Vitellogenic Cycle Research Articles

From Mosquito Ovaries to Ecdysone; from Ecdysone to Wolbachia: One Woman's Career in Insect Biology.

Simple SummaryMy career in science, and specifically in insect biology, benefitted from post-war efforts to improve science education in the 1950s and 1960s and increased opportunities for women students and faculty; teachers, mentors, associates, colleagues and friends of all sorts helped along the way. Here, I recount the highlights of my research, focusing on studies involving the insect steroid hormone ecdysone, which regulates molting in insect larvae, and reproduction in adults. Ecdysone was the unknown “ovarian factor” involved in egg production in Aedes aegypti mosquitoes. In addition, ecdysone affects the process of cell division in cultured mosquito cells, causing a cell cycle arrest that enhances growth of an obligate symbiotic bacterium, Wolbachia pipientis. Since the mid-1920s, Wolbachia was known to infect ovaries of Culex pipiens mosquitoes. After Wolbachia sequences were discovered in DNA extracted from Drosophila, this obligate intracellular alpha proteobacterium was found to be widespread among insect species, and its effects on reproduction have important applications for control of insect pests. Most recently, effects of ecdysone on Wolbachia replication in mosquito cell lines led to insights that enhance production of infectious bacteria at levels suitable for microinjection into eggs and eventual genetic manipulation.In anautogenous mosquitoes, synchronous development of terminal ovarian follicles after a blood meal provides an important model for studies on insect reproduction. Removal and implantation of ovaries, in vitro culture of dissected tissues and immunological assays for vitellogenin synthesis by the fat body showed that the Aedes aegypti (L.) (Diptera, Culicidae) mosquito ovary produces a factor essential for egg production. The discovery that the ovarian factor was the insect steroid hormone, ecdysone, provided a model for co-option of the larval hormones as reproductive hormones in adult insects. In later work on cultured mosquito cells, ecdysone was shown to arrest the cell cycle, resulting in an accumulation of diploid cells in G1, prior to initiation of DNA synthesis. Some mosquito species, such as Culex pipiens L. (Diptera, Culicidae), harbor the obligate intracellular bacterium, Wolbachia pipientis Hertig (Rickettsiales, Anaplasmataceae), in their reproductive tissues. When maintained in mosquito cell lines, Wolbachia abundance increases in ecdysone-arrested cells. This observation facilitated the recovery of high levels of Wolbachia from cultured cells for microinjection and genetic manipulation. In female Culex pipiens, it will be of interest to explore how hormonal cues that support initiation and progression of the vitellogenic cycle influence Wolbachia replication and transmission to subsequent generations via infected eggs.

Metabolic and Hormonal Changes Associated with Vitellogenesis in Salvator merianae Lizards.

Vitellogenesis in oviparous vertebrates is a critical marker of the restart of seasonal reproductive activity. During this process of multihormonal regulation, females allocate a considerable amount of organic and mineral reserves to the synthesis of yolk, with changes in their plasma values. In this work, we determined plasma levels of various metabolites and steroid hormones throughout the reproductive cycle in females of Salvator merianae who developed vitellogenic and non-vitellogenic follicular cycles. We worked for two consecutive years with 20 adult females from the Experimental Hatchery of the Facultad de Agronomía y Zootecnia of the Universidad Nacional de Tucumán. Values of metabolites: glucose, triglycerides, cholesterol, calcium, phosphorus, albumin, total proteins, and hormones: estradiol, progesterone, and testosterone, were determined during the following stages of the annual cycle: hibernation, hibernation emergence, courtship-mating, oviposition, and incubation. Vitellogenic females showed significantly higher plasma levels of triglycerides, calcium, phosphorus, and albumin than non-vitellogenic females, mainly in the courtship-mating stage (advanced vitellogenesis). In contrast, annual cholesterol averages were lower in vitellogenic females. Glucose showed changes throughout the annual cycle regardless of the vitellogenic condition. Total proteins plasma levels had very few fluctuations during the cycle. Among the hormones studied, only testosterone showed differences related to vitellogenic condition, with higher levels in non-vitellogenic females during the entire reproductive cycle. The knowledge of these changes associated with vitellogenesis will improve zootechnical management and will allow optimizing the reproductive efficiency of Salvator lizards in captivity.

Fat and Happy: Profiling Mosquito Fat Body Lipid Storage and Composition Post-blood Meal.

