Amphibian Reproduction Research Articles

The effects of atrazine on spotted salamander embryos and their symbiotic alga

Worldwide amphibian declines have been a concern for biologists for the past several decades. The causes of such declines may include habitat loss, invasive species, pathogens, and man-made chemicals. Agricultural herbicides, in particular, are known to interfere with reproduction in amphibians and are likely contributing to population declines. We tested the effects of the herbicide atrazine on developing spotted salamanders (Ambystoma maculatum) and their symbiotic green alga Oophila amblystomatis. We exposed spotted salamander egg masses to atrazine at concentrations of 0 microg/L (control), 50, 100, 200, and 400 microg/L. Algae were eliminated in all atrazine treatments. Hatching success was significantly lower for atrazine-treated egg masses than for the controls, and was inversely related to atrazine concentration. The highest developmental stage reached by the embryos was significantly lower in the atrazine treatments than in the controls, and was inversely related to atrazine concentration. These results indicate that atrazine exposure affected spotted salamanders both directly by causing pathologies and mortality in embryos and indirectly by eliminating their symbiotic alga.

Lethal and Sublethal Effects of Embryonic and Larval Exposure of Hyla versicolor to Stormwater Pond Sediments

Stormwater ponds are common features of modern stormwater management practices. Stormwater ponds often retain standing water for extended periods of time, develop vegetative characteristics similar to natural wetlands, and attract wildlife. However, because stormwater ponds are designed to capture pollutants, wildlife that utilize ponds might be exposed to pollutants and suffer toxicological effects. To investigate the toxicity of stormwater pond sediments to Hyla versicolor, an anuran commonly found using retention ponds for breeding, we exposed embryos and larvae to sediments in laboratory microcosms. Exposure to pond sediments reduced survival of embryos by approximately 50% but did not affect larval survival. Larvae exposed to stormwater pond sediment developed significantly faster (x = 39 days compared to 42 days; p = 0.005) and were significantly larger at metamorphosis (x = 0.49 g compared to 0.36 g; p < 0.001) than controls that were exposed to clean sand. Substantial amounts (712-2215 mg/l) of chloride leached from pond sediments into the water column of treatment microcosms; subsequently, survival of embryos was negatively correlated (r (2) = 0.50; p < 0.001) with water conductivity during development. Our results, along with the limited number of other toxicological studies of stormwater ponds, suggest that road salt contributes to the degradation of stormwater pond habitat quality for amphibian reproduction and that future research should focus on understanding interactions among road salts and other pollutants and stressors characteristic of urban environments.

Size-structured effects of common carp on reproduction of pond-breeding amphibians

The role of fish in driving amphibian communities has been widely recognized. However, little is known about size-structured interactions between amphibian and fish populations. This study compared the taxonomic occurrence and densities of larval amphibians between unstocked ponds and ponds stocked with different age cohorts of common carp Cyprinus carpio differing in average body size. The average total densities of early and late breeding anurans known to be vulnerable to fish were by 1–2 orders of magnitude greater in the presence of young-of-the-year carp than that of older cohorts. The probabilities of occurrence of the most common taxa did not differ between ponds stocked with young-of-the-year fish and ponds free of carp, but were significantly larger in those ponds than in ponds stocked with large-size cohorts. No significant differences between pond categories were found for densities of unpalatable Bufo bufo larvae. In aquatic systems harbouring size-structured fish populations, a fish age/size gradient may explain differential habitat suitability for breeding amphibians better than the fish presence/absence dichotomy. When dominated by young cohorts incapable of predation or of adverse habitat alteration, fish-abundant waters are suitable for amphibian reproduction. Conversely, even a ‘non-predatory’ fish, after attaining large body size, may exert a detrimental impact on amphibian breeding success. These findings may be particularly important for amphibian conservation at pond fisheries characterized by spatial separation of age/size distributed stocks.

Can Myxosporean parasites compromise fish and amphibian reproduction?

