak) on day 2 of embryonic development, and the fourth during post-embryonic development. The second wave defined the boundaries of maternal-to-zygotic transition, with maternal mRNAs being cleared, presumably by Mir-309 and associated scavenger miRNAs. CONCLUSION: miRNAs follow well-defined patterns of expression over hemimetabolan ontogeny, patterns that are more diverse during embryonic development than during the nymphal stages. The results suggest that miRNAs play important roles in the developmental transitions between the embryonic stages of development (starting with maternal loading), during which they might influence the germ-band type and metamorphosis mode.This work was supported by the Spanish Ministry of Economy and Competitiveness (grants CGL2012–36251 and CGL2015–64727-P to X.B.), the Spanish Ministry of Science and Innovation (grants BFU2011–22404 and CGL2016–76011-R to M.D.P.) and the Catalan Government (2014 SGR 619). It also received financial assistance from the European Fund for Economic and Regional Development (FEDER funds to X.B. and M.D.P.

The notch pathway regulates both the proliferation and differentiation of follicular cells in the panoistic ovary of Blattella germanica

The Notch pathway is an essential regulator of cell proliferation and differentiation during development. Its involvement in insect oogenesis has been examined in insect species with meroistic ovaries, and it is known to play a fundamental role in cell fate decisions and the induction of the mitosis-to-endocycle switch in follicular cells (FCs). This work reports the functions of the main components of the Notch pathway (Notch and its ligands Delta and Serrate) during oogenesis in Blattella germanica, a phylogenetically basal species with panoistic ovary. As is revealed by RNAi-based analyses, Notch and Delta were found to contribute towards maintaining the FCs in an immature, non-apoptotic state. This ancestral function of Notch appears in opposition to the induction of transition from mitosis to endocycle that Notch exerts in Drosophila melanogaster, a change in the Notch function that might be in agreement with the evolution of the insect ovary types. Notch was also shown to play an active role in inducing ovarian follicle elongation via the regulation of the cytoskeleton. In addition, Delta and Notch interactions were seen to determine the differentiation of the posterior population of FCs. Serrate levels were found to be Notch-dependent and are involved in the control of the FC programme, although they would appear to play no crucial role in panoistic ovary oogenesis.This work was supported by the Ministry of Science and Innovation, Spain (grant no. BFU2011-22404), by the Catalan Regional Government (grant no. 2014 SGR 619), and by CONICYT + PAI Concurso Nacional Apoyo al Retorno de Investigadores desde el Extranjero, Convocatoria 2013 + 821320046. P.I. is the recipient of a postdoctoral research grant (BECAS CHILE from CONICYT. N.E. is the recipient of a JAE pre-doctoral research grant from the Consejo Superior de Investigaciones Científicas (CSIC

Expansions of key protein families in the German cockroach highlight the molecular basis of its remarkable success as a global indoor pest

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.We thank the Deutsche Forschungsgemeinschaft for providing funding for this research to M.C.H. via grant BO2544/11‐1 to E.B.B., and acknowledge United States National Science Foundation grant IOS1557864 to C.S. X.B. & M.‐D.P. were supported by the Spanish Ministry of Economy and Competitiveness (grants CGL2015‐64727‐P to X.B. and CGL2016‐76011‐R to M.‐D.P., both with FEDER funds) and Catalan Government (grant 2017 SGR 1030).Peer reviewe