Morphology,ultrastructure, and germ cell cluster formation in ovarioles of aphids

The structure of aphid ovaries, including ovipare and virginopare morphs of five species, was investigated by light and electron microscopy. Aphids contain telotrophic meroistic ovarioles. The amount and distribution of cytoplasmic components of nurse cells, nutritive cords, and young oocytes are nearly identical to those known from scale insects and heteropterans. Each ovariole has a constant number of nurse cells and oocytes. In ovaries of ovipare morphs, the nurse cell nuclei enlarge by endomitosis (n = 2⁸n?2¹⁰n), whereas in virginopare morphs the nurse cell nuclei remain small (n = 2²n?2⁴n). Furthermore, in virginoparae the previtellogenic growth of oocytes is highly reduced, and vitellogenesis and chorionogenesis are blocked totally. Embryogenesis starts immediately after the shortened previtellogenic growth. In each ovariole, all germ cell descendants belong to one germ cell cluster that follows the 2ⁿ rule. The cluster normally contains 2⁵ = (32) cells, but other mostly smaller numbers also occur. In contrast to polytrophic meroistic ovarioles, more than one cell of each cluster will develop into an oocyte. In Drepanosiphum platanoides, 16 (2^n?1) nurse cells and 16 (2^n?1) oocytes exist in each cluster, whereas, in Metopolophium dirhodum, 8 (2^n?2) oocytes and 24 (2^n?1 + 2^n?2) nurse cells are normally found. In many ovarioles of Macrosiphum rosae, 21 nurse cells nourish 11 oocytes. Models of germ cell cluster formation in aphid ovaries are discussed.     

Structure and development of the telotrophic ovariole in ensign scale insects (Hemiptera, Coccomorpha: Ortheziidae)

The paired ovaries of young larva of the 3rd instar of Orthezia urticae are filled with numerous germ cell clusters that can be regarded as ovariole anlagen. Germ cells (cystocytes) belonging to one cluster form a rosette, in the centre of which a polyfusome occurs. Staining with rhodamine-phalloidin has revealed that polyfusomes contain numerous microfilaments. The number of cystocytes per cluster is not stable and varies considerably. The ovaries of older larva become elongated with numerous young ovarioles protruding into the body cavity. The ovarioles are not subdivided into the tropharium and vitellarium. In this stage germ cells differentiate into oocytes and trophocytes (nurse cells). The ovaries of adult females are composed of about 20 (Newsteadia floccosa) or 30 (O. urticae) ovarioles. Their trophic chambers contain trophocytes and arrested oocytes. In the vitellarium, at the given moment, only one oocyte develops. It has been observed that after maturation of the first egg the arrested oocytes may develop.     

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.     

Germ cell cluster formation and ovariole structure in viviparous and oviparous generations of the aphid Stomaphis quercus

The developing ovaries of S. quercus contain a limited number of oogonial cells which undergo a series of incomplete mitotic divisions resulting in the formation of clusters of cystocytes. Ovaries of viviparous generations contain 6 to 9 clusters, containing 32 cystocytes each, whereas ovaries of oviparous generations contain 5 clusters containing 45-60 cystocytes. During further development, clusters become surrounded by a single layer of follicular cells, and within each cluster the cystocytes differentiate into oocytes and trophocytes (nurse cells). Concurrently, cysts transform into ovarioles. The anterior part of the ovariole containing the trophocytes becomes the tropharium, whereas its posterior part containing oocytes transforms into the vitellarium. The vitellaria of viviparous females are composed of one or two oocytes, which develop until previtellogenesis. The nuclei of previtellogenic oocytes enter cycles of mitotic divisions which lead to the formation of the embryo. Ovarioles of oviparous females contain a single oocyte which develops through three stages: previtellogenesis, vitellogenesis and choriogenesis. The ovaries are accompanied by large cells termed bacteriocytes which harbor endosymbiotic microorganisms.     

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.     

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.     