Research into fish and amphibian reproduction has increased exponentially in recent years owing to the expansion of the aquaculture industry, the need to recover fishery populations, the impact of endocrine disruptors on the aquatic environment and the global decline of amphibian populations. This review focuses on a group of parasites, the Myxozoa, that affect fish and amphibian reproduction. Lists of the myxosporeans that specifically infect gonads are provided. Most of these are parasitic of freshwater hosts, and most amphibian cases are reported from testes. Sex specificity and sex reversal are discussed in relation to gonadal parasitism. The immune response of the fish to the infection is described, and the contribution of the immunoprivilege of gonads to host invasion is emphasized. The pathological effect of these parasites can be significant, especially in aquacultured broodstocks, on some occasions, leading to parasitic castration. Although myxosporean parasites are currently not very frequent in gonads, their impact could increase in the future owing to the transactions in the global market. Their easy release into the aquatic environment with spawning could make their spreading even more feasible. In the absence of commercial drugs or vaccines to treat and prevent these infections, there is an urgent need to develop specific, rapid and reliable diagnostic tools to control and manage animal movements. In addition, much effort is still to be made on deciphering the life cycle of these organisms, their invasion strategies and their immune evasion mechanisms.

Endocrine Disruption in Aquatic Vertebrates

Environmental compounds can interfere with endocrine systems of wildlife and humans. The main sink of such substances, called endocrine disrupters (ED), are surface waters. Thus, aquatic vertebrates, such as fish and amphibians, are most endangered. ED can adversely affect reproductive biology and the thyroid system. ED act by (anti)estrogenic and (anti)androgenic modes of action, resulting in abnormal sexual differentiation and impaired reproduction. These effects are mainly driven by direct interferences of ED with sex steroid receptors rather than indirectly by impacting synthesis and bioavailability of sex steroids, which in turn might affect the hypothalamic-pituitary-gonadal axis. Recent findings reveal that, in addition to the human-produced waste of ED, natural sources, such as parasites and decomposition of leaves, also might act as ED, markedly affecting sexual differentiation and reproduction in fish and amphibians. Although the thyroid system has essential functions in both fish and amphibians, amphibian metamorphosis has been introduced as the most sensitive model to detect thyroidal ED; no suitable fish model exists. Whereas ED may act primarily on only one specific endocrine target, all endocrine systems will eventually be deregulated as they are intimately connected to each other. The recent ecotoxicological issue of pharmaceutically active compounds (PhACs) present in the aquatic environment indicates a high potential for further endocrine modes of action on aquatic vertebrates by ED derived from PhACs, such as glucocorticoids, progestins, and beta-agonists.

Using habitat models to determine conservation priorities for pond-breeding amphibians in a privately-owned landscape of northern Idaho, USA

As rural landscapes experience increasing levels of residential development, the persistence of species that rely on them will depend on informed planning and management decisions. An understanding of habitat requirements is essential for setting priorities and developing landscape-level plans for the survival of these species. In many temperate rural landscapes, artificially created ponds may be the only wetlands available for aquatic reproduction by amphibians. The introduction of non-native fish into these ponds reduces survival and prohibits successful reproduction of many native pond-breeding amphibians. We surveyed 105 randomly selected wetlands in a primarily privately-owned, rural landscape in north Idaho, USA, for pond-breeding amphibian larvae in 2004 and 2005. We used an information theoretic multimodel inference and an algorithmic (random forests) approach to model habitat for each species based on local and landscape characteristics. We also used a mail survey to quantify how landowners value fish in their wetlands and their plans for future wetland development and fish stocking. Sixty-seven percent of pond owners reported that fishing in their pond was at least slightly important to them and 36% of owners indicated that they were at least 50% likely to add fish to their ponds in the next 5–10 years. Landscape change predictions for this area indicate that forests will become more open due to thinning; habitat models indicated that this is likely to be detrimental to long-toed salamanders and beneficial to Pacific treefrogs. Habitat models also indicate that Columbia spotted frog breeding sites consist of wetlands on flat ground with high solar insolation and that this species is sensitive to nearby development, indicating that as this landscape becomes further developed, this species may require habitat protection for persistence.

Are human progestagens among the causes of amphibian extinction?

We hypothesised that massive elimination of human and animal progestagens through wastewater can be toxic for reproduction of amphibians. Several medical test using amphibian bioassays (i.e. Hogben test) have demonstrated that human chorionic gonadotropin is toxic for amphibians. Nowadays, a huge amount of sexual hormones from women, animal farms and aquaculture arrive to inland water systems and could be a cause of amphibians extinction.