The fat body is considered the insect analog of vertebrate liver and fat tissue. In mosquitoes, a blood meal triggers a series of processes in the fat body that culminate in vitellogenesis, the process of yolk formation. Lipids are stored in the fat body in specialized organelles called lipid droplets that change in size depending on the nutritional and metabolic status of the insect. We surveyed lipid droplets in female Aedes aegypti fat body during a reproductive cycle using confocal microscopy and analyzed the dynamic changes in the fat body lipidome during this process using LC/MS. We found that lipid droplets underwent dynamic changes in volume after the mosquito took a blood meal. The lipid composition found in the fat body is quite complex with 117 distinct lipids that fall into 19 classes and sublcasses. Our results demonstrate that the lipid composition of the fat body is complex as most lipid classes underwent significant changes over the course of the vitellogenic cycle. This study lays the foundation for identifying unknown biochemical pathways active in the mosquito fat body, that are high-value targets for the development of novel mosquito control strategies.

Dynamics of oogenesis in ghost shrimp Callichirus major (Crustacea: Axiidea): a morphofunctional and histochemical study

Callichirus major, popularly known as ghost shrimp, is a species of great importance in the fishing industry, because of its use as live bait. This study aimed to describe the different stages of the developing ovaries in C. major. Shrimps were collected along the Corujão beach, Piuma, Brazil (20°50'41.6“S 40°44'15.7“W), and the gonads were dissected for histological and histochemical analysis. The ovary consists of two elongated filaments covered by a connective tissue that divides the organ into lobules, where somatic and germ cells are found. It was possible to classify five types of germ cells: Oogonia (Oog), previtellogenic oocyte (Oc1), early vitellogenic oocyte (Oc2), late vitellogenic oocyte (Oc3) and mature oocyte (Oc4) based on their vitellogenic stage, cytoplasmic, nuclear and morphometric characteristics. The histochemical analysis demonstrated an intense reaction for proteins and polysaccharides in peripheral cytoplasm of Oc3 comparing to others cell types. According to size, volume, color intensity and distribution of oocyte types the gonads were classified into: immature, developing, developed and spent, being in females at this last stage, observed empty follicles and oocytes in reabsorption process. During oogenesis was observed a gradual increase in cytoplasmic acidophilia due to accumulation of yolk granules and the intense histochemical reaction in periphery of Oc3, which indicate the beginning of an extravitellogenic source of nutrients. Based on the microscopic analysis of the vitellogenesis, shrimp C. major showed the initial short phase of oocyte growth following with a fast vitellogenic cycle.

Signals of vitellogenesis and estrus in female hawksbill turtles.

This study reports a viable means of identifying the vitellogenic cycle and limited estrus period in hawksbill turtles for the purposes of developing captive breeding program, based on the combination of blood metabolite parameters (triglyceride, total protein, and calcium levels), feeding status, and ovary condition. Follicle size of two focal captive females showed clear seasonal changes, with major development occurring between March and May (19.0-24.4 mm), and exceeding 25 mm between June and September. Triglyceride, total protein, and calcium levels dropped with follicular development and maintenance (March to October), and then began to rise when follicular retraction occurred from October onwards. The two focal turtles reduced food intake during intensive follicular development (April to May). These findings suggest that blood metabolite parameters and feeding conditions are inferred by the vitellogenic cycle. An additional 10 females exhibiting follicular development were mated with a single male for 7-day period between May and June. Follicle size was measured immediately prior to pairing, and a statistically significant difference in follicle size of 10 females was recorded between the seven failed (20.9 mm) and three successful (23.6 mm) mating events. This indicates follicle development is essential to successful mate and monitoring of vitellogenic cycle may help improve the success rates of captive hawksbill breeding programs.

Non-lethal assessment of the reproductive status of broadnose sevengill sharks (Notorynchus cepedianus) to determine the significance of habitat use in coastal areas.

