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.     

Some Physiological Effects of Viviparous Genes vp(1) and vp(5) on Developing Maize Kernels

The effects of two viviparous genes, vp₁ and vp₅, on development of the maize (Zea mays L.) embryo and endosperm were investigated. Differences between viviparous and normal embryos first appeared at 25 to 30 days after pollination. Increases in fresh weights indicated that viviparous began to grow more rapidly than normal embryos at that time. Amino acids and ethanol-soluble carbohydrates also accumulated more rapidly in viviparous, but a reserve material (lipid) was lower in viviparous than in normal embryos.     

Cover Caption

The social aphid Pseudoregma bambucicola can produce morphologically and behaviorally specialized sterile soldiers and reproductively normal nymphs, and represents a good model system for studying social evolution. The cover photo shows a colony of this species on bamboo, in which several parthenogenetic viviparous mothers are producing offspring. Remarkable reproductive plasticity in parthenogenetic mothers and a novel strategy of regulating caste ratio via maternal reproductive differentiation were found in this social hemipteran (see pages 582-586). Photo provided by Xiaolei Huang.     

Chromosome numbers of parthenogenetic females of fifty-five species of Aphididae (Homoptera) new to cytology

The numbers of somatic chromosomes of 55 species of Aphididae are presented. Squashed embryos from apterous viviparous females are stained with crystalviolet. Some relationships between aphid chromosomes and aphid systematics are discussed.     

Structure of ovaries in ensign scale insects,the most primitive representatives of Coccomorpha (Insecta,Hemiptera)

The ovaries of Orthezia urticae and Newsteadia floccosa are paired and composed of numerous short ovarioles. Each ovariole consists of an anterior trophic chamber and a posterior vitellarium that contains one developing oocyte. The trophic chamber contains large nurse cells (trophocytes) and arrested oocytes. The total number of germ cells per ovariole (i.e., cluster) is variable, but it is always higher than 32 and less than 64. This suggests that five successive mitotic cycles of a cystoblast plus additional divisions of individual cells are responsible for the generation of the cluster. Cells of the trophic chamber maintain contact with the oocyte via a relatively broad nutritive cord. The trophic chamber and oocyte are surrounded by somatic cells that constitute the inner epithelial sheath around the former and the follicular epithelium around the latter. Anagenesis of hemipteran ovarioles is discussed in relation to the findings presented. © 1995 Wiley-Liss, Inc.     

Ovariole number and fecundity in aphids

Ovariole number in both the sycamore aphid and the bird cherry-oat aphid was shown to be a constant for a particular generation. This generation specific ovariole number remained constant, despite varied nymphal experiences. The number of embryos per ovariole in the sycamore aphid was dependent on the size of the adult aphid. In the bird cherry-oat aphid, the number of embryos per ovariole was a constant, but the number of pigmented embryos present was a function of the size of the aphid at adult moult. Ovariole number, an index of reproductive potential, was thus concluded to be the result of an intrinsic and programmed factor.

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Zusammenfassung Individuen der ersten und zweiten Generation der Ahornzierlaus, Drepanosiphum platanoidis, und der ersten drei apteren Generationen von Migranten der Traubenkirschenlaus, Rhopalosiphum padi, wurden unter verschiedenen Temperatur- und Ernährungs-bedingungen gehalten. Die Imagines wurden seziert. Die Anzahl der Ovariolen war konstant, unabhängig von den Bedingungen, denen die Larven ausgesetzt waren. Tiere der ersten Generation der Ahornzierlaus hatten zwölf Ovariolen während Tiere der zweiten und dritten Generation acht bzw. sechs Ovariolen aufwiesen.Vergleiche von Läusen derselben Generation zeigten, daß die großen Individuen der Ahornzierlaus ein größeres Vermehrungspotential hatten als kleine Individuen. Die Bedingungen, unter denen sie gehalten wurden, beeinflußte die Gesamtzahl der Embryos pro Ovariole. Bei Rhopalosiphum padi dagegen war die Gesamtzahl der Embryos sowohl bei kleinen als auch bei großen Läusen gleich; jedoch erhöhte sich die Anzahl der reifen Embryos pro Ovariole, wenn die Larven unter verbesserten Bedingungen aufgezogen wurden.Die Anzahl der Ovariolen, ein Index für das Vermehrungspotential der Blattläuse, ist teilweise das Resultat eines internen, programmierten Faktors.