Effects of paraquat and glyphosate on steroidogenesis in gonads of the frog Rana esculenta in vitro

Herbicides are toxic for amphibians, influencing various developmental stages. In particular, paraquat and glyphosate have teratogenic effects on tadpole development, though little information is available on how they affect reproduction in amphibians. In the present work, ovarian tissue and testis of the water frog Rana esculenta were incubated in vitro in presence of different concentrations of the two herbicides. 17β-estradiol and testosterone levels were measured in the incubation medium by radioimmunoassay. The data showed that paraquat inhibited testosterone and 17β-estradiol production, with the lowest-observed-effect concentration (LOEC) of 10 −4 M, while paraquat inhibition of steroidogenesis in amphibian gonads was reversed by the presence of epigallocatechin gallate in the culture medium. These result indicates that paraquat acts on gonadal steridogenesis through a mechanism involving reactive oxygen species. Glyphosate showed no effect on gonadal steroidogenesis. These results suggest that paraquat may interfere in amphibian reproductive processes.

The effect of beaver (Castor fiber Linnaeus, 1758) activity on amphibian reproduction

The effect of beaver (Castor fiber Linnaeus, 1758) activity on amphibian reproduction was studied in the small river valleys in Novgorod oblast. At sites not subjected to beaver activity, the reproduction rate of amphibians was low. In spring the water in beaver ponds warms up quickly, which attracts amphibians for reproduction. The maximal abundance of tadpoles due to a high reproduction rate was observed in partially drained ponds. However, a high larvae mortality rate was often observed there. Tadpole survival in flooded and empty ponds depends on factors that are not controlled by beavers, such as geomorphology and climate.

Sex-Related Differences in Hematological Stress Indices of Breeding Paedomorphic Mole Salamanders

Abstract Reproduction in amphibians is stressful and perhaps more so in females than males because of the higher energetic costs of producing eggs than sperm. The ratio of two white blood cells, neutrophils and lymphocytes, has been shown in birds, turtles, and amphibians to increase with stress. We captured breeding and nonbreeding, paedomorphic Mole Salamanders (Ambystoma talpoideum), to determine whether the stress of reproduction is reflected by neutrophil:lymphocyte ratios in this species. In blood smears of all individuals, we observed approximately 25.5 leukocytes per 1000 erythrocytes, with 13% of the leukocytes being neutrophils, 41% lymphocytes, and 46% eosinophils. Less than 1% of leukocytes were basophils and monocytes. Neutrophil:lymphocyte ratios of gravid females were significantly higher (and also more variable) than those of reproductive males and of nonreproductive individuals, indicating a higher degree of physiological stress in reproductive females. Reproductive males did not have hig...

Effects of Atrazine on Fish, Amphibians, and Aquatic Reptiles: A Critical Review

The herbicide atrazine is widely used in agriculture for the production of corn and other crops. Because of its physical and chemical properties, atrazine is found in small concentrations in surface waters—habitats for some species. A number of reports on the effects of atrazine on aquatic vertebrates, mostly amphibians, have been published, yet there is inconsistency in the effects reported, and inconsistency between studies in different laboratories. We have brought the results and conclusions of all of the relevant laboratory and field studies together in this critical review and assessed causality using procedures for the identification of causative agents of disease and ecoepidemiology derived from Koch's postulates and the Bradford–Hill guidelines. Based on a weight of evidence analysis of all of the data, the central theory that environmentally relevant concentrations of atrazine affect reproduction and/or reproductive development in fish, amphibians, and reptiles is not supported by the vast majority of observations. The same conclusions also hold for the supporting theories such as induction of aromatase, the enzyme that converts testosterone to estradiol. For other responses, such as immune function, stress endocrinology, parasitism, or population-level effects, there are no indications of effects or there is such a paucity of good data that definitive conclusions cannot be made.

