Germline specification and axis determination in viviparous and oviparous pea aphids: conserved and divergent features.

Aphids are hemimetabolous insects that undergo incomplete metamorphosis without pupation. The annual life cycle of most aphids includes both an asexual (viviparous) and a sexual (oviparous) phase. Sexual reproduction only occurs once per year and is followed by many generations of asexual reproduction, during which aphids propagate exponentially with telescopic development. Here, we discuss the potential links between viviparous embryogenesis and derived developmental features in the pea aphid Acyrthosiphon pisum, particularly focusing on germline specification and axis determination, both of which are key events of early development in insects. We also discuss potential evolutionary paths through which both viviparous and oviparous females might have come to utilize maternal germ plasm to drive germline specification. This developmental strategy, as defined by germline markers, has not been reported in other hemimetabolous insects. In viviparous females, furthermore, we discuss whether molecules that in other insects characterize germ plasm, like Vasa, also participate in posterior determination and how the anterior localization of the hunchback orthologue Ap-hb establishes the anterior-posterior axis. We propose that the linked chain of developing oocytes and embryos within each ovariole and the special morphology of early embryos might have driven the formation of evolutionary novelties in germline specification and axis determination in the viviparous aphids. Moreover, based upon the finding that the endosymbiont Buchnera aphidicola is closely associated with germ cells throughout embryogenesis, we propose presumptive roles for B. aphidicola in aphid development, discussing how it might regulate germline migration in both reproductive modes of pea aphids. In summary, we expect that this review will shed light on viviparous as well as oviparous development in aphids.

Epicatechin isolated from Tripterygium wilfordii extract reduces tau-GFP-induced neurotoxicity in zebrafish embryo through the activation of Nrf2.

Tau plays important roles in the assembly and stabilization of the microtubule structure to facilitate axonal transport in mammalian brain. The intracellular tau aggregates to form paired helical filaments leading to neurodegenerative disorders, collectively called tauopathies. In our previous report, we established a zebrafish model to express tau-GFP to induce neuronal death, which could be directly traced in vivo. Recently, we used this model to screen 400 herbal extracts and found 45 of them to be effective on reducing tau-GFP-induced neuronal death. One of the effective herbal extracts is the Tripterygium wilfordii stem extract. HPLC analysis and functional assay demonstrated that epicatechin (EC) is the major compound of Tripterygium wilfordii stem extract to decrease the neurotoxicity induced by tau-GFP. Using a luciferase reporter assay in the zebrafish, we confirmed that EC could activate Nrf2-dependent antioxidant responses to significantly increase the ARE-controlled expression of luciferase reporter gene. These data suggest that EC from the Tripterygium wilfordii stem extract could diminish tau-GFP-induced neuronal death through the activation of Nrf2.

Multiple signaling factors and drugs alleviate neuronal death induced by expression of human and zebrafish tau proteins in vivo.

BACKGROUND: The axonal tau protein is a tubulin-binding protein, which plays important roles in the formation and stability of the microtubule. Mutations in the tau gene are associated with familial forms of frontotemporal dementia with Parkinsonism linked to chromosome-17 (FTDP-17). Paired helical filaments of tau and extracellular plaques containing beta-amyloid are found in the brain of Alzheimer's disease (AD) patients. RESULTS: Transgenic models, including those of zebrafish, have been employed to elucidate the mechanisms by which tau protein causes neurodegeneration. In this study, a transient expression system was established to express GFP fusion proteins of zebrafish and human tau under the control of a neuron-specific HuC promoter. Approximately ten neuronal cells expressing tau-GFP in zebrafish embryos were directly imaged and traced by time-lapse recording, in order to evaluate the neurotoxicity induced by tau-GFP proteins. Expression of tau-GFP was observed to cause high levels of neuronal death. However, multiple signaling factors, such as Bcl2-L1, Nrf2, and GDNF, were found to effectively protect neuronal cells expressing tau-GFP from death. Treatment with chemical compounds that exert anti-oxidative or neurotrophic effects also resulted in a similar protective effect and maintained human tau-GFP protein in a phosphorylated state, as detected by antibodies pT212 and AT8. CONCLUSIONS: The novel finding of this study is that we established an expression system expressing tau-GFP in zebrafish embryos were directly imaged and traced by time-lapse recording to evaluate the neurotoxicity induced by tau-GFP proteins. This system may serve as an efficient in vivo imaging platform for the discovery of novel drugs against tauopathy.