     

Embryogenesis in parthenogenetic and sexual females of Aphis fabae

Embryogenesis and oogenesis in the black bean aphid, Aphis fabae, was studied by dissecting viviparous and oviparous individuals and examining whole mounts of the ovaries in Ringer's solution. Apteriform and alatiform virginoparae, gynoparae, and oviparae were raised on broadbean leafdiscs at 17.5° and either 16 h or 10 h light/day.Each of the two ovaries of these forms consists, with few exceptions, of six ovarioles. Development of embryos in virginoparous aphids can commence, in the most developed embryos within their grandmothers, but not before the latter reached the 4th instar. At birth viviparous larvae contain about two embryos in each ovariole. In apteriform larvae, additional embryos are generated at a rate of about one per instar. Adult apterae raised at 16 h and 10 h light/day contain about six and five embryos per ovariole, respectively. Alatiform larvae (virginoparae or gynoparae) raised under these conditions contain no more than four embryos per ovariole when they become adult. Ovulation in oviparae is evident only in the 2nd instar of these sexual females. Only one egg develops per ovariole during their larval development. A second egg occasionally develops later in the adult life of oviparae that have not mated and hence have not oviposited.

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Zusammenfassung Die Embryogenese und Oogenese der Schwarzen Bohnenlaus, Aphis fabae, wurde untersucht durch Aufpräparieren von viviparen und oviparen Tieren und Überprüfung der Ovarien als Totalpräparate in Ringerlösung. Ungeflügelte und geflügelte Virginopare, Gynopare und Ovipate wurden an Blattscheibchen von Dicker Bohne bei 17,5° und 16/8 oder 10/14 HD gezogen.Jedes der beiden Ovarien dieser Formen besteht, mit wenigen Ausnahmen, aus sechs Ovariolen. Die Entwicklung der Embryonen von virginoparen Tieren kann bei den am weitesten entwickelten Embryonen in ihren Großmüttern beginnen aber nicht bevor die letzteren das 4. Larvenstadium erreicht haben. Bei der Geburt enthalten vivipare Larven etwa zwei Embryonen in jeder Ovariole. Bei apteriformen Larven entstehen weitere Embryonen mit einer Rate von etwa einem je Stadium. Adulte Ungeflügelte enthalten bei Aufzucht im 16/8 und im 10/14 HD-Tag etwa sechs bzw. fünf Embryonen je Ovariole. Alatiforme Larven (virginopare und gynopare) enthalten bei solchen Bedingungen nicht mehr als vier Embryonen je Ovariole, wenn sie adult werden. Die Ovulation ist bei den Oviparen erst im 2. Stadium dieser Sexualweibchen erkennbar. Nur ein Ei entwickelt sich je Ovariole während der Larvenentwicklung. Ein zweites Ei entwickelt sich gelegentlich später im adulten Leben von Oviparen, die sich nicht gepaart haben und demnach keine Eier abgelegt haben.

     

Impact of a parasitoid on the bacterial symbiosis of its aphid host

Embryo production in aphids is absolutely dependent on the function of symbiotic bacteria, mainly Buchnera, and the growth and development of koinobiont parasitoids in aphids requires the diversion of nutrients from aphid embryo production to the parasitoid. The implication that the bacterial symbiosis may be promoted in parasitized aphids to support the growing parasitoid was explored by analysis of the number and biomass of mycetocytes, and the aphid cells bearing Buchnera, in the pea aphid Acyrthosiphon pisum Harris (Hemiptera: Aphididae) parasitized by the wasp Aphidius ervi Haliday (Hymenoptera: Braconidae). Aphids hosting a young larval parasitoid bore more mycetocytes of greater total biomass, and embryos of lower biomass than unparasitized aphids. Furthermore, one of the three aphid clones tested, which limited teratocyte growth (giant cells of parasitoid origin having a trophic role), bore smaller mycetocytes and larger embryos, than one or both of the two aphid clones with greater susceptibility to the parasitoid. These data suggest that susceptibility of the aphid‐Buchnera symbiosis to parasitoid‐mediated manipulation may, directly or indirectly, contribute to aphid susceptibility to parasitoid exploitation.     

Embryo nutrition in parthenogenetic viviparous aphids

Abstract The reproductive potential of aphids is well known and is the basis for their considerable economic importance. Reproductive output is ultimately fuelled by nutrients derived from the maternal haemolymph but mechanisms of embryo provisioning are poorly understood. Here, the morphological features of the reproductive system that have a critical role in nutrient transfer are reviewed, and the contribution of the symbiotic bacteria Buchnera to embryo nutrition is considered. In particular, the importance of the ovariole sheath as the selective barrier for nutrient exchange between the maternal haemolymph and the embryo tissue is highlighted, along with the division of the bacterial population into distinct embryo and maternal compartments. Three models are proposed to explain the relative contribution of the symbiotic compartments to embryo nutrition and the review concludes by highlighting experimental studies that have directly examined nutrient transfer from the maternal haemolymph to the developing embryos.     