Structure of ovaries of scale insects: ii. margarodidae (INSECTA,hemiptera, COCCINEA)

The paired, spindle-shaped ovaries of the second instar of the Polish cochineal, Porphyrophora polonica (L.) (Hemiptera: Coccinea) are filled with cystocytes that are arranged into rosettes. In the centre of each rosette, there is a polyfusome. During the third instar, cystocytes differentiate into oocytes and trophocytes (nurse cells) and ovarioles are formed. Ovaries of adult females are composed of about 300 ovarioles of the telotrophic type. Each of them is subdivided into a tropharium (trophic chamber) and vitellarium. The tropharium consists of trophocytes and arrested oocytes that may develop. The number of germ cells in the trophic chambers varies from 11 to 18 even between the ovarioles of the same ovary. The obtained results seem to confirm the concept of a monophyletic origin of the primitive scale insects (Archaeococcoidea).     

Female reproductive system of the first-instar nymphs of Machilis helleri (verh.) (Thysanura : Machilidae) with special reference to the segmental arrangement of ovarioles in arthropods

The anatomy, histology and ultrastructure of immature ovarioles of the 1st-instar nymphs of Machilis helleri (Thysanura : Machilidae) are described. The nymphs have 7 pairs of segmentally arranged panoistic ovariole primordia in which the germarium and previtellarium can be distinguished. The germarium contains oogonia, young oocytes, and prefollicular cells. The previtellarium is filled with previtellogenic oocytes, prefollicular cells, and a pyramid-like group of somatic cells representing the primordium of the pedicel and oviduct. The ultrastructure of individual types of cells correlates to a great extent with the respective cells of ovarioles of adult machilids. Oogonia undergo mitosis in the germarium and transform into young oocytes. These grow and develop into previtellogenic oocytes characterized by changes in the nucleolus and by emission of ribonucleoproteinaceous bodies from the nucleus into cytoplasm. Segmental arrangement of ovarioles in Archaeognatha is discussed in view of contemporary hypotheses on the anagenesis of the reproductive system of Articulata.     

Microsporidia infect the Liophloeus lentus (Insecta, Colepotera) ovarioles, developing oocytes and eggs

In the ovarioles of Liophloeus lentus (Insecta, Coleoptera, Curculionidae) two types of bacteria and parasitic microorganisms belonging to Microsporidia have been found. This study shows that the different microsporidian life stages (meronts, sporonts, sporoblasts and spores) infect the outer ovariole sheath, trophic chambers, follicular cells, late previtellogenic and vitellogenic oocytes and eggs. In trophic chambers the parasites are very abundant and are distributed unevenly, i.e. their large mass occupies the syncytial cytoplasm between the nurse cell nuclei, whereas the neck region of the trophic chamber (which houses young oocytes, prefollicular cells and trophic cords) is almost free of parasites. The developing oocytes and eggs contain a lower number of parasites which are usually distributed in the cortical ooplasm. The gross morphology of the ovaries is similar in infected and non-infected specimens. Similarly, the presence of a parasite seems to not disturb the course of oogensis. The only difference was found in the ultrastructure of mitochondria in young previtellogenic oocytes. In the infected females they are unusual i.e. bigger and spherical with tubullar cristae, whereas in the non-infected insects they are elongated and have lamellar cristae. As oogenesis progresses the unusual mitochondria rapidly change their morphology and become similar to the mitochondria in non-infected females. Taking into account the distribution of parasites within the ovarioles, it is suggested that they infect growing oocytes via outer ovariole sheath and follicular epithelium rather than via trophic cords.     

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.     

Ovary structure and transovarial transmission of endosymbiotic microorganisms in Marchalina hellenica (Insecta,Hemiptera, Coccomorpha: Marchalinidae)

Szklarzewicz, T., Kalandyk‐Kolodziejczyk, M., Kot, M. and Michalik, A. 2011. Ovary structure and transovarial transmission of endosymbiotic microorganisms in Marchalina hellenica (Insecta, Hemiptera, Coccomorpha: Marchalinidae). —Acta Zoologica (Stockholm) 00 :1–9. The paired ovaries of Marchalina hellenica are composed of about 200 ovarioles of telotrophic type. In each ovariole, a trophic chamber, vitellarium and ovariolar stalk can be distinguished. The tropharia comprise trophocytes and early previtellogenic oocytes (termed arrested oocytes) or trophocytes only. The arrested oocytes are not capable of further development. In the vitellaria, single oocytes develop that are connected to the tropharium by means of broad nutritive cords. The number of germ cells (trophocytes and oocytes) constituting ovarioles is not constant and may range between 25 and 32. Numerous endosymbiotic bacteria occur in the cytoplasm of trophocytes. The endosymbionts are transported via nutritive cords to the developing oocyte. The obtained results are discussed in a phylogenetic context.     