Enhancing protein signal detection in asexual and viviparous pea aphids: A guided protocol for tissue dissection and proteinase K treatment.

Aphids, as hemipteran insects, reproduce via parthenogenesis and viviparity, resulting in rapid and exponential offspring production. To investigate the molecular mechanisms underlying parthenogenetic viviparity in asexual aphids, precise protein detection through immunostaining is essential. Our previous research demonstrated the need for proteinase K (PK) treatment to improve tissue permeability, enabling antibodies targeting the germ-cell marker Ap-Vas1 to access gastrulating and later-stage embryos. However, optimal PK digestion protocols have not been thoroughly explored. In this study, we propose strategies to optimize PK digestion conditions for early, middle, and late-stage pea aphid embryos, which have varying tissue thicknesses. Additionally, we extend the application of PK treatment to salivary glands, a representative somatic tissue, by optimizing conditions for antibody penetration against the salivary gland marker C002. To enhance spatial precision in signal detection, we provide a detailed protocol for tissue dissection specific to pea aphids, focusing on the preservation of tissue integrity. These comprehensive guidelines, covering tissue dissection and PK titration, are expected to improve the specificity and intensity of protein signals in pea aphids and other aphid species.•Provide aphid-specific dissection methods to obtain intact embryos and salivary glands.•Present strategies for optimizing PK treatment conditions across different tissue types.

Identification and characterization of alternative promoters of zebrafish Rtn-4/Nogo genes in cultured cells and zebrafish embryos.

In mammals, the Nogo family consists of Nogo-A, Nogo-B and Nogo-C. However, there are three Rtn-4/Nogo-related transcripts were identified in zebrafish. In addition to the common C-terminal region, the N-terminal regions of Rtn4-n/Nogo-C1, Rtn4-m/Nogo-C2 and Rtn4-l/Nogo-B, respectively, contain 9, 25 and 132 amino acid residues. In this study, we isolated the 5'-upstream region of each gene from a BAC clone and demonstrated that the putative promoter regions, P1-P3, are functional in cultured cells and zebrafish embryos. A transgenic zebrafish Tg(Nogo-B:GFP) line was generated using P1 promoter region to drive green fluorescent protein (GFP) expression through Tol2-mediated transgenesis. This line recapitulates the endogenous expression pattern of Rtn4-l/Nogo-B mRNA in the brain, brachial arches, eyes, muscle, liver and intestines. In contrast, GFP expressions by P2 and P3 promoters were localized to skeletal muscles of zebrafish embryos. Several GATA and E-box motifs are found in these promoter regions. Using morpholino knockdown experiments, GATA4 and GATA6 were involved in the control of P1 promoter activity in the liver and intestine, while Myf5 and MyoD for the control of P1 and P3 promoter activities in muscles. These data demonstrate that zebrafish Rtn4/Nogo transcripts might be generated by coupling mechanisms of alternative first exons and alternative promoter usage.

Optical phase modulators using deformable waveguides actuated by micro-electro-mechanical systems.

An optical phase modulator is presented by using micro-electro-mechanical systems to actuate deformable silicon waveguides. Via mechanically stretching the waveguide length, the optical path is extended, resulting in a phase shift. The experimental results show that a phase shift of near 0.4π is achieved at 200 V for both TE- and TM-polarized waves by cascading six phase modulation units, agreeing well with the theoretical prediction. The power consumption is estimated to be smaller than 0.2 mW at 200 V, mainly resulting from the leakage current.

