Genetic Basis for Developmental Homeostasis of Germline Stem Cell Niche Number: A Network of Tramtrack-Group Nuclear BTB Factors

The potential to produce new cells during adult life depends on the number of stem cell niches and the capacity of stem cells to divide, and is therefore under the control of programs ensuring developmental homeostasis. However, it remains generally unknown how the number of stem cell niches is controlled. In the insect ovary, each germline stem cell (GSC) niche is embedded in a functional unit called an ovariole. The number of ovarioles, and thus the number of GSC niches, varies widely among species. In Drosophila, morphogenesis of ovarioles starts in larvae with the formation of terminal filaments (TFs), each made of 8–10 cells that pile up and sort in stacks. TFs constitute organizers of individual germline stem cell niches during larval and early pupal development. In the Drosophila melanogaster subgroup, the number of ovarioles varies interspecifically from 8 to 20. Here we show that pipsqueak, Trithorax-like, batman and the bric-à-brac (bab) locus, all encoding nuclear BTB/POZ factors of the Tramtrack Group, are involved in limiting the number of ovarioles in D. melanogaster. At least two different processes are differentially perturbed by reducing the function of these genes. We found that when the bab dose is reduced, sorting of TF cells into TFs was affected such that each TF contains fewer cells and more TFs are formed. In contrast, psq mutants exhibited a greater number of TF cells per ovary, with a normal number of cells per TF, thereby leading to formation of more TFs per ovary than in the wild type. Our results indicate that two parallel genetic pathways under the control of a network of nuclear BTB factors are combined in order to negatively control the number of germline stem cell niches.     

Reproductive characteristics of Drosophila hibisci in the Northern Territory, Australia

Reproductive traits of Drosophila hibisci collected at 18 sites in the Northern Territory (NT) of Australia in May, 1998, as well as at two sites in north Queensland, in June, 1998, were compared to those from earlier work on a cline in ovariole number in D. hibisci along the east coast of Australia. The flies in the NT were considerably smaller, but had more ovarioles than comparably-sized flies on the east coast. Although the flies on the east coast showed an increasing number of ovarioles in populations at increasing distances from the equator, these new populations, both on the east coast and in the NT, reversed this trend, producing a generally U-shaped pattern of ovariole number with latitude among all populations. The northernmost and southernmost populations allocate more to ovariole numbers than populations in intermediate latitudes. Ovariole number is closely related to body size of females in all populations, but the regression coefficient is small at intermediate latitudes and increases at the northern and southern ends of the distribution. Egg volumes primarily varied with body size of the female (positive) and number of ovarioles per female (negatively), producing a generally inverted U-shaped pattern of egg volumes with latitude. Reproductive allocation patterns, but not thorax size or ovariole number, varied significantly in two samples taken 10 days apart at one NT site. This variation probably results from environmental differences across generations of developing larvae and is consistent with our earlier suggestion of substantial effects of the environment, primarily rainfall and temperature, on reproductive allocation in D. hibisci.     

A test of three hypotheses for ovariole number determination in the grasshopper Romalea microptera

Ovariole number in insects determines potential fecundity and can be influenced by genes, environmental conditions during development and parental effects. In the present study, three hypotheses are tested for ovariole number determination in the grasshopper Romalea microptera (Beauvois), which exhibits both intra‐ and interpopulation variation in ovariole number. In hypothesis 1, variation in ovariole number is a result of genetic variation. In hypothesis 2, ovariole number is influenced by nutrition during development. In hypothesis 3, ovariole number is influenced by maternal nutritional status. Females from four treatments are compared: low‐food, high‐food, daughters of low‐food, and daughters of high‐food. There is a relationship between parent and offspring ovariole number despite different environments, supporting hypothesis 1. Also, ovariole numbers are slightly, but significantly lower in individuals fed a low‐food diet compared with a high‐food diet, supporting hypothesis 2. Hypothesis 3 is not supported: starved and well‐fed females produce eggs of similar mass, as well as offspring with similar numbers of ovarioles, suggesting that the nutritional status of mothers does not influence offspring mass or offspring ovariole number. The results imply that genetic variation and developmental conditions determine ovariole number in this species but maternal environment does not. These results conflict with previous studies of ovariole determination in grasshoppers and locusts.     