Distribution of cytoskeletal elements in the ovarioles of Mutilla sp. (Insecta,Hymenoptera)

The ovaries of Mutilla sp., as those of other hymenopterans, consist of meroistic-polytrophic ovarioles. Within each ovariole, a terminal filament, a germarium, and a vitellarium can be distinguished. The germaria contain numerous dividing and/or differentiating groups (clusters) of germ cells. The vitellaria are composed of several, linearly arranged, ovarian follicles; each follicle consists of an oocyte and a group of nurse cells. Distribution of cytoskeletal elements (microfilaments and microtubules) throughout the ovarioles of Mutilla sp. has been studied on whole mount preparations stained with rhodamine-conjugated phalloidin and FITC-labelled anti-tubulin.     

Structure of ovaries in scale insects. i. pseudococcidae,kermesidae, eriococcidae,and cryptococcidae (INSECTA,hemiptera, COCCINEA)

Developing ovaries of scale insects (Hemiptera : Coccinea) Nipaecoccus nipae (Pseudococcidae) and Cryptococcus fagisuga (Cryptococcidae) contain clusters of interconnected cells (cystocytes) that are arranged into rosettes; polyfusomes occur in the centres of the rosettes. Ovaries of the investigated adult scale insects are composed of numerous short telotrophic ovarioles. Tropharia (trophic chambers) of Dysmicoccus newsteadi (Pseudococcidae), Eriococcus buxi (Eriococcidae), Cryptococcus fagisuga and Pseudochermes fraxini (Cryptococcidae) comprise only trophocytes (nurse cells), whereas those of Kermes quercus (Kermesidae) and Gossyparia spuria (Eriococcidae) also contain arrested oocytes. The latter probably degenerate. It is suggested that during evolution of scale insects a gradual reduction of germ cells to 4 per cluster (3 trophocytes and 1 oocyte) took place. In light of the obtained results, anagenesis of scale insects ovarioles is discussed.     

Ultrastructural studies of the ovary of Palaeococcus fuscipennis (Burmeister) (Insecta, Hemiptera, Coccinea: Monophlebidae)

Ovaries of Palaeocoocus fuscipennis are composed of about 100 telotrophic ovarioles that are devoid of terminal filaments. In the ovariole a tropharium ( = trophic chamber) and vitellarium can be distinguished. The tropharium contains 7 trophocytes. A single oocyte develops in the vitellarium. The oocyte is surrounded by follicular cells that do not undergo diversification into subpopulations. The obtained results are discussed in a phylogenetic context.     

Structure of the ovaries and oogenesis in Cixius nervosus (Cixiidae), Javesella pellucida and Conomelus anceps (Delphacidae) (Insecta, Hemiptera, Fulgoromorpha)

Ovary organization in representatives of two families of Fulgoromorpha, Cixiidae (Cixius nervosus) and Delphacidae (Javesella pellucida and Conomelus anceps), was examined by light and transmission electron microscopy. Ovaries of studied fulgoromorphans consist of telotrophic ovarioles. From apex to base individual ovarioles have four well defined regions: a terminal filament, tropharium (trophic chamber), vitellarium and pedicel (ovariolar stalk). Tropharia are not differentiated into distinct zones and consist of syncytial lobes containing multiple trophocyte nuclei embedded in a common cytoplasm. Lobes are radially arranged around a branched, cell-free trophic core. Early previtellogenic (arrested) oocytes and prefollicular cells are located at the base of the tropharium. The vitellarium houses linearly arranged developing oocytes each of which is connected to the trophic core by a broad nutritive cord. Each oocyte is surrounded by a single layer of follicular cells that become binucleate at the beginning of vitellogenesis.     