Morphological and molecular studies of a microsporidium (Nosema sp.) isolated from the thee spot grass yellow butterfly, Eurema blanda arsakia (Lepidoptera: Pieridae).

A microsporidium possessing molecular and morphological characteristics of the genus Nosema was isolated from larvae of the thee-spot grass yellow butterfly, Eurema blanda arsakia. The complete rRNA gene sequences of the E. blanda isolate contained 4,428 base pairs (GenBank Accession No. EU338534). The organization of the rRNA genes is LSU rRNA-ITS-SSU rRNA-IGS-5S, which corresponds with that of Nosema species closely related to Nosema bombycis. Phylogenetic analysis based on rRNA gene sequences show that this isolate is closely related to Nosema bombycis, Nosema plutellae, Nosema spodopterae, and Nosema antheraeae. The ultrastructure of all developmental stages of this microsporidium confirmed its placement in the genus Nosema. The isolate was successfully propagated in cell lines IPLB-LD652Y (Lymantria dispar) and NTU-LY (Lymantria xylina) and, in the in vitro system, it was frequently found to develop in the nuclei of the host cells, a circumstance that seldom occurs in other Nosema species. An extra-cellular vegetative stage of this microsporidium was also observed in the culture medium after 14 days of infection. The ECMDFs might be released from disrupted host cells.

Molecular evolutionary trends and feeding ecology diversification in the Hemiptera, anchored by the milkweed bug genome.

BACKGROUND: The Hemiptera (aphids, cicadas, and true bugs) are a key insect order, with high diversity for feeding ecology and excellent experimental tractability for molecular genetics. Building upon recent sequencing of hemipteran pests such as phloem-feeding aphids and blood-feeding bed bugs, we present the genome sequence and comparative analyses centered on the milkweed bug Oncopeltus fasciatus, a seed feeder of the family Lygaeidae. RESULTS: The 926-Mb Oncopeltus genome is well represented by the current assembly and official gene set. We use our genomic and RNA-seq data not only to characterize the protein-coding gene repertoire and perform isoform-specific RNAi, but also to elucidate patterns of molecular evolution and physiology. We find ongoing, lineage-specific expansion and diversification of repressive C2H2 zinc finger proteins. The discovery of intron gain and turnover specific to the Hemiptera also prompted the evaluation of lineage and genome size as predictors of gene structure evolution. Furthermore, we identify enzymatic gains and losses that correlate with feeding biology, particularly for reductions associated with derived, fluid nutrition feeding. CONCLUSIONS: With the milkweed bug, we now have a critical mass of sequenced species for a hemimetabolous insect order and close outgroup to the Holometabola, substantially improving the diversity of insect genomics. We thereby define commonalities among the Hemiptera and delve into how hemipteran genomes reflect distinct feeding ecologies. Given Oncopeltus's strength as an experimental model, these new sequence resources bolster the foundation for molecular research and highlight technical considerations for the analysis of medium-sized invertebrate genomes.

Whole-mount identification of gene transcripts in aphids: protocols and evaluation of probe accessibility.

In situ hybridization has become a powerful tool for detecting the temporal and spatial distribution of gene transcripts in prokaryotes and eukaryotes. We report an efficient protocol for whole-mount identification of the expression of mRNAs in the parthenogenetic pea aphid Acyrthosiphon pisum, an emerging model organism with a growing accumulation of genome sequencing data. In addition to steps common for most animal in situ hybridization protocols, we describe processing methods specific to aphids, the accessibility of antisense riboprobes of different lengths in whole-mounted aphids, and signal intensity versus probe lengths. To find substrate combinations that clearly contrast single and double in situ signals in A. pisum, we tested our protocols using riboprobes constructed from two conserved germline markers, Apvasa and Apnanos, and examined colocalized signals in the germaria and developing oocytes. Finally, we propose conditions for stringent permeabilization that may be applied to tissues deep within the aphid embryo.