The role of intracellular symbiotic bacteria in the amino acid nutrition of embryos from the black bean aphid,Aphis fabae

Aphids are well‐known for their symbiotic relationship with intracellular bacteria of the genus Buchnera (γ‐Proteobacteria). The symbiosis has a nutritional basis in that the bacteria supplement the aphid diet of phloem sap through the provision of essential amino acids. To date, few studies have considered the spatial complexity of the association, particularly the delineation of the symbiosis into embryo and maternal compartments. Here, we generate aposymbiotic (bacteria‐free) embryos of the black bean aphid, Aphis fabae (Scopoli) (Hemiptera: Aphididae), as our experimental model and demonstrate that embryos reared in culture media require an external supply of essential amino acids. Analysis of individual amino acid deletions from the culture medium indicate that the key individual amino acids for embryo growth are phenylalanine and valine derived from the maternal tissues, and tryptophan derived from Buchnera. These results are discussed in relation to our current limited understanding of nutrient supply to aphid embryos.     

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.     

Temperature prediction and the timing of sex in aphids

Summary The aphid life cycle contains a series of parthenogenetic, viviparous generations, followed usually by a sexual generation that produces resistant overwintering eggs. Since the decision to produce sexually-reproducing offspring ends the period of rapid growth of the clone, it should be postponed as late as is compatible with successful oviposition. The time of leaf fall determines the latest possible time of oviposition, and is itself determined mainly by daylength. The time required for the development of the final generations of aphid depends on temperature. The decision to end the sequence of parthenogenetic generations should thus depend on temperature and photoperiod. This paper calculates the optimal combination of daylength and temperature for the cueing of this decision in Rhopalosiphum padi. It is shown that the experimental data of Dixon and Glen (1971) are in agreement with the predictions, and that, in the field, the time of production of male R. padi does vary adaptively with July temperature.     

Redescription of Neoamphorophora ledi (Wahlgren, 1938) (Hemiptera: Aphididae) from Japan

Apterous viviparous females of Neoamphorophora ledi (Wahlgren, 1938) living on Rhododendron degronianum Carr. (Ericaceae) on Honshu island, Japan, are redescribed and the hitherto unknown alate viviparous females are here described. Rhododendron degronianum is a new host plant species for this aphid. This species is recorded for aphid fauna of Eastern Palearctic for the first time. Keys to species of Neoamphorophora based on apterous and alate viviparous females are given. A key to aphid genera with swollen siphunculi living on species of Rhododendron worldwide based on apterous viviparous females is also provided.     

The ovaries of aphids (Hemiptera,Sternorrhyncha, Aphidoidea): morphology and phylogenetic implications

The ovaries of aphids belonging to the families Eriosomatidae, Anoeciidae, Drepanosiphidae, Thelaxidae, Aphididae, and Lachnidae were examined at the ultrastructural level. The ovaries of these aphids are composed of several telotrophic ovarioles. The individual ovariole is differentiated into a terminal filament, tropharium, vitellarium, and pedicel (ovariolar stalk). Terminal filaments of all ovarioles join together into the suspensory ligament, which attaches the ovary to the lobe of the fat body. The tropharium houses individual trophocytes and early previtellogenic oocytes termed arrested oocytes. Trophocytes are connected with the central part of the tropharium, the trophic core, by means of broad cytoplasmic processes. One or more oocytes develop in the vitellarium. Oocytes are surrounded by a single layer of follicular cells, which do not diversify into distinct subpopulations. The general organization of the ovaries in oviparous females is similar to that of the ovaries in viviparous females, but there are significant differences in their functioning: (1) in viviparous females, all ovarioles develop, whereas in oviparous females, some of them degenerate; (2) the number of germ cells per ovariole is usually greater in females of the oviparous generation than in females of viviparous generations; (3) in oviparous females, oocytes in the vitellarium develop through three stages (previtellogenesis, vitellogenesis, and choriogenesis), whereas in viviparous females, the development of oocytes stops after previtellogenesis; and (4) in the oocyte cytoplasm of oviparous females, lipid droplets and yolk granules accumulate, whereas in viviparous females, oocytes accrue only lipid droplets. Our results indicate that a large number of germ cells per ovariole represent the ancestral state within aphids. This trait may be helpful in inferring the phylogeny of Aphidoidea.     

The trophic chamber (germanium) of the ovary in oviparae of the vetch aphid,Megoura viciae Buckton (Homoptera : Aphididae)

In the germaria of oviparae of the vetch aphid, Megoura viciae Buckton (Homoptera : Aphididae), the trophocytes are syncytial and arranged around a trophic core, to which they are all joined. Resting oocytes occur in the posterior region of the germaria, and encircle the basal region of the nutritive cord. The trophocytes contain mitochondria, ribosomes, vesicles, electron-dense spheres and a single large nucleus that is highly lobed and has many nucleopores. Electron-dense, “nuage-like” materials are confluent between nucleoplasm and cytoplasm, suggesting nucleocytoplasmic transport. Exterior to the trophocytes, a unicellular sheath surrounds each germarium, bordered to the exterior by a tunica propria. The cells of the sheath are continuous with the prefollicular tissue. Only one oocyte in each ovariole undergoes vitellogenesis at a time.     