The larval development of the telotrophic meroistic ovary in the bug Dysdercus intermedius (Heteroptera, Pyrrhocoridae)

Bug ovaries are of the telotrophic meroistic type. Nurse cells are restricted to the anterior tropharium and are in syncytial connection with the oocytes via the acellular trophic core region into which cytoplasmic projections of oocytes and nurse cells open. The origin of intercellular connections in bug ovaries is not well understood. In order to elucidate the cellular processes underlying the emergence of the syncytium, we analysed the development of the ovary of Dysdercus intermedius throughout the five larval instars. Up to the third instar, the germ cell population of an ovariole anlage forms a single, tight rosette. In the center of the rosette, phosphotyrosine containing proteins and f-actin accumulate. This center is filled with fusomal cytoplasm and closely interdigitating cell membranes known as the membrane labyrinth. With the molt to the fourth instar germ cells enhance their mitotic activity considerably. As a rule, germ cells divide asynchronously. Simultaneously, the membrane labyrinth expands and establishes a central column within the growing tropharium. In the fifth instar the membrane labyrinth retracts to an apical position, where it is maintained even in ovarioles of adult females. The former membrane labyrinth in middle and posterior regions of the tropharium is replaced by the central core to which nurse cells and oocytes are syncytially connected. Germ cells in the most anterior part of the tropharium, i.e. those in close proximity to the membrane labyrinth remain proliferative. The posterior-most germ cells enter meiosis and become oocytes. The majority of the ovarioles' germ cells, located in between these two populations, endopolyploidize and function as nurse cells. We conclude that the extensive multiplication of germ cells and their syncytial assembly during larval development is achieved by incomplete cytokineses followed by massive membrane production. Membranes are degraded as soon as the trophic core develops. For comparative reasons, we also undertook a cursory examination of early germ cell development in Dysdercus intermedius males. All results were compatible with the known basic patterns of early insect spermatogenesis. Germ cells run through mitotic and meiotic divisions in synchronous clusters emerging from incomplete cytokineses. During the division phase, the germ cells of an individual cluster are connected by a polyfusome rich in f-actin.     

Germ cell cluster formation in insect ovaries

Three different ovariole types exist in insects: panoistic, polytrophic- and telotrophic-meroistic. Their ontogenetic development is comparable to all insect orders. Each ovariole is composed of somatic tissues and germ cells.Panoistic ovarioles can be developed: (1) by totally blocking germ cell cluster division (e.g. in “primitive” insect orders; and (2) after germ cell cluster formation by final cleavage of cystocytes, which develop as oocytes (e.g. in stoneflies or thrips).Polytrophic-meroistic ovaries, showing a set of identical characters, are found among hemirnetabolous and holometabolous insects, indicating a “basic type” of common origin. One characteristic feature is the differentiation of only one oocyte, which is derived from one central cell of the cluster, whereas all other siblings are transformed into nurse cells.Telotrophic ovaries differ from polytrophic ovaries by retention of all nurse cells in the anterior trophic chamber. In addition, oocyte-nurse cell determination can be shifted towards more oocytes in a cluster, and clusters or subclusters can fuse by cell membrane reduction among nurse cells. This type of ovary developed independently 3 times from polytrophic ancestors and once in mayflies directly from panoistic ancestors.     

Ontogenesis of the ovary in a moth midge,Tinearia alternata Say (Diptera: Psychodidae)