Identification of the importance of habitats that are frequently used by any species is essential to a complete understanding of the species' biology and to incorporate their ecological role into conservation and management programmes. In this context, the present study investigated whether Tasmanian coastal waters have any reproductive relevance for the broadnose sevengill shark (Notorynchus cepedianus). Although this species is a large coast-associated apex predator in these areas, there is a complete gap in understanding the role that these coastal systems could play in its reproduction. Reproductive hormones were used as a non-lethal method to address the reproductive biology of this species. Females seemed to have at least a bi-annual reproductive cycle, being pregnant for ∼1 year and spending at least 1 year non-pregnant, with the ovulatory cycle separated from gestation. Mature females were found to be ovulating, in the initial stages of pregnancy, resting or starting a new vitellogenic cycle. Notorynchus cepedianus did not use these coastal habitats for mating or as a pupping ground. Although the mating season was distinguished between September to April, only 22% of males showed mating scars during the peak of the mating period and no near-term pregnant females were observed. Thus, despite these coastal waters being an important foraging ground for this species, these areas did not have any reproductive relevance. In consequence, future management and conservation planning programmes need to identify whether there are other areas in Tasmania that play a critical role for reproductive purposes in this species. Finally, although previous studies have linked reproductive hormones with external examination of the gonads to validate the use of steroids as a non-lethal tool to address reproduction, the present study used this methodology without killing any animals. This has important implications for conservation programmes of threatened and endangered species worldwide where the methodology cannot be validated.

Reproductive Strategies of Leopard Toad and Mascarene Frog from Giza, Egypt

I examined the reproductive strategies of leopard toad and mascarene frog by studying their annual vitellogenic cycle, monthly changes of masses of ovary, liver and fat bodies as well as egg size and number in two study areas, Abo Roash and El Mansuriya, and in the years 2001, 2005, and 2008-2009, particularly during the final two years of that period. Based on the presence of the mature ova, I found that vitellogenic cycle is continuous in toad, but discontinuous in frog. Further, leopard body reserves allocated more energy to vitellogenesis than did mascarene frog. Hence, fecundity in toad was higher than that in frog, as associated with higher egg number and size. During oviposition, female mascarene retained a small portion of a clutch, whereas toad shed all egg mass at once. Over the study period, both body and reproductive conditions reacted positively in toad, but negatively in frog. Warm temperature and long photoperiod elucidated ovarian development under high relative humidity in frog. In contrast, in toad, low relative humidity may be an environmental cue for the increase in ovarian mass. Thus, higher sexual activities occurred in spring for toad (dry environment), but in moist summer for frog. Ovarian mass and egg number were temperature-dependent in frog, but independent in toad. Relative humidity correlated significantly and negatively to egg size in both populations. It also related inversely to egg number in toad, but not in frog. Hence, eggs of the frog are controlled by both temperature and humidity in summer season. Rainfall had no effect on sexual parameters in both species.

Effects of juvenile hormone analogue treatment in adult females of the ovoviviparous cockroach, Blaptica dubia (Dictyoptera, Blaberidae)

Adult females of the ovoviviparous Argentinian cockroach, Blaptica dubia, were repeatedly treated with either 100 μg methoprene or 100 μg pyriproxyfen in 5 μL acetone either during the first vitellogenic cycle or during the period of gestation. Treatment during the first vitellogenic cycle (days 2–20 of adult life) did not inhibit vitellogenesis and oocyte growth, but prevented the formation of an ootheca. This was accompanied with a significant reduction of the titer of juvenile hormone (JH) III and an increased amount of ecdysone (E) and 20-hydroxyecdysone (20E) in the haemolymph of the animals. Treatment of adult females during the period of gestation (days 30–70) resulted in a complete degradation and resorption of the ootheca and induced another vitellogenic cycle. Again, this was associated with a decrease in haemolymph JH III titer, but an increase in the concentrations of free ecdysteroids.

Endocrine aspects of ovary growth and gestation in the Argentinian cockroach, Blaptica dubia (Dictyoptera, Blaberidae)

Titers of juvenile hormone (JH) III and free ecdysteroids were studied in the hemolymph of the ovoviviparous Argentinian cockroach, Blaptica dubia, related to fat body depletion and reproduction. Adult females were analyzed during the first (days 5–25) and second vitellogenic cycle (days 80–100) and during the periods of gestation. Body weight changes of adult females were closely related to ovarian growth, ootheca formation, ootheca deposition, and hatching of the nymphs. Biochemical analysis of the fat body revealed lipids as the main storage compounds, followed by glycogen, proteins, and free carbohydrates. Changes in the fat body weight and in the chemical constituents of the fat body correlated with the processes of vitellogenesis and gestation. Concentrations of JH and free ecdysteroids in the hemolymph were measured by high pressure liquid chromatography-mass spectrometry. JH III was the only JH homolog found. JH III titers were high during vitellogenesis as well as toward the end of the gestation period. Changes in the concentrations of ecdysone and 20-hydroxyecdysone were less clear. The results reveal JH III as the major gonadotropic hormone in adult females of B. dubia.

