A COMPARATIVE STUDY OF THE OVARIES IN SOME BRAZILIAN BEES ( HYMENOPTERA ; APOIDEA )

The present paper concerns the morphological features of ovaries in 33 species of bees with different social behavior patterns. The ovaries of bees were examined under light microscope. They are polytrophicmeroistic ovaries formed for an anterior germarium and a basal vitellarium. The germarium houses the germ cells and in the vitellarium there are follicles arranged linearly. In general the follicle is constituted by a nutritive chamber (a cluster of nurse cells) and an oocyte chamber, both covered by a single epithelial layer of follicular cells. The number of ovarioles per ovary and the number of mature oocyte per ovary were analyzed. Measurements of ovariole length, oocyte size, oocyte width, follicular epithelial height and the intertegular distance were made to support the comparative study. Statistical analysis showed that representatives of Meliponini and Apini have the largest ovaries. On the other hand, in solitary bees were found the bigger oocytes. Furthermore, our results suggest that there is a tendency for increase in ovary size and ovariole number, with increasing level of sociality.


INTRODUCTION
Insect ovaries are formed of several functional and elongated units called ovarioles (Bilinsk, 1998) and in Hymenoptera they are of meroistic polytrophic type (Chapman, 1998).A typical polytrophic ovariole consists of three regions: terminal filament, germarium and vitellarium (Chapman, 1998).The germarium contains the germ cells and their derivatives, the terminal filament continues from the anterior end of the germarium and constitute a suspensory apparatus of the ovarioles while the vitellarium occupies the proximal region of the ovariole where yolk uptake and The number of ovarioles per ovary is variable and shows interspecific differences (Jaglarz, 1998).The ovary morphology and its phylogenetic relationships have been studied by various authors (Simiczyjew et al., 1998;Szklarzewicz, 1998;Jaglarz, 1998;Kubrakiewicz et al., 1998;Bilinsk et al., 1998).Iwata (1955;1965) studied the polymorphism of the ovaries in bees considering the change that may occur on the ovary structure, observing the number of mature and immature oocytes, the size of mature oocyte and the speed of oocyte maturation, showing that these characteristics have a distinct correlation with the different behavioral patterns found in this insect group.The following study reports on the variation of ovary morphology in bees to support a comparative study, in order to test the hypothesis that structure is indeed related to their sociality.
Bees were collected in the field in Viçosa, MG, Brazil, while for Meliponini and Apini were analyzed physogastric queens obtained from the Central Apiary, Universidade Federal de Viçosa, MG.
The specimens were dissected in insect saline solution and the pieces were removed from mated bees, what was determined by the presence of spermatozoa in their spermathecae.The ovaries were isolated from the dissected reproductive tract and transferred to 4% paraformaldehyde in phosphate buffer 0.1M, pH 7.2.
The samples were dehydrated in an ethanol series, embedded in historesin (Leica) and cut at 4 µm serial sections, which were stained with Dominici solution.
Some sections were submitted to the following histochemical tests: mercury-bromophenol blue for protein, Nile blue for lipids and methyl green-pyronin for cell death.These tests were performed as described by Pearse (1968) with few variations for historesin embedded tissues.
For each bee the following parameters were analyzed: number of ovarioles per ovary, ovariole length, number of mature oocytes per ovary, oocyte length, oocyte width, follicular epithelium thickness and the intertegular distance (as representative of the body size).
Measurements were performed with aid of the software Image Pro-Plus™, 4.0 version for Windows.For determination of the body size of bees, measurements of the intertegular distance were made with the same software.The number of mature oocyte per ovary was determined as proposed for Iwata (1955;1965).
To determine the degree of dependency of body size and morphological parameters, we used a linear regression procedure following standard statistical tests described by Snedecor & Cochran (1980).