Egg envelopes of Baetis rhodani and Cloeon dipterum (Ephemeroptera, Baetidae): a comparative analysis between an oviparous and an ovoviviparous species

A comparative ultrastructural investigation on the eggshell (vitelline and chorionic envelopes) has been carried out in the nymphs of two mayfly species encompassed into the Baetidae, namely Baetis rhodani and Cloeon dipterum. During oocyte differentiation in the meroistic telotrophic ovarioles, gametes are connected to discrete nurse cells by trophic cords. In B. rhodani, each ovariole contains several eggs arranged in sequence, whereas in C. dipterum each contains a single egg. Follicle cells are competent for vitelline and chorionic envelope synthesis. Baetis rhodani is an oviparous species and the chorion is fairly thick, formed by an alveolate endochorion and a fibrillar exochorion delimited by a honey‐comb roof. Cloeon dipterum stands out among Ephemeroptera for being ovoviviparous. In B. rhodani, ovulation starts in the older nymphs with dark wing‐pads, whereas in C. dipterum choriogenesis ends in the imaginal stage. Here the chorion is very thin and laid eggs hatch almost immediately, allowing the larvulae to move out. The maturation of a single egg per ovariole is synchronized with the achievement of the adult stage. The acquisition of ovoviviparity has led to remarkable changes in the ovariole organization along with a simplification of the eggshell structure.     

Juvenile Hormone Stimulation of Oocyte Development and Embryogenesis in the Parthenogenetic Ovaries of an Aphid,Aphis fabae

Summary

The parthenogenetic ovaries of the black bean aphid, Aphis fabae, contain developing embryos. When reared at 15°C in long days (LD 16:8) oocyte development begins within the ovaries of the largest embryos of a fourth instar mother 24–48 hr after her ecdysis from the third instar. Starvation, decapitation and precocene III treatment inhibit embryonic oocyte development; juvenile hormone treatment reverses this inhibition. A method for the in vitro culture of embryos is described and under these conditions juvenile hormone again stimulates oogenesis. Embryogénie growth in vivo, as measured by the increase in length of the oldest daughter embryos, is also stimulated by juvenile hormone treatment. The results are discussed in relation to other roles proposed for juvenile hormone in aphid development.     

Isotopic (δ15N) relationship of pregnant females and their embryos: Comparing placental and yolk-sac viviparous elasmobranchs

Nitrogen stable isotopes ratios (δ¹⁵N) were determined for selected tissues (muscle, liver, blood and yolk) of pregnant females and their embryos of a placental viviparous species, the Pacific sharpnose shark (Rhizoprionodon longurio), and a yolk-sac viviparous species, the speckled guitarfish (Pseudobatos glaucostigmus). The R. longurio embryo tissues were ¹⁵N enriched compared to the same tissues in the pregnant female, using the difference in δ¹⁵N (Δδ¹⁵N) between embryo and adult. Mean Δδ¹⁵N was 2.17‰ in muscle, 4.39‰ in liver and 0.80‰ in blood. For P. glaucostigmus, embryo liver tissue was significantly ¹⁵N enriched in comparison with liver of the pregnant female (Δδ¹⁵N mean = 1.22‰), whereas embryo muscle was ¹⁵N depleted relative to the muscle of the pregnant female (Δδ¹⁵N mean = −1.22‰). Both species presented a significant positive linear relationship between Δδ¹⁵N and embryo total length (L_T). The results indicated that embryos have different Δδ¹⁵N depending on their reproductive strategy, tissue type analysed and embryo L_T.     

Morphogenesis of the ovarian follicular epithelium during initial stages of embryogenesis of the viviparous earwig,Hemimerus talpoides

Representatives of the highly specialized earwig family Hemimeridae are epizoic and viviparous. Their embryos develop inside terminal ovarian follicles (termed also embryonic follicles) and rely solely on nutrients transferred from mother tissues. In this report, we present results of ultrastructural and histochemical studies of the initial stage of Hemimerus talpoides development. Our results show that the follicular cells surrounding fully grown oocyte of Hemimerus do not degenerate after initiation of embryogenesis, but transform and gradually form the wall of the incubation chamber in which the embryo develops. We also show that amniotic cells of the early embryo remain in direct contact with transformed follicular cells. In the region of contact, short outgrowths of the amniotic cells associate with irregular surface specializations of the transformed follicular cells. We suggest that extended “postfertilization” activity of hemimerid follicular cells represents an adaptation to viviparity and matrotrophy in this insect lineage.