Vitellogenin in Asian catfish, Clarias batrachus: Purification, partial characterization and quantification during the reproductive cycle by ELISA

A study on vitellogenin (Vtg), the egg yolk precursor protein from Asian catfish, Clarias batrachus was undertaken. Vtg synthesis was induced in male C. batrachus by three intraperitoneal injections of 17β-estradiol (E2). It was purified from E2-induced plasma by precipitation with EDTA and MgCl2 followed by gel filtration chromatography on Sephacryl S-300-HR. The purified protein was run in native gradient PAGE that appeared as a single band with apparent molecular weight of ~482kDa. Further, the protein in electrophoresed gels was stained separately and found positive for carbohydrate, lipid and phosphorus; and thus was confirmed as vitellogenin. In SDS-PAGE, two major polypeptides corresponding to ~97 and ~67kDa could be observed. Polyclonal antiserum to C. batrachus vitellogenin (Vtg) was raised in a rabbit. It was found to be specific for C. batrachus Vtg in western blot, after adsorption with male C. batrachus liver homogenate. Using this antiserum, Vtg was found to be localized in liver and ovary of vitellogenic female C. batrachus by immunohistochemistry. Further, a competitive ELISA was developed for the quantification of Vtg in plasma samples of C. batrachus. The developed ELISA showed a detection range between 7.8 and 500ng/ml. The intra- and inter-assay coefficients of variation of the assay were 7.7% (n=10) and 12.6% (n=6), respectively. The ELISA was used to measure the Vtg concentration during the annual vitellogenic cycle of female C. batrachus. The average maximum plasma Vtg concentration (2187.51±228.8μg/ml) was observed approximately 1month before spawning (May) and the concentration was found maintained at significantly higher level during the majority of the spawning period (June and July).

Nuclear receptor FTZ: partial cloning and expression pattern in fat body of the American cockroach Periplaneta americana (Linnaeus, 1758) during vitellogensis

The aim of the present study was to understand the importance of the nuclear receptor Fushi tarazu factor 1 (FTZ-F1) in relation to vitellogenin genes expression in the fat body of Periplaneta mericana during the first vitellogenic cycle. Initially, a 342 bp fragment was cloned from the female fat body through degenerate primer RTPCR method. Its sequence analysis revealed high homology with other insects and animals FTZ-F1. In order to find the relation between the P. americana FTZ and vitellogenins, we analyzed the expression pattern of the latter in the fat body during vitellogensis. The PamFTZ transcript was detected during all stages of vitellogenesis in the fat body with a little increase in day 5, preceding the bulk of vitellogenin genes expression. We assumed that nuclear protein FTZ in the hemimetabolous insect, P. americana might functions as a competence factor that facilitates juvenile hormonal activation of gene expression as in holometabolous insect, Drosophila melanogaster.

How are the oocytes recruited in an indeterminate fish? Applications of stereological techniques along with advanced packing density theory on European hake ( Merluccius merluccius L.)

Early cycle oocyte recruitment has previously been described and partly quantified in teleosts with a determinate reproductive style. This recruitment is, however, less understood in indeterminate fish due to additional factors further complicating quantification by traditional means. These factors include the extremely broad size range of oocytes, where gap formation between previtellogenic and vitellogenic oocytes cannot be identified, an important number of previtellogenic oocytes close to the threshold of vitellogenesis, and a lack of knowledge of the importance of atresia during vitellogenesis. These problems were largely solved by combining advanced packing density theory and stereological measurements aided by image analysis, which allowed for a much better understanding of the early cycle oocyte recruitment pattern in European hake ( Merluccius merluccius). This novel type of analysis demonstrated that the European hake presents a continuous, indeterminate fecundity style in which the steady recruitment of oocytes from the large pool of previtellogenic oocytes (about 500,000 g −1 at the perinucleolar stage) leads to multiple vitellogenic cycles during the extensive reproductive season.