Germ-plasm specification and germline development in the parthenogenetic pea aphid Acyrthosiphon pisum: Vasa and Nanos as markers.

The germarium, oocytes and embryos of the parthenogenetic viviparous pea aphid Acyrthosiphon pisum are contained within a single ovariole. This species provides an excellent model for studying how maternally-inherited germ plasm is specified and how it is transferred to primordial germ cells. Previous studies have shown that germ cells are first segregated at the embryonic posterior after formation of the blastoderm. We used two cross-reacting antibodies against the conserved germline markers Vasa and Nanos, which specifically identified these presumptive germ cells, to investigate whether germ cells were determined during early development. We observed randomly-distributed weak expression of Vasa signals in the developing oocyte but no localization in the oocyte segregated from the germarium. Localized Vasa was not apparent until it was detected at the posterior in the embryo undergoing the second nuclear division. Nanos, on the other hand, was localized to a nuage-like structure surrounding the nucleus in the developing and segregated oocytes. At the beginning of the oocyte maturation division, Nanos localization shifted to the posterior and could be identified in successive stages until it was incorporated into the germ cells. Taken together, our results suggest that germ plasm is specified in the developing oocyte and that Nanos is an earlier germline marker than Vasa. Germ cells stained for Vasa remained at a dorsal location in the egg during mid-development and then were guided into abdominal segments A1 to A6 during germ-band retraction. We infer that germ cells coalesce with segmented gonadal mesoderm during this period.

Re-epithelialization of large wound in paedomorphic and metamorphic axolotls.

Axolotls (Ambystoma mexicanum) may heal their skin wounds scar-free in both paedomorphs and metamorphs. In previous studies on small punch skin wounds, rapid re-epithelialisation was noted in these two axolotl morphs. However, large wound size in mammals may affect wound healing. In this study, large circumferential full thickness excision wounds on the hind limbs were created on juvenile paedomorphic and metamorphic axolotls. The results showed re-epithelialisation was more quickly initiated in paedomorphs than in metamorphs after wounding. The migrating rate of epidermis on the wound bed was faster in paedomorphs than in metamorphs and thus completion of re-epithelialisation was faster in paedomorphs than in metamorphs. Within these re-epithelialisation periods, neither basement membrane nor dermis was reformed. Epidermal cell proliferation was detected by EdU-labelling technique. In the normal unwounded skin, epidermal proliferation rate was higher in paedomorphs than in metamorphs. After wounding, the epidermal proliferation rate was significantly lower in the migrating front on the wound bed than in the normal skin in paedomorphs. The EdU-labelling rate between normal skin and migration front was not different in metamorphs. Lacking of more proliferating epidermal cells on the wound bed indicated that the new epidermis here derived rather from migrating epidermal cells than from cell proliferation in situ. In conclusion, re-epithelialisation in the large wound might be fully completed in both morphs despite it was initiated earlier and with faster rate in paedomorphs than in metamorphs. The new epidermis on the wound bed derived mainly from cell migration than by cell proliferation in the re-epithelialisation period. J. Morphol. 278:228-235, 2017. © 2016 Wiley Periodicals,Inc.

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining.

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 µg/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

Amino acid transporters implicated in endocytosis of Buchnera during symbiont transmission in the pea aphid.