RESULTS
The general morphology of the ovaries is almost equal to that was described before for some species (Snodgrass, 1956;Cruz-Landim et al., 1998), and therefore only a brief description will be given, emphasizing only the features that have not been detailed before.
A sheath, which is made up of two layers, encloses each ovariole: an outer peritoneal membrane and an inner non-cellular tunica propria.The former is constituted by a network of cells, including muscle cells, fat body cells, and tracheoles that do not penetrate the tunica propria 7,9).
The height of the follicular epithelium of egg chamber changes according to the species, with measurements varying from 6.6 µm in Plebeia sp. to 45 µm in Xylocopa frontalis in mature basal follicles.In Epicharis flava and E. affinis extensive cell projections were found (64.08 ± 8.6 µm length) in follicular tissue and these structures penetrate the corion that is very thick (102.0 ± 7.8 µm length) (Figs. 7, 9).
The cytoplasm of nurse and follicular cells was strongly stained by methyl green-pyronin, mercurybromophenol blue and nile blue, marking the following constituents in the cytoplasm: RNA, proteins and neutral-lipid (Figs. 3, 7).The follicular cells however show different staining tonalities for bromophenol blue in the mid-vitellogenic follicles (Fig. 7).
Among nurse cells there is the presence of groups of small cells, which are smaller than nurse cells, the somatic-like cells (Fig. 6).
Presence of accessory nuclei was observed in the oocytes during the previtellogenic and vitellogenic stages.In Meliponini accessory nuclei were found in the previtellogenic region (Fig. 8) while in Centridini and Halictidae they were found in the vitellogenic  region (Figs.3-5).In Halictidae this structure was more developed in comparison with other species (Figs. 4,5).
Apidae species have four ovarioles per ovary, while Andrenidae, Halictidae and Megachilidae have three ovarioles per ovary.These numbers were constant in both ovaries and bee group, except A. mellifera.However, in Apidae the number of mature oocytes per ovariole varied from 1 to many (Table 1).
Morphometrical data of the ovarioles showed some differences among the studied bees, with Meliponini and Bombini presenting the longest ovarioles (Table 1).In addition the germarium in solitary bees is reduced in comparison with the ones found in primitively and highly eusocial bees (Figs. 1, 2).
Ovarioles in solitary bees contain 1 to 3 follicles (nutritive plus egg chambers) and the sizes greatly differ among them, while in the ovaries of primitively and highly eusocial bees many follicles were found arranged in linear series with small differences in their size.Meliponini have the proximal region of the ovarioles with an accumulation of follicles with degenerative nutritive chamber.