The Amphibian (Rana esculenta) Brain Progesterone Receptor: Relationship to Plasma Steroids and Vitellogenic Cycle during the Gonadal Recovery Phase

We report the presence of a progesterone receptor (PR) in the brain of the female amphibian Rana esculenta and changes in the levels of this PR during the gonadal recovery phase in relation to different circulating steroid levels and steroid treatment. The highest level of the PR in the nuclear brain extract corresponded to a low level of plasma progesterone and occurred when vitellogenin synthesis was at a minimum. The lowest level was found during follicular growth, concomitant with increased plasma 17beta-estradiol and progesterone levels. The PR levels were significantly higher in ovariectomized female R. esculenta than in intact and sham-operated female frogs. Treatment with 17beta-estradiol and progesterone downregulated the frog brain PR, indicating that a possible progesterone-receptor interaction is involved in the modulation of vitellogenin.

Natural History of the Vine SnakeOxybelis Fulgidus(Serpentes, Colubridae) From Brazil

Aspects of natural history of the vine snake Oxybelis fulgidus from the northern region of Brazil were inferred based on the analysis of 106 preserved specimens (55 males and 51 females), and from a review of records in the published literature. Males mature at smaller size than females. Differences in body length and shape were also found among adult females and males: adult females are larger in mean snout-vent length and have relatively larger heads and shorter tails. Females have an extended seasonal vitellogenic cycle from April to December (mainly throughout the dry season). Oviductal eggs and egg-laying were recorded from September to December, while births occurred from January to April. Fecundity varied from four to twelve eggs or vitellogenic follicles (n = 13), and was positively correlated with female body length. Testicle volume is significantly larger from February to July (mainly in April and May; end of the rainy season), however the deferent ducts diameter do not differ significantly throughout the year. We hypothesized that both males and females may have an associated reproductive pattern, both synchronized in the end of the rainy season. Oxybelis fulgidus feeds on lizards (mainly iguanian) and passerine birds. Apparently, females feed more frequently on birds than males.

Timing of reproduction of a cold desert viperid snake from North America, the Great Basin rattlesnake ( Crotalus lutosus)

In this study, we examined the reproductive tracts of preserved specimens of the Great Basin rattlesnake ( Crotalus lutosus) to investigate the reproductive strategies used by the only species of rattlesnakes widely occurring in a cold desert. The vitellogenic cycle of female rattlesnakes spanned over two consecutive years: females started vitellogenesis in late spring/early summer, and ovulated in the summer of the next year. This contrasts with the female reproductive cycle of other rattlesnake species from warmer regions of North America, which typically initiate vitellogenesis in summer/fall, and ovulate in late spring of the next year. On the other hand, the reproductive cycle of male C. lutosus conformed well to those described for other rattlesnakes; that is, testes were active (i.e., sperm formation) in summer. The sexual segments of the kidneys of males were hypertrophied in summer, suggesting an unimodal summer mating season. Our findings suggests that the relatively short active season (i.e., 4–5 months) experienced by these snakes in the Great Basin of North America has implications for the timing of female reproductive events, such as an early onset of vitellogenesis, a late time of ovulation, and possibly a restricted period for giving birth.

Sexual size dimorphism, diet, and reproductive biology of the Afro-Asian Sand Snake, Psammophis schokari (Psammophiidae)

AbstractWe measured and dissected 226 museum specimens of Psammophis schokari (Schokari Sand Snake), a widespread, common “whipsnake” occurring in North Africa and southwest Asia. Three aspects of its ecology were investigated: sexual size dimorphism (SSD), diet, and reproductive biology. There was no significant SSD in mean body size or shape for the traits measured. The species is an opportunistic feeder, but preys predominately on lizards. Males and females exhibit their highest levels of sexual activity in synchrony and exhibit prenuptial spermiogenesis and a Type 1 vitellogenic cycle, respectively. Mating occurs at the end of the rainy season and clutch sizes are small. These findings from a Northern Hemisphere temperate sand snake are consistent with previous results for both tropical and south temperate Psammophis. These traits thus appear to be highly conserved within the genus and also exhibit convergence with respect to unrelated colubroid “whipsnakes”.