BACKGROUND: Many insects host their obligate, maternally transmitted symbiotic bacteria in specialized cells called bacteriocytes. One of the best-studied insect nutritional endosymbioses is that of the aphid and its endosymbiont, Buchnera aphidicola. Aphids and Buchnera are metabolically and developmentally integrated, but the molecular mechanisms underlying Buchnera transmission and coordination with aphid development remain largely unknown. Previous work using electron microscopy to study aphid asexual embryogenesis has revealed that Buchnera transmission involves exocytosis from a maternal bacteriocyte followed by endocytotic uptake by a blastula. While the importance of exo- and endocytic cellular processes for symbiont transmission is clear, the molecular mechanisms that regulate these processes are not known. Here, we shed light on the molecular mechanisms that regulate Buchnera transmission and developmental integration. RESULTS: We present the developmental atlas of ACYPI000536 and ACYPI008904 mRNAs during asexual embryogenesis in the pea aphid, Acyrthosiphon pisum. Immediately before Buchnera invasion, transcripts of both genes were detected by whole-mount in situ hybridization in the posterior syncytial nuclei of late blastula embryos. Following Buchnera invasion, expression of both genes was identified in the region occupied by Buchnera throughout embryogenesis. Notably during Buchnera migration, expression of both genes was not concomitant with the entirety of the bacterial mass but rather expression colocalized with Buchnera in the anterior region of the bacterial mass. In addition, we found that ACYPI000536 was expressed in nuclei at the leading edge of the bacterial mass, joining the bacterial mass in subsequent developmental stages. Finally, quantitative reverse transcription real-time PCR suggested that early in development both transcripts were maternally provisioned to embryos. CONCLUSIONS: We venture that ACYPI000536 and ACYPI008904 function as nutrient sensors at the site of symbiont invasion to facilitate TOR-pathway-mediated endocytosis of Buchnera by the aphid blastula. Our data support earlier reports of bacteriocyte determination involving a two-step recruitment process but suggest that the second wave of recruitment occurs earlier than previously described. Finally, our work highlights that bacteriocyte-enriched amino acid transporter paralogs have additionally been retained to play novel developmental roles in both symbiont recruitment and bacteriome development.

Ontogenetic differences in localization of glutamine transporter ApGLNT1 in the pea aphid demonstrate that mechanisms of host/symbiont integration are not similar in the maternal versus embryonic bacteriome.

BACKGROUND: Obligate intracellular symbionts of insects are metabolically and developmentally integrated with their hosts. Typically, reproduction fails in many insect nutritional endosymbioses when host insects are cured of their bacterial symbionts, and yet remarkably little is known about the processes that developmentally integrate host and symbiont. Here in the best studied insect obligate intracellular symbiosis, that of the pea aphid, Acyrthosiphon pisum, with the gammaproteobacterium Buchnera aphidicola, we tracked the expression and localization of amino acid transporter ApGLNT1 gene products during asexual embryogenesis. Recently being characterized as a glutamine transporter, ApGLNT1 has been proposed to be a key regulator of amino acid biosynthesis in A. pisum bacteriocytes. To determine when this important mediator of the symbiosis becomes expressed in aphid embryonic bacteriocytes, we applied whole-mount in situ hybridization and fluorescent immunostaining with a specific anti-ApGLNT1 antibody to detect the temporal and spatial expression of ApGLNT1 gene products during asexual embryogenesis. RESULTS: During embryogenesis, ApGLNT1 mRNA and protein localize to the follicular epithelium that surrounds parthenogenetic viviparous embryos, where we speculate that it functions to supply developing embryos with glutamine from maternal hemolymph. Unexpectedly, in the embryonic bacteriome ApGLNT1 protein does not localize to the membrane of bacteriocytes, a pattern that leads us to conclude that the regulation of amino acid metabolism in the embryonic bacteriome mechanistically differs from that in the maternal bacteriome. Paralleling our earlier report of punctate cytoplasmic localization of ApGLNT1 in maternal bacteriocytes, we find ApGLNT1 protein localizing as cytoplasmic puncta throughout development in association with Buchnera. CONCLUSIONS: Our work that documents ontogenetic shifts in the localization of ApGLNT1 protein in the host bacteriome demonstrates that maternal and embryonic bacteriomes are not equivalent. Significantly, the persistent punctate cytoplasmic localization of ApGLNT1 in association with Buchnera in embryos prior to bacteriocyte formation and later in both embryonic and maternal bacteriomes suggests that ApGLNT1 plays multiple roles in this symbiosis, roles that include amino acid transport and possibly nutrient sensing.