DISCUSSION
The small cells scattered among nurse cells are similar to those found in the beetle Badister bipustulatus, which present characteristics of somatic cells derived from pre follicular cells (Jaglarz, 1998), in spite of their role is unknown.
In youngest follicles, the follicular cells acquire different bromophenol blue staining tonalities, suggesting that protein synthesis is an asynchronous process, similar to the one observed in the ant Neoponera villosa (Camargo-Mathias, 1993).It means that some cells start protein synthesis before the others.On the other hand, in post-vitellogenic follicles all follicular cells are equally stained, suggesting that all cells are synthesizing protein.Fleig (1995) observed that after    coriogenesis, follicular cell degenerates in A. mellifera that is in agreement with the results obtained in this study for others species of bees.
Accessories nuclei have been studied in Eomenacanthus stramineus (Mallophaga) (Bilinsk, 1989) and in the bee M. quadrifasciata anthidioides and in the ant Atta sexdens rubropilosa (Cruz-Landim, 1991) and their formation were very similar among them.It is evident that these accessories nuclei arise from the protrusion of the nuclear envelope of the germinal vesicle as described in ants by Billen (1985) following migration to the anterior pole of the peripheral region of the oocyte cytoplasm.The accessories nuclei are involved in the transport and deposition of RNA and proteins into ooplasm (Büning, 1994;Cruz-Landim, 1991), but the function of accessory nuclei in bees needs further investigations.
In Apini, Meliponini and Bombini all ovarioles have mature oocyte suggesting a synchronism in the oocyte production in these ovaries what did not occur in the solitary bees that have only one mature oocyte per ovary.Social bees have an elongated germarium followed by many follicles in a linear arrangement while the solitary ones have short germarium followed by few mature follicles which can be correlated with egg production, because social bees lay many eggs, while solitary bees lay only few eggs.
Number of ovarioles per ovary was found to be a multi-state character.We, Iwatta (1955), Rozen (1986) and Alexander & Rozen (1987) found that Andrenidae, Megachilidae, Halictidae, Colletidae and Melittidae have three ovarioles/ovary, while Apidae presents four or more.Thus, three ovarioles per ovary is the plesiomorphic condition, the others being derived states.Whether these are ordered or unordered states is not known for sure, but it seems reasonable to believe that an increased feature derived from a smaller one, hardly would evolve to decrease, by the criterion of similarity.It seems more likely that four ovarioles/ovary, five ovarioles/ovary found in Nomadinae (Alexander & Rozen, 1987), six to 18 ovarioles/ovary in parasitic Bombini (Cumber, 1949) and hundreds ovarioles/ ovary derived independently from the three ovarioles/ ovary condition, because they are very discontinuous.
Among corbiculate Apinae, Euglossini is the most similar to the ancestor because this tribe presents few mature oocytes, such as found in others non-corbiculate Apinae and bee families, so that orchid bees can be considered as sister-group of other corbiculate Apinae, which corroborates the hypothesis of Roig-Alsina & Michener (1993), based on external morphology, and FIGURE 10.Relationship between the ovariole size and intertegular distance of bees (Linear regression).Numbers regards to number of the species listed in the Table 1.FIGURE 12. Relationship between the oocyte size and intertegular distance of bees (Linear regression).Numbers regards to number of the species listed in the Table 1 FIGURE 11.Relationship between the ovariole size and intertegular distance of bees (Linear regression).Data on Meliponini and Apini were excluded.Numbers regards to number of the species listed in the Table 1.FIGURE 13.Relationship between the oocyte width and intertegular distance of bees (Linear regression).Numbers regards to number of the species listed in the Table 1.Snodgrass (1956).*** Classification from Roubik (1992).Serrão (2001) and Peixoto & Serrão (2001) based on digestive tract features, that placed Euglossini in a more basal position in their trees.In this sense, synchronism of egg production represented by accumulation of mature oocytes in the proximal region is a synapomorphy for Apini, Bombini and Meliponini.
Females of the solitary bee, Andrena erythronii lay approximately 8 diploid eggs in the reproductive lifetime (Michener & Rettenmeyer, 1956), while Megachile rotundata lay approximately 20 diploid eggs (Gerber & Klostermeyer, 1970).Primitively eusocial Lasioglossum laevissimum lay approximately 75 diploid eggs (Packer, 1992) and L. marginatum lay over 2000 eggs (Plateaux-Quénu, 1960).In the high social bees, A. mellifera queens lay hundreds of thousands of diploid eggs in the reproductive lifetime (Snodgrass, 1956).In Meliponini, M. compressipes fasciculata lay 25.6 to 30.43 eggs/day, M. quadrifasciata anthidioides lay 10-22 eggs/ day (Kerr, 1949) and P. remota probably produce 60-180 eggs/day (Van Benthem et al., 1995).According to Iwata (1955;1964), Iwata & Sakagami (1966) and Alexander & Rozen (1987) an increased number of ovarioles functions to increase the reproductive potential of an individual.In this sense, Cruz-Landim et al. (1998) stated that the achievement of reproductive efficiency in bees is attained through the increase of ovariole numbers and length.In Meliponini it seems that the chosen mechanism is mostly that of the ovarioles length, because there is a negative correlation between body size and ovary length in this bee group (see Figs. 10,11), while in Apis the achievement of reproductive efficiency is attained through the increase of ovariole number and length (see Table 1).
We suggest that the queen ovary of highly eusocial species are more efficient than that of the solitary and primitively eusocial ones, because in eusocial bees the germarial zone is more developed, housing a higher numbers of germinative cells, ovarioles and mature follicles.
The primitively eusocial Bombus morio have not the same reproductive efficiency in comparison to eusocial species, because its ovary has 4 ovarioles per ovary, one mature oocyte per ovariole, and are shorter than those found in the highly eusocial honey bee and stinglessbees.
We are in agreement with Cruz-Landim et al. (1998) with the possibility that the number of ovarioles and their length are related to the oviposition rate.Our results showed that the increasing of ovariole number, ovariole size, number of follicles per ovary and size of the germarium, follows the increasing of egg number laying and degree of sociability.