Reptile (Podarcis sicula) brain progesterone receptor: relationship to plasma steroids and vitellogenic cycle during Gonadal recrudescence

Reptile (Podarcis sicula) brain progesterone receptor: relationship to plasma steroids and vitellogenic cycle during Gonadal recrudescence

Folliculogenesis and ovarian histology of the oviparous gecko, Hemidactylus mabouia (Sauria: Gekkonidae)

Histological changes during the ovarian cycle of the oviparous gekkonid lizard, Hemidactylus mabouia are described. The ovaries each contained a single germinal epithelium (bed) with numerous oogonia and primordial follicles. Similar to other squamates, the ovarian stroma was characterized by follicles in different stages of development, and may contain corpora lutea, and corpora atretica. The seasonal study revealed that vitellogenic ovarian hypertrophy commenced in spring (September) and ovulation of the first clutch (two eggs) occurred in summer (November). A second vitellogenic cycle followed, the second clutch being ovulated in late‐summer (January). Ovarian quiescence, marked by predominance of pre‐vitellogenic (hydration stage) follicles, was evident in females collected from February to July. Although oocyte growth was accompanied by changes in the granulosa layer, zona radiata and thecal layers, the zona pellucida remained unchanged throughout. The small cells of the granulosa layer differentiated into three distinct cell types ‐ small, intermediate and the conspicuous large pyriform cells. Similar to most other squamates, the intermediate and pyriform cells at the onset of vitellogenesis regressed to a single cuboidal epithelium. Following ovulation, the granulosa layer hypertrophied forming luteal tissue. The theca layer differentiated into two layers and septal invasion of the corpus luteum took place. At the time of oviposition, corpora lutea regressed to form ovarian scars (corpora albicantia). Follicular artresia occurred in previtellogenic follicles (hydration stage) and seldom in vitellogenic follicles. The highest incidence of atresia occurred in the post‐breeding period (February). The gross morphology, oogenesis and folliculogenesis of the ovaries of H. mabouia correspond to the general squamate pattern described for oviparous reptiles.

Reproductive and thyroid hormone profiles in captive Western fence lizards (Sceloporus occidentalis) after a period of brumation

Seasonal fluctuation in serum concentrations of sex steroid (testosterone [T] and 17beta-estradiol [E(2)]) and thyroid (triiodothyronine [T(3)] and thyroxine [T(4)]) hormones was determined in captive Western fence lizards (Sceloporus occidentalis). Samples were collected from male and female breeding pairs weekly for a 4-month period after their emergence from artificial brumation. Circulating levels of E(2) corresponded with the expected vitellogenic and ovulatory cycles in females, and surprisingly, E(2) in males followed a similar pattern, indicating a possible role in breeding behavior. Serum T was elevated in male lizards for the first 6 weeks after emergence from brumation, possibly related to an increase in the onset of active spermatogenesis. Thyroid hormones showed little cyclical activity throughout the breeding period, with the exception of small increases of T(3) at weeks 8 and 16, possibly implying an active role of this hormone with ovulation in females. Overall, these baseline hormone data are not only useful in developing this animal as a laboratory reptile model for assessment of endocrine-mediated toxicity, but also of value for understanding herpetological endocrinology and for application in the conservation of threatened species. Zoo Biol 27:36-48, 2008. (c) 2007 Wiley-Liss, Inc.

Gametogenesis and gonad mass cycles in the common circumpolar Antarctic echinoid Sterechinus neumayeri

Polar conditions (low and stable seawater temperature coupled with highly seasonal primary productivity) constrain reproduction in benthic invertebrates. The reproductive cycle of the common Antarctic sea urchin Sterechinus neumayeri was investigated at monthly intervals over a 2 yr period in relation to seasonality of chlorophyll standing stock and ability to feed. Competence to spawn increased gradually throughout the austral winter to peaks in September to November. Gonad mass varied strongly, both seasonally and among 3 geographically close sites. Spawning occurred between October and January at each site, and was strongest in November of both years. Spawning was not always accompanied by a reduction in gonad mass because of simultaneous feeding and deposition of nutritive tissue for the forthcoming winter. Spawning was annual but there was also strong evidence for longer, multi-year periodicity in reproductive output. Histological analysis indicated an 18 to 24 mo vitellogenic cycle, with oocytes developing from 20 to 50 μm in the first winter, and from 50 to 120 μm in the second winter. Of total oocyte volume increase (vitellogenesis), 93% occurred within the last 12 mo. Spermatogenesis took 12 mo. S. neumayeri does not feed during the austral winter, and vitellogenesis is therefore predominantly decoupled from energy intake. Much of the cost of reproductive synthesis is met at a time of severe energy limitation.