Germ plasm localisation of the HELICc of Vasa in Drosophila: analysis of domain sufficiency and amino acids critical for localisation.

Formation of the germ plasm drives germline specification in Drosophila and some other insects such as aphids. Identification of the DEAD-box protein Vasa (Vas) as a conserved germline marker in flies and aphids suggests that they share common components for assembling the germ plasm. However, to which extent the assembly order is conserved and the correlation between functions and sequences of Vas remain unclear. Ectopic expression of the pea aphid Vas (ApVas1) in Drosophila did not drive its localisation to the germ plasm, but ApVas1 with a replaced C-terminal domain (HELICc) of Drosophila Vas (DmVas) became germ-plasm restricted. We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation. Similarly, HELICc of the grasshopper Vas could be recruited to the germ plasm in Drosophila. Nonetheless, germ-plasm localisation was not seen in the Drosophila oocytes expressing HELICcs of Vas orthologues from aphids, crickets, and mice. We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species. This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

Posterior localization of ApVas1 positions the preformed germ plasm in the sexual oviparous pea aphid Acyrthosiphon pisum.

BACKGROUND: Germline specification in some animals is driven by the maternally inherited germ plasm during early embryogenesis (inheritance mode), whereas in others it is induced by signals from neighboring cells in mid or late development (induction mode). In the Metazoa, the induction mode appears as a more prevalent and ancestral condition; the inheritance mode is therefore derived. However, regarding germline specification in organisms with asexual and sexual reproduction it has not been clear whether both strategies are used, one for each reproductive phase, or if just one strategy is used for both phases. Previously we have demonstrated that specification of germ cells in the asexual viviparous pea aphid depends on a preformed germ plasm. In this study, we extended this work to investigate how germ cells were specified in the sexual oviparous embryos, aiming to understand whether or not developmental plasticity of germline specification exists in the pea aphid. RESULTS: We employed Apvas1, a Drosophila vasa ortholog in the pea aphid, as a germline marker to examine whether germ plasm is preformed during oviparous development, as has already been seen in the viviparous embryos. During oogenesis, Apvas1 mRNA and ApVas1 protein were both evenly distributed. After fertilization, uniform expression of Apvas1 remained in the egg but posterior localization of ApVas1 occurred from the fifth nuclear cycle onward. Posterior co-localization of Apvas1/ApVas1 was first identified in the syncytial blastoderm undergoing cellularization, and later we could detect specific expression of Apvas1/ApVas1 in the morphologically identifiable germ cells of mature embryos. This suggests that Apvas1/ApVas1-positive cells are primordial germ cells and posterior localization of ApVas1 prior to cellularization positions the preformed germ plasm. CONCLUSIONS: We conclude that both asexual and sexual pea aphids rely on the preformed germ plasm to specify germ cells and that developmental plasticity of germline specification, unlike axis patterning, occurs in neither of the two aphid reproductive phases. Consequently, the maternal inheritance mode implicated by a preformed germ plasm in the oviparous pea aphid becomes a non-canonical case in the Hemimetabola, where so far the zygotic induction mode prevails in most other studied insects.

Reliable protocols for whole-mount fluorescent in situ hybridization (FISH) in the pea aphid Acyrthosiphon pisum: a comprehensive survey and analysis.