Reproduction and Larval Rearing of Amphibians

Reproduction technologies for amphibians are increasingly used for the in vitro treatment of ovulation, spermiation, oocytes, eggs, sperm, and larvae. Recent advances in these reproduction technologies have been driven by (1) difficulties with achieving reliable reproduction of threatened species in captive breeding programs, (2) the need for the efficient reproduction of laboratory model species, and (3) the cost of maintaining increasing numbers of amphibian gene lines for both research and conservation. Many amphibians are particularly well suited to the use of reproduction technologies due to external fertilization and development. However, due to limitations in our knowledge of reproductive mechanisms, it is still necessary to reproduce many species in captivity by the simulation of natural reproductive cues. Recent advances in reproduction technologies for amphibians include improved hormonal induction of oocytes and sperm, storage of sperm and oocytes, artificial fertilization, and high-density rearing of larvae to metamorphosis. The storage of sperm in particular can both increase the security and reduce the cost of maintaining genetic diversity. It is possible to cryopreserve sperm for millennia, or store it unfrozen for weeks in refrigerators. The storage of sperm can enable multiple parentages of individual females' clutches of eggs and reduce the need to transport animals. Cryopreserved sperm can maintain the gene pool indefinitely, reduce the optimum number of males in captive breeding programs, and usher in new generations of Xenopus spp. germ lines for research. Improved in vitro fertilization using genetic diversity from stored sperm means that investigators need the oocytes from only a few females to produce genetically diverse progeny. In both research and captive breeding programs, it is necessary to provide suitable conditions for the rearing of large numbers of a diverse range of species. Compared with traditional systems, the raising of larvae at high densities has the potential to produce these large numbers of larvae in smaller spaces and to reduce costs.

Influence of land use on metal concentrations in playa sediments and amphibians in the Southern High Plains

The Southern High Plains (SHP) is a semi-arid region in which playa wetlands are the focal points of biodiversity. Playas are highly influenced by surrounding land use. Most of the SHP is in agricultural production (primarily cotton) with a history of arsenic-containing herbicide use. Metals influence reproduction and development in amphibians. We analyzed metal residues in playa sediment and whole body tissue of Spea spp. and Bufo cognatus metamorphs from two land uses: cropland and native grassland. Cd and Ni concentrations in B. cognatus tissues differed between land uses. Metal concentrations in Spea spp. tissues did not differ between land uses. Ba was higher in Spea spp. than B. cognatus collected from the same grassland playas, indicating differential habitat use. No correlations between sediment and tissue concentrations were found. Land use appeared to have little influence on metal concentrations and levels were below those known to cause effects in amphibians.

Effects of atrazine on sexual maturation in female Japanese quail induced by photostimulation or exogenous gonadotropin

The herbicide atrazine has gained recent attention for its reported effects on reproduction in amphibians. The present study examined the putative effects of atrazine during sexual maturation in the photostimulated female Japanese quail. Furthermore, the effects of atrazine on birds administered exogenous gonadotropin (pregnant mare serum gonadotropin [PMSG]) were investigated. Atrazine was administered up to 1,000 ppm in the diet to female quail undergoing photoperiodically induced sexual maturation. At high dietary concentrations, atrazine exhibits signs of overt toxicity with reductions in growth, feed intake, and liver weights, but these effects were dependent on the timing of treatment administration. Atrazine did not influence the weights of reproductive tissues (ovary and oviduct) or circulating concentrations of luteinizing hormone (LH). However, high concentrations of atrazine depressed circulating concentrations of estradiol. Treatment with atrazine for four weeks during sexual maturation inhibited growth but did not affect any other parameter assessed (feed intake, liver, ovary, or oviduct weights or the circulating concentrations of LH and estradiol). In birds receiving daily injections of PMSG, atrazine reduced growth, feed intake, and liver weights. However, PMSG-induced gonadal and oviduct growth was not affected by atrazine. The present results suggest that dietary atrazine exhibits limited reproductive toxicity in female quail during sexual maturation and only at concentrations above ecological relevance.

Reproduction, embryonic development, and maternal transfer of contaminants in the amphibian Gastrophryne carolinensis.

Although many amphibian populations around the world are declining at alarming rates, the cause of most declines remains unknown. Environmental contamination is one of several factors implicated in declines and may have particularly important effects on sensitive developmental stages. Despite the severe effects of maternal transfer of contaminants on early development in other vertebrate lineages, no studies have examined the effects of maternal transfer of contaminants on reproduction or development in amphibians. We examined maternal transfer of contaminants in eastern narrow-mouth toads (Gastrophryne carolinensis) collected from a reference site and near a coal-burning power plant. Adult toads inhabiting the industrial area transferred significant quantities of selenium and strontium to their eggs, but Se concentrations were most notable (up to 100 μg/g dry mass). Compared with the reference site, hatching success was reduced by 11% in clutches from the contaminated site. In surviving larvae, the frequency of developmental abnormalities and abnormal swimming was 55–58% higher in the contaminated site relative to the reference site. Craniofacial abnormalities were nearly an order of magnitude more prevalent in hatchlings from the contaminated site. When all developmental criteria were considered collectively, offspring from the contaminated site experienced 19% lower viability. Although there was no statistical relationship between the concentration of Se or Sr transferred to eggs and any measure of offspring viability, our study demonstrates that maternal transfer may be an important route of contaminant exposure in amphibians that has been overlooked.