In a psychodid, Tinearia alternata, the initial differentiation of the polytrophic ovary occurs during the early larval stages. Early in development, each ovary anlage is a solid organ subdivided into three distinct zones: the cortex houses germ cells and somatic interstitial tissue, while two other somatic regions will give rise to the oviduct calyx and anterior part of the lateral oviduct. Germ cell cluster formation precedes the development of ovarioles. Each ovariole houses only one functional egg chamber. All ovarioles within paired ovaries are developmentally synchronized. In the larval ovaries, the newly formed egg chambers and then the ovarioles are intermingeled with and surrounded by the somatic interstitial tissue of the ovary cortex. The interstitial cells give rise to all the somatic elements of the ovarioles. In the pupal ovaries, the remaining interstitial tissue degenerates; thus, the ovarioles protrude into the body cavity. The ovaries in psychodids develop relatively large and swollen oviduct calyxes that are equivalent to receptaculum seminis (spermatheca). The morphological differentiation of germ cells within the egg chambers starts during late larval/early pupal stages. Nurse cell nuclei contain prominent nucleoli and polytene chromosomes. Oocyte growth results from accumulation of yolk and then, in the final stages of oogenesis, from an inflow of cytoplasm from the nurse cells. J. Morphol. 236:167–177, 1998. © 1998 Wiley-Liss, Inc.     

Germ-cell cluster formation in the telotrophic meroistic ovary of <Emphasis Type="Italic">Tribolium castaneum</Emphasis> (Coleoptera,Polyphaga, Tenebrionidae) and its implication on insect phylogeny

Tribolium castaneum has telotrophic meroistic ovarioles of the Polyphaga type. During larval stages, germ cells multiply in a first mitotic cycle forming many small, irregularly branched germ-cell clusters which colonize between the anterior and posterior somatic tissues in each ovariole. Because germ-cell multiplication is accompanied by cluster splitting, we assume a very low number of germ cells per ovariole at the beginning of ovariole development. In the late larval and early pupal stages, we found programmed cell death of germ-cell clusters that are located in anterior and middle regions of the ovarioles. Only those clusters survive that rest on posterior somatic tissue. The germ cells that are in direct contact with posterior somatic cells transform into morphologically distinct pro-oocytes. Intercellular bridges interconnecting pro-oocytes are located posteriorly and are filled with fusomes that regularly fuse to form polyfusomes. Intercellular bridges connecting pro-oocytes to pro-nurse cells are always positioned anteriorly and contain small fusomal plugs. During pupal stages, a second wave of metasynchronous mitoses is initiated by the pro-oocytes, leading to linear subclusters with few bifurcations. We assume that the pro-oocytes together with posterior somatic cells build the center of determination and differentiation of germ cells throughout the larval, pupal, and adult stages. The early developmental pattern of germ-cell multiplication is highly similar to the events known from the telotrophic ovary of the Sialis type. We conclude that among the common ancestors of Neuropterida and Coleoptera, a telotrophic meroistic ovary of the Sialis type evolved, which still exists in Sialidae, Raphidioptera, and a myxophagan Coleoptera family, the Hydroscaphidae. Consequently, the telotrophic ovary of the Polyphaga type evolved from the Sialis type. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

The roles of cell size and cell number in determining ovariole number in Drosophila

All insect ovaries are composed of functional units called ovarioles, which contain sequentially developing egg chambers. The number of ovarioles varies between and within species. Ovariole number is an important determinant of fecundity and thus affects individual fitness. Although Drosophila oogenesis has been intensively studied, the genetic and cellular basis for determination of ovariole number remains unknown. Ovariole formation begins during larval development with the morphogenesis of terminal filament cells (TFCs) into stacks called terminal filaments (TFs). We induced changes in ovariole number in Drosophila melanogaster by genetically altering cell size and cell number in the TFC population, and analyzed TF morphogenesis in these ovaries to understand the cellular basis for the changes in ovariole number. Increasing TFC size contributed to higher ovariole number by increasing TF number. Similarly, increasing total TFC number led to higher ovariole number via an increase in TF number. By analyzing ovarian morphogenesis in another Drosophila species we showed that TFC number regulation is a target of evolutionary change that affects ovariole number. In contrast, temperature-dependent plasticity in ovariole number was due to changes in cell-cell sorting during TF morphogenesis, rather than changes in cell size or cell number. We have thus identified two distinct developmental processes that regulate ovariole number: establishment of total TFC number, and TFC sorting during TF morphogenesis. Our data suggest that the genetic changes underlying species-specific ovariole number may alter the total number of TFCs available to contribute to TF formation. This work provides for the first time specific and quantitative developmental tools to investigate the evolution of a highly conserved reproductive structure.