Germ cell cluster formation and cell death are alternatives in caste-specific differentiation of the larval honey bee ovary

Summary

Caste-specific differentiation of the female honey bee gonad takes place in the fifth larval instar. In queen larvae most ovarioles exhibit almost simultaneous formation of numerous germ cell clusters within the first 20 h after the last larval molt. Ultrastructurally distinctive fusomal cytoplasm connects these cystocytes. Germ cell differentiation is accompanied by morphological changes in somatic components of the ovarioles, the follicle and the terminal filament cells. Subsequently, queen ovarioles elongate and differentiate basal stalks that coalesce in a basal calyx. A second round of mitotic activity was found to occur in the late prepupal and early pupal queen ovary. This round may elevate germ cell numbers composing each cluster to levels observed in follicles of adult honey bee queens. In contrast, germ cell cluster formation does not occur in most of the 120–160 ovarioles of the larval worker ovary, but instead many cells in such ovarioles show signs of impending degeneration, such as large autophagic bodies. DNA extracted from worker ovaries did not reveal nucleosomal laddering, and ultrastructurally, chromatin in germ cell nuclei appeared intact. In the 4–7 surviving ovarioles of the small worker ovary, germ cell clusters were found with ultrastructural characteristics identical to those in queen ovarioles. The temporal window during which divergence in developmental pathways of the larval ovaries initiates shortly after the last larval molt coincides with caste-specific differences in juvenile hormone titer which have long been considered critical to caste-specific morphogenesis.     

Cell death in ovarioles causes permanent sterility in Frieseomelitta varia worker bees

Frieseomelitta varia worker bees do not lay eggs even when living in queenless colonies, a condition that favors ovary development and oviposition in the majority of highly social bees. The permanent sterility of these worker bees was initially attributed to a failure in ovary morphogenesis and differentiation. Using transmission electron microscopy we found that at the beginning of the pupal phase the ovaries of F. varia workers are formed by four ovarioles, each of them composed of 1) a terminal filament at the apex of the ovarioles, containing juxtaposed and irregularly shaped cells, 2) a germarium with clusters of cystocytes and prefollicular cells showing long cytoplasmic projections that envelop the cystocyte clusters, 3) fusiform interfollicular and basal stalk precursor cells, and 4) globular, irregularly contoured basal cells with large nuclei. However, during the pupal phase an accentuated and progressive process of cell death takes place in the ovarioles. The dying cells are characterized by large membrane bodies, electron-dense apoptotic bodies, vacuoles, vesiculation, secondary lysosomes, enlarged rough endoplasmic reticulum cisternae, swollen mitochondria, pycnotic nuclei, masses of chromatin adjacent to the convoluted nuclear envelope, and nucleoli showing signs of fragmentation. Cell death continues in ovarioles even after the emergence of the workers. Once they become nurse bees, the ovaries have become transformed into a cell mass in which structurally organized ovarioles can no longer be identified. In F. varia workers, ovariole cell death most certainly is part of the program of caste differentiation.     

SIMILARITIES AND DIFFERENCES IN ALTITUDINAL VERSUS LATITUDINAL VARIATION FOR MORPHOLOGICAL TRAITS IN DROSOPHILA MELANOGASTER

Understanding how natural environments shape phenotypic variation is a major aim in evolutionary biology. Here, we have examined clinal, likely genetically based variation in morphology among 19 populations of the fruit fly (Drosophila melanogaster) from Africa and Europe, spanning a range from sea level to 3000 m altitude and including locations approximating the southern and northern range limit. We were interested in testing whether latitude and altitude have similar phenotypic effects, as has often been postulated. Both latitude and altitude were positively correlated with wing area, ovariole number, and cell number. In contrast, latitude and altitude had opposite effects on the ratio between ovariole number and body size, which was negatively correlated with egg production rate per ovariole. We also used transgenic manipulation to examine how increased cell number affects morphology and found that larger transgenic flies, due to a higher number of cells, had more ovarioles, larger wings, and, unlike flies from natural populations, increased wing loading. Clinal patterns in morphology are thus not a simple function of changes in body size; instead, each trait might be subject to different selection pressures. Together, our results provide compelling evidence for profound similarities as well as differences between phenotypic effects of latitude and altitude.     

The ovaries of Mecoptera: basic similarities and one exception to the rule

Two entirely different types of ovaries (ovarioles) have been described in mecopterans. In the representatives of Meropeidae, Bittacidae, Panorpodidae and Panorpidae the ovarioles are of the polytrophic-meroistic type. Four regions: a terminal filament, germarium, vitellarium and ovariole stalk can be distinguished in the ovarioles. The germaria house numerous germ cell clusters. Each cluster arises as a result of 2 consecutive mitoses of a cystoblast and consists of 4 sibling cells. The oocyte always differentiates from one of the central cells of the cluster, whereas the remaining 3 cells develop into large, polyploid nurse cells. The vitellaria contain 7-12 growing egg chambers (= oocyte-nurse cell complexes). In contrast, the ovaries of the snow flea, Boreus hyemalis, are devoid of nurse cells and therefore panoistic (secondary panoistic). The ovarioles are composed of terminal filaments, vitellaria and ovariole stalks only; in adult females functional germaria are absent. Histochemical tests suggest that amplification of rDNA takes place in the oocyte nuclei. Resulting dense nucleolar masses undergo fragmentation into multiple polymorphic nucleoli. The classification of extant mecopterans as well as the phylogenetic relationships between Mecoptera and Siphonaptera are discussed in the context of presented data.     