RNA in situ hybridization (ISH), including chromogenic ISH (CISH) and fluorescent ISH (FISH), has become a powerful tool for revealing the spatial distribution of gene transcripts in model organisms. Previously, we developed a robust protocol for whole-mount RNA CISH in the pea aphid Acyrthosiphon pisum, an emerging insect genomic model. In order to improve the resolving capacity of gene detection, we comprehensively surveyed current protocols of whole-mount RNA-FISH and developed protocols that allow, using confocal microscopy, clearer visualization of target messenger RNAs (mRNAs) - including those subcellularly localized and those with spatially overlapping expression. We find that Fast dye-based substrate fluorescence (SF), tyramide signal amplification (TSA), and TSA Plus all enable identifying gene expression thanks to multiplex amplification of fluorescent signals. By contrast, methods of direct fluorescence (DF) do not allow visualizing signals. Detection of a single gene target was achieved with SF and TSA Plus for most mRNAs, whereas TSA only allowed visualization of abundant transcripts such as Apvas1 and Appiwi2 in the germ cells. For detection of multiple gene targets using double FISH, we recommend: (i) TSA/TSA, rather than TSA Plus/TSA Plus for colocalized mRNAs abundantly expressed in germ cells, as proteinase K treatment can be omitted; and (ii) SF/TSA Plus for other gene targets such as Apen1 and Apen2 as inactivation of enzyme conjugates is not required. SF/SF is not ideal for double FISH experiments due to signal blurring. Based on these new conditions for RNA-FISH, we have obtained a better understanding of germline specification and embryonic segmentation in the pea aphid. We anticipate that the RNA-FISH protocols for the pea aphid may also be used for other aphids and possibly other insect species, thus expanding the range of species from which useful insights into development and evolution may be obtained.

Expansion of genes encoding piRNA-associated argonaute proteins in the pea aphid: diversification of expression profiles in different plastic morphs.

Piwi-interacting RNAs (piRNAs) are known to regulate transposon activity in germ cells of several animal models that propagate sexually. However, the role of piRNAs during asexual reproduction remains almost unknown. Aphids that can alternate sexual and asexual reproduction cycles in response to seasonal changes of photoperiod provide a unique opportunity to study piRNAs and the piRNA pathway in both reproductive modes. Taking advantage of the recently sequenced genome of the pea aphid Acyrthosiphon pisum, we found an unusually large lineage-specific expansion of genes encoding the Piwi sub-clade of Argonaute proteins. In situ hybridisation showed differential expressions between the duplicated piwi copies: while Api-piwi2 and Api-piwi6 are "specialised" in germ cells their most closely related copy, respectively Api-piwi5 and Api-piwi3, are expressed in the somatic cells. The differential expression was also identified in duplicated ago3: Api-ago3a in germ cells and Api-ago3b in somatic cells. Moreover, analyses of expression profiles of the expanded piwi and ago3 genes by semi-quantitative RT-PCR showed that expressions varied according to the reproductive types. These specific expression patterns suggest that expanded aphid piwi and ago3 genes have distinct roles in asexual and sexual reproduction.

Developmental expression of Apnanos during oogenesis and embryogenesis in the parthenogenetic pea aphid Acyrthosiphon pisum.

Among genes that are preferentially expressed in germ cells, nanos and vasa are the two most conserved germline markers in animals. Both genes are usually expressed in germ cells in the adult gonads, and often also during embryogenesis. Both nanos-first or vasa-first expression patterns have been observed in embryos, implying that the molecular networks governing germline development vary among species. Previously we identified Apvasa, a vasa homologue expressed in germ cells throughout all developmental stages in the parthenogenetic and viviparous pea aphid Acyrthosiphon pisum. In asexual A. pisum, oogenesis is followed by embryogenesis, and both occur within the ovarioles. In order to understand the temporal and spatial distribution of nanos versus vasa during oogenesis and embryogenesis, we isolated a nanos homologue, Apnanos, and studied its expression. In adults, Apnanos is preferentially expressed in the ovaries. In early embryos, Apnanos transcripts are localized to the cytoplasm of cellularizing germ cells, and soon thereafter are restricted to the newly segregated germ cells in the posterior region of the cellularized blastoderm. These results strongly suggest that the Apnanos gene is a germline marker and is involved in germline specification in asexual A. pisum. However, during the middle stages of development, when germline migration occurs, Apnanos is not expressed in the migrating germ cells expressing Apvasa, suggesting that Apnanos is not directly associated with germline migration.