FIGURE 1 .
FIGURE 1. Longitudinal section of the germarium position of Melipona quadrifasciata.The anterior region have undifferentiated cells and the posterior region have many oocyte-nurse cell complexes; o: young oocyte; on: oocyte nucleus; uc: undifferentiated cells; pm: peritoneal membrane; T: trachea; tp: tunica propria.Bar = 10 µm.

FIGURE 2 .
FIGURE 2. Longitudinal section of the anterior region of the ovariole of the Xylocopa frontalis, showing a short terminal filament (arrow) followed by a short germarium with few oocyte-nurse cell complexes followed by a nutritive chamber with the nurse cells (N); on: oocyte nucleus; pm: peritoneal membrane; tp: tunica propria; T: trachea.Bar = 10 µm.

FIGURE 3 .
FIGURE 3. Longitudinal section of a follicle of Epicharis affinis, observed under phase contrast microscope, Methyl Green-Pyronin stained, showing one vitellogenic growing oocyte (o) that have the nucleus (on) and the accessory nuclei (a) placed in the peripheral region of the cytoplasm; E: follicular epithelium covering the oocyte chamber; e: follicular layer of the nutritive chamber; m: muscle; N: nurse cells; pm: peritoneal membrane; T: trachea; tp: tunica propria.Bar = 10 µm.

FIGURE 5 .
FIGURE 5. Longitudinal section of mature oocyte of Pseudaugochlora graminea with accessory nuclei placed in the peripheral oocyte region.The follicular cells are flattened; E: follicular epithelium; a: accessory nucleus; en: follicular epithelial cell nucleus.Bar = 10 µm.

FIGURE 6 .
FIGURE 6. Longitudinal section of the nutritive chamber of Pseudaugochlora graminea, showing the nurse cells (N) with irregular nucleus (n) and multiple nucleoli.Between the nurse cells there are somatic-like cells (sc).Bar = 10 µm.

FIGURE 7 .
FIGURE 7. Longitudinal section of the ovariole of Epicharis affinis obeserved under phase contrast microscope, Bromophenol Blue stained, showing the follicular epithelium (E) of two successive follicles with different developmental stages.Covering the mature oocyte, the follicular projection (p) that penetrate the corion (c).Notice that the epithelium cover the vitellogenic growing oocyte (o) without projections and with cells presenting different staining tonalities (*); en: follicular epithelial cell nucleus; (tp) tunica propria; (pm) peritoneal membrane; en: follicular epithelial cell nucleus.Bar = 10 µm.

FIGURE 8 :
FIGURE 8: Oocyte-nurse cell complex in the posterior region of the germarium of Melipona bicolor.Notice the presence of accessory nuclei (a) into the oocyte cytoplasm.The nurse cells present a linear arrangement and they are connected with the oocyte; tp: tunica propria; (o): oocyte; (on): oocyte nucleus; (N): nurse cell; (n): nurse cell nucleus.Bar = 10 µm.

FIGURE 9 :
FIGURE 9: Longitudinal section of a mature follicle of Epicharis affinis, Methyl Green Pyronin, observed under phase contrast microscope.Notice the follicular cells (E) with extensive cellular projections (p) that penetrate the thick corion (c); en: follicular epithelial cell nucleus; F: fat body cells.Bar = 10 µm.
in parenthesis indicates those plotted in the Figures 10-13.** Data from

TABLE 1 :
Morphometric parameters of ovaries in different species of bees.