What determines the number of ovarioles in a fly ovary?

The relation between the size of a fly and the number of ovarioles in its ovary was investigated in Phormia regina and Sarcophaga bullata. Small flies of varying size were produced by taking larvae prematurely off the food. The smallest flies thus obtained were derived from larvae only $\text{1}\text{8}$ of the weight of a normal larva. The number of ovarioles in an ovary is directly proportional to the size of the fly and, in the extreme case, is about $\text{1}\text{5}$ the normal number in Sarcophaga and about $\text{1}\text{3}$ in Phormia. Larvae prematurely taken off the food, but fed again after starving for several days, grow to normal or almost normal size and develop ovaries with about the normal number of ovarioles. Small or re-fed Sarcophaga do not show any changes in the anatomy of individual ovarioles but in Phormia disorders in ovariole development and a consequent reduction of fertility are frequent. The number of ovarioles remains identical from the early pupal stage all through the development of the pharate fly and then through ovarian development in the adult fly: it is determined by the size of the larva when it was taken off the food. This shows that it is not lack of space in a small adult fly abdomen which determines the number nor the occurrence of degenerative processes during ovarian development.     

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.

     

The ovaries of scale insects (Hemiptera, Coccinea). Morphology and phylogenetic conclusions

Coccoids (Coccinea, Coccoidea, Coccomorpha, scale insects, scales) are a highly diverse group of ectoparasitic insects. They comprise 2 subgroups: primitive archaeococcoids (= Orthezioidea sensu Koteja) and advanced neococcoids (= Coccoidea sensu Koteja). The ovaries of coccoids consist of numerous short telotrophic-meroistic ovarioles. The ovarioles of all investigated species share common characters (e.g. the same mechanism of ovariole development, lack of terminal filaments, occurrence of single oocytes in the vitellaria) supporting the concept of monophyletic origin of this group. Despite these characteristics, the ovaries of archaeococcoids and neococcoids differ in the number of germ cells (oocytes + trophocytes) constituting a single ovariole. In primitive families (Ortheziidae, Margarodidae), this number is relatively large (15-58), whereas in advanced ones (Pseudococcidae, Kermesidae, Eriococcidae, Cryptococcidae, Coccidae, Diaspididae) it is small and usually does not exceed 8. The comparative analysis of the ovary structure in the representatives of Coccinea and closely related Aphidinea (aphids) has revealed that: (1) the organization of archaeococcoid ovaries is more similar to those of aphids than to neococcoids and (2) during the evolution of Coccinea a gradual reduction in the number of germ cells in ovarioles took place.     

Oocyte maturation and ovariole number in lines of Drosophila melanogaster selected for postponed senescence

1. Postponed senescence lines of Drosophila melanogaster have previously been generated by selection for delayed female reproduction.
2. This study addresses the question of whether the selection regime has differentiated lines with respect to oocyte maturation or ovariole number.
3. Oocyte stage and ovariole number were characterized in replicate postponed senescence lines (O) and in replicate control lines (B).
4. Oocyte maturation is delayed in O females.
5. The delay in oocyte maturation in O lines corresponds to reduced early age fecundity.
6. Selection may have resulted in an increased number of ovarioles in O females.     

Juvenile-hormone-dependent interaction of actin and spectrin is crucial for polymorphic differentiation of the larval honey bee ovary

Programmed cell death in the worker ovary of Apis mellifera reduces the number of ovarioles during metamorphosis from 150-200 primordia to less than 10. In contrast, practically all ovarioles in the ovary of queens survive to the adult stage. The correct formation and persistence of polyfusomes has been suggested as a critical factor for ovariole survival. We have analyzed the developmental dynamics of F-actin and alpha-spectrin in fusomes of queen and worker larvae, and in juvenile-hormone-treated worker larvae. Small fusomes containing actin and spectrin can be detected in the ovaries of fourth instar larvae in both castes. After molting to the fifth instar, the actin-spectrin association persists in the enlarged fusomes of queen ovarioles. In workers, actin dissociates from the fusomal and cortical alpha-spectrin. Coinciding with the appearance of apoptosis markers, large agglomerates of actin are detectable in worker ovarioles. Treatment of fourth-instar worker larvae with juvenile hormone rescues ovarioles from apoptosis and maintains the actin-spectrin association. Juvenile-hormone-dependent actin-spectrin interaction is thus one of the earliest steps in the differentiation of a polymorphic ovary. Plasticity in ovariole numbers as a result of hormone-dependent fusome formation may be a more widespread phenomenon in insects, extending beyond caste polymorphism in highly eusocial Hymenoptera.     