INFLUENCE OF LAND USE ON POSTMETAMORPHIC BODY SIZE OF PLAYA LAKE AMPHIBIANS

Agricultural land use may indirectly affect the body size of amphibians by altering the hydroperiods of nearby wetlands and influencing amphibian densities—both factors which can limit the larval and postmetamorphic growth rates of amphibians. We measured postmetamorphic body size for 4 species (Spea multiplicata, S. bombifrons, Bufo cognatus, Ambystoma tigrinum mavortium) and 3 age classes (metamorph, subadult, adult) of amphibians captured at playa wetlands surrounded by one of 2 general land-use types (cultivation, grassland) in the Southern High Plains. Sixteen playas (4 per land-use type in 1999 and 2000) were partially enclosed with drift fence and pitfall traps, and mass and snout-vent length (SVL) were measured from a subsample of captured individuals. Mass and SVL were 10–148% greater for amphibians captured at grassland wetlands than at cropland wetlands for most species and age classes. Mass and SVL also were 3–124% greater in 1999 than in 2000 for most species and age classes. We attribute differences in body size between land-use types to differences in the hydroperiods of the associated wetlands, and potentially to variation in the density of terrestrial conspecifics and aquatic predators. We attribute differences in body size between years to differences in rainfall. Body size is positively related to the probability of survival, reproduction, and evolutionary fitness in amphibians. Thus, if cultivation of landscapes surrounding wetlands negatively influences postmetamorphic body size of amphibians, restoration of native grasslands surrounding playa wetlands may help prevent local amphibian declines.

IDENTIFICATION OF REPRODUCTIVE STATUS IN FEMALE FROGS—A QUANTITATIVE COMPARISON OF NINE METHODS

Many techniques commonly used by herpetologists for monitoring amphibian populations and communities yield censuses of the total adult population size (N). However, for many studies, e.g., of reproductive output, development of populations and potential for evolutionary changes, the effective population size (Ne) must be known. While modern molecular techniques make it possible to measure Ne, they are expensive, work-intensive and may not be possible or necessary for many questions on amphibian reproduction. For females of two species of water frog (Rana lessonae and R. esculenta), we investigate the effectiveness of several techniques to determine the presence or absence of eggs. The direct methods are (1) dissection of dead frogs and (2) a small skin incision into the abdominal side of live females. The indirect methods, all applied to live frogs, include (3) visual inspection of body shape, (4) tactile inspection of the epidermis, (5) transillumination with a strong cold light source, (6) ultrasound, (7) electromagnetic measurement of total body electrical conductivity (TOBEC), (8) calculation of body condition index and (9) analysis of blood plasma testosterone titers. Only two indirect methods were somewhat successful at predicting whether females were gravid. Testosterone titers (9) yielded the best results (ca. 80–90% effective). Body condition (8) was also significantly related to egg presence or absence, but predicted gravidity only weakly. We suggest that a combination of skin incision and hormone analysis provides a fairly good estimate of gravidity. When complemented by mark-recapture techniques and performed on the same individuals at different times of the season, this combination yields estimates not only of the reproductive output of the study population but also of the relative contribution of different females.