Reproductive characteristics of the flower breeding Drosophila hibisci Bock (Drosophilidae) along a latitudinal gradient in eastern Australia: relation to flower and habitat features

For many insect species, egg and larval substrate characteristics are significantly correlated with interspecific differences in female reproductive allocation and egg size-number tradeoffs. We tested the hypothesis that a similar pattern occurred within the Australian drosophilid, Drosophila hibisci, that is restricted throughout its life cycle to flowers of species in the genus Hibiscus. These plants occur as small, isolated, normally monospecific stands that should facilitate differentiation of the fly populations in relation to specific oviposition and larval substrates. Data from 38 sites ranging from 20.8̀ to 34.4̀ S latitude in eastern Australia indicated no relationship between female body size, egg size, or ovariole numbers and floral size or mass among four species of Hibiscus. However, the flies did show a latitudinal cline in ovariole number that was independent of floral variation. Females averaged 15–20 ovarioles per female in the south (32–34̀ S latitude) and 10–12 ovarioles in the north (21–22̀ S latitude). The increase in ovariole number with latitude was due to divergence in the ovariole number of the largest females. In contrast, small females in the north and south had the same number of ovarioles. Reproductive allocation of female flies in the northern region was less than females in the southern region. The latitudinal divergence in ovariole number was not associated with habitat differences (density of trees, density of flies and beetles), nor with differences in floral characteristics (flower weight, petal length, yeast species present). Short term weather patterns in daily temperature and rainfall preceding collections pardy explain the variation in ovariole number. These observations in conjunction with preliminary genetic results suggest the cline is associated with genetic differences that interact with environmental determinants such as the temperature during larval development.     

Structure of the female reproductive system of the lac insect,Kerria chinensis (Sternorrhyncha,Coccoidea: Kerridae)

The ovaries of female lac insects, Kerria chinensis Mahd (Sternorrhyncha: Coccoidea: Kerridae), at the last nymphal stage are composed of several balloon‐like clusters of cystocytes with different sizes. Each cluster consists of several clusters of cystocytes arranging in rosette forms. At the adult stage, the pair of ovaries consists of about 600 ovarioles of the telotrophic‐meroistic type. An unusual feature when considering most scale insects is that the lateral oviducts are highly branched, each with a number of short ovarioles. Each ovariole is subdivided into an anterior trophic chamber (tropharium) containing six or seven large trophocytes and a posterior vitellarium harbouring one oocyte which is connected with the trophic chamber via a nutritive cord. No terminal filament is present. Late‐stage adult females show synchronized development of the ovarioles, while in undernourished females, a small proportion of ovarioles proceed to maturity.     

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.     

Abnormal ovarian morphogenesis in Drosophila melanogaster after injection of embryos with conditioned media from the Daudi cell line

Summary Drosophila melanogaster embryos were injected before the blastoderm stage with conditioned media from several male Burkitt's lymphoma human cell lines and the Daudi cell line. Such injections do not have any effect on the male genital apparatus or on the female tract. The Daudi conditioned medium modifies the ovarian morphogenesis of the flies and the rudimentary ovaries obtained look like nymphal gonads. Moreover, they have a drastically reduced number of germ cells. The ovaries that looked functional contain numerous necrotic germ cells and the mean number of ovarioles per fly is significantly smaller than that of the controls. The abnormalities observed resemble the results of experimental and genetic lack of germ cells. They disappear at very high dilution (1×10^–6).     

The ovary ofCatajapyx aquilonaris (Insecta,Entognatha): ultrastructure of germarium and terminal filament

Summary Each of the two ovaries ofCatajapyx aquilonaris is composed of seven segmentally (metamerically) arranged ovarioles. The two lateral oviducts that join and bear ovarioles extend throughout the abdomen. In the ovariole three regions can be recognized: the terminal filament, the germarium and the vitellarium. The terminal filaments do not fuse with each other but attach separately (by means of muscle fibres) to the closest lobes of the fat body. Germ cells in the germarium are not joined by intercellular bridges and do not form clusters. Thus the ovarioles ofC. aquilonaris are interpreted as being primarily panoistic. The results obtained support the hypothesis that both dipluran subgroups (Campodeina and Japygina) do not form a monophyletic unit.     

Ovariole development in Staphylinidae (Coleoptera)

Summary

Twenty percent of the species from 88% of the genera, which comprise the British Staphylinidae fauna, were dissected and their ovaries attributed to one often defined types. Three-quarters of the species studied possessed six ovarioles in each ovary although the eight species of Bledius examined were found to have only a single ovary, a condition previously thought to be unique to the family Scarabaeidae within the Coleoptera. The type of ovariole development within most staphylinid subfamilies is remarkably constant. Any inconsistencies are described. Species of Aleochara, known to have parasitic larvae, were found to have a larger number of ovarioles per ovary than any other member of the Staphylinidae examined. They also exhibited a greater variation in ovariole number within each species which is correlated with the size of the adult beetle.