AGRICULTURAL PONDS SUPPORT AMPHIBIAN POPULATIONS

In some agricultural regions, natural wetlands are scarce, and constructed agricultural ponds may represent important alternative breeding habitats for amphibians. Properly managed, these agricultural ponds may effectively increase the total amount of breeding habitat and help to sustain populations. We studied small, constructed agricultural ponds in southeastern Minnesota to assess their value as amphibian breeding sites. Our study examined habitat factors associated with amphibian reproduction at two spatial scales: the pond and the landscape surrounding the pond. We found that small agricultural ponds in southeastern Minnesota provided breeding habitat for at least 10 species of amphibians. Species richness and multispecies reproductive success were more closely associated with characteristics of the pond (water quality, vegetation, and predators) compared with characteristics of the surrounding landscape, but individual species were associated with both pond and landscape variables. Ponds surrounded by row crops had similar species richness and reproductive success compared with natural wetlands and ponds surrounded by nongrazed pasture. Ponds used for watering livestock had elevated concentrations of phosphorus, higher turbidity, and a trend toward reduced amphibian reproductive success. Species richness was highest in small ponds, ponds with lower total nitrogen concentrations, tiger salamanders (Ambystoma tigrinum) present, and lacking fish. Multispecies reproductive success was best in ponds with lower total nitrogen concentrations, less emergent vegetation, and lacking fish. Habitat factors associated with higher reproductive success varied among individual species. We conclude that small, constructed farm ponds, properly managed, may help sustain amphibian populations in landscapes where natural wetland habitat is rare. We recommend management actions such as limiting livestock access to the pond to improve water quality, reducing nitrogen input, and avoiding the introduction of fish.

Weather-related effects on woodland vernal pool hydrology and hydroperiod

Woodland vernal pools occur commonly throughout northeastern North America. These pools provide preferred breeding habitat for mole salamanders (Ambystoma spp.) and wood frogs (Rana sylvatica) and support an abundant and diverse macroinvertebrate fauna. Vernal pool hydrology, and especially hydroperiod or duration of the wet phase, affects the composition and productivity of pool fauna. The hydrology of ephemeral wetlands is dominated by local weather conditions. In this paper, I report a ten-year record of the relationships between precipitation and evapotranspiration and water-level change and hydroperiod in four typical southern New England vernal pools. Long-term average precipitation is evenly distributed throughout the year in the Northeast; potential evapotranspiration peaks in the summer months and exceeds precipitation from mid-June through mid-September. This period of water deficit causes the period of maximum vernal pool drying. Vernal pool hydroperiods were shorter and pools dried earlier in those years with larger cumulative water deficits, especially when early spring ground-water resources were below long-term means and late winter snowpack was reduced or absent. Weekly water-level change in vernal pools was significantly related to precipitation and potential evapotranspiration, with precipitation having 2–5 times greater effect than evapotranspiration. Under elimate-change predictions of more episodic precipitation and increased evapotranspiration, vernal pools would dry earlier in the year and remain dry longer. These changes would adversely affect the successful reproduction of pool-breeding amphibians and isolate the remaining productive pools.

Ovarian follicle cells in torpedo marmorata synthesize vitellogenin.

The pattern of vitellogenesis is similar in all non-mammalian vertebrates: the liver, under oestrogenic stimulus, synthesizes vitellogenin (VTG) that, via the maternal circulation, is delivered to the oocyte and here internalized by receptor-mediated endocytosis (Wallace, 1985: Development Biology. A comprehensive synthesis. Vol. 1 Oogenesis:127-177; Schneider, 1996: Int Rev Cytol 166:103-134; LaFleur, 1999: Encyclopedia of Reproduction Vol. 4:985-992). The contribution to vitellogenesis of different components of the ovarian follicle has also been reported in amphibians (Wallace, 1985), squamate reptiles (Ghiara and Limatola, 1980: Acta Embryol Morphol Exper 1:5-6; Andreuccetti, 1992: J Morphol 212:1-11), and recently, supporting previous reports (Chieffi and Pierantoni, 1987: Hormones and Reproduction in Fishes, Amphibians and Reptiles Single vol.:117-144), in Torpedo marmorata (Prisco et al., 2001: Perspective in comparative endocrinology: Unity and diversity Single vol.:1197-1201; Prisco et al., 2002b: Gen Comp Endocrinol 128:171-179). The present investigation, performed with immunoblotting, immunohistochemical, and in situ hybridization techniques during different stages of follicular growth in T. marmorata, shows that, as previously supposed (Prisco et al., 2002b), granulosa cells in both previtellogenic and vitellogenic phases actively synthesize VTG. This is the first time among vertebrates that the synthesis of this protein has been found to occur also within the ovarian follicle. The present data also demonstrate that the contribution of granulosa cells becomes particularly evident during vitellogenesis. Indeed, in vitellogenic follicles, small, intermediate, and pyriform-like cells cross-react with an anti-VTG antibody and are positive to a hybridization signal with a VTG mRNA probe. By contrast, in previtellogenesis only the enlarged cells, i.e., intermediate and pyriform-like cells, are involved in VTG synthesis.