2n Females Research Articles

Oogenesis and Spermatogenesis of the Triploid Black Tiger Shrimp, Penaeus monodon

Triploidy (3n) induction of Penaeus monodon was performed separately by Australian and Thai groups of researchers 14 years ago, incidentally at the same period. The Australian group employed the chemical induction method, while the Thai group the cold shock one, and both groups obtained 3n P. monodon in their attempts. The success has led to several studies on the physiology of the 3n P. monodon, including growth, survival and reproductive functions. Both groups reported sterility of the 3n shrimp. The Australian group reported defected oogenesis and spermatogenesis of the 3n shrimp examined at subadult stage. Since the evaluation of the reproductive functions should be performed at the adult stage of the animals, we, therefore, performed in-depth studies on oogenesis and spermatogenesis of adult 3n P. monodon induced by cold shock. The studies include gross observation of the ovary, light (LM) and transmission electron (TEM) microscopy and image analysis of sex cells. The ovary of the 3n P. monodon females could not develop to full-maturation and their gonadosomatic index (GSI) was significantly lower than that of the 2n shrimp. Oogenesis of the 3n females proceeded from oogonia to cortical rod oocytes, but with significantly lower percentage than that of the 2n shrimp. The GSI of the 3n males was significantly lower than that of the 2n shrimp. Their spermatogenesis, however, proceeded normally from spermatogonia to spermatozoa, but with low density of spermatozoa in the seminiferous tubule and vas deferens, and none in the terminal ampoule. The average nuclear area of the 3n spermatozoa was significantly lower than that of the 2n shrimp as well. Altogether, these results suggest that 3n P. monodon induced by cold shock had defect in both oogenesis and spermatogenesis, probably more in quantity than in quality. HIGHLIGHTS Oogenesis and spermatogenesis of the triploid black tiger shrimp, monodon, induced by cold shock were studied. The study found abnormalities in the spermatogenesis, but not oogenesis, of the triploid P. monodon induced by cold shock. It may be concluded that triploidy condition of P. monodon induced by cold shock affects meiotic, but not mitotic activity of the sex cells. GRAPHICAL ABSTRACT

Ploidy influences on metabolic substrate deposition of rainbow trout

Triploid fish are usually sterile. Thus, the energy and nutrients intended for sexual maturation may be available to enhance flesh quality and physical growth. The present study aimed to investigate differences in the metabolic substrates, lipids and proteins, between storage tissues from diploid and triploid female rainbow trout. Monthly, metabolic substrates were quantified in liver, muscle, and ovaries, which were collected during the first reproductive cycle. In general, it was possible to identify a seasonal and similar deposition of metabolites in different tissues of 2n and 3n females, mainly at early stages of gonadal maturation. However, from the stages 5-6, the ovaries showed great differences between ploidies, with higher concentration of lipids and protein in 2n females. This result reflects the incorporation of vitellogenin in oocytes, which is a process that does not occur in 3n females. It was possible to observe seasonal hepato-somatic index changes in 2n females, with higher values observed in the post-ovulatory stage, and the triploid animals showed lower values compared to 2n, with no seasonal difference. Viscero-somatic index can reflect the mobilization of substrates, with higher values found for 2n females in stage 5-6, which is the period of active mobilization of tissue substrates.

The concentration of 17β-estradiol in tissue of diploid and triploid Carassius gibelio (Bloch, 1782) females during the reproduction season

The concentration of 17β-estradiol in tissue of diploid and triploid Carassius gibelio (Bloch, 1782) females during the reproduction season

Expression of genes associated with fatty acid metabolism during maturation in diploid and triploid female rainbow trout

Abstract To study the effects of sexual maturation on fatty acid metabolism in fish fed to satiation daily, expression of thirty-five genes involved in fatty acid metabolism was determined in sexually maturing diploid (2N; fertile) and triploid (3N; sterile) female rainbow trout. Gene expression was assessed in liver, white muscle, and visceral adipose tissues for fish that were 16 to 24 M of age. Previously, we reported minimal differences in most growth measurements between maturing 2N and 3N fish, but there were disparate changes in muscle proximate composition, visceral fat stores, and fatty acid contents of energy stores at 21 M with 3N females having greater lipid stores. Here, we report that gene expression profiles of liver and white muscle corresponded to the phenotypes with significant differences in expression at 20 M. Triploid females had increased expression of genes involved in fatty acid synthesis; including gpat , srebp1 , acyl , acc , fas , and scd1 in liver and fas in muscle. Conversely, 2N muscle had increased expression of β-oxidation genes cpt1b , cpt2 , ehhadh , and acat2 and TORC1 inhibitors redd1 , erk , mo25 , and pras40 . Diploid muscle also had increased expression of pparβ along with increased expression of the fatty acid transporter gene cd36 , and β-oxidation genes cpt1a , cpt1c , aco , and acdhvl at 20 M. Additionally, 2N visceral adipose tissue had increased cpt1a expression at 21 M. Overall, data suggest that 3N females are undergoing higher levels of fatty acid synthesis while 2N females have higher levels of β-oxidation during sexual maturation. Phenotypic data supports these findings with decreasing fatty acid stores in 2N females during this time period. Additionally, changes in gene expression are associated with altered expression within the mTOR and PPARβ signaling pathways.

Effects of sexual maturation and feeding level on fatty acid metabolism gene expression in muscle, liver, and visceral adipose tissue of diploid and triploid rainbow trout, Oncorhynchus mykiss.

In many cultured fish species, such as salmonids, gonadal development occurs at the expense of stored energy and nutrients, including lipids. However, mechanisms regulating nutrient repartitioning during sexual maturation are not well understood. This study compared sexually maturing diploid (2N) and sterile triploid (3N) female rainbow trout to investigate effects of sexual maturation on expression of 35 genes involved in fatty acid metabolism, including genes within fatty acid synthesis, β-oxidation, and cofactors of the mTOR and PPAR signaling pathways, in liver, white muscle, and visceral adipose tissue. Diploid fish were fed at different rations (0.25% and 0.50% tank biomass, and satiation) to determine effects of ration on gene expression. Gene expression was affected by ration level only in white muscle; erk and acat2 had higher expression in fish fed higher rations. On the other hand, sexual maturation affected gene expression across all three tissue types. Data indicate 2N fish have higher expression of β-oxidation genes within white muscle and within visceral adipose tissue. These findings support enhanced fatty acid mobilization within these tissues during sexual maturation. Higher expression of fatty acid synthesis genes in 3N female liver is associated with higher expression of mTOR cofactors and pparγ, which reflects continued deposition of lipids in these fish. Furthermore, greater expression of genes involved in β-oxidation pathways across ration levels in 2N females suggests that sexual maturation and the associated maturation-related signals are stronger regulators of lipid metabolism-related genes rather the rations applied in the current study.

Ploidy-dependent survival of progeny arising from crosses between natural allotriploid Cobitis females and diploid C. taenia males (Pisces, Cobitidae).

Crosses between 21 triploid hybrid Cobitis females and 19 C. taenia (2n=48) males led to viable progeny; whereas no embryonic development was observed in crosses with tetraploid males (4n=98). The ploidy status of 491 progenies randomly selected with flow cytometry (316) or chromosome analysis (175) revealed an average of 55.2% triploids and 44.8% tetraploids, but the ratio of 3n versus 4n fish did change during development. In the first 2days after hatching, approximately 65.1% of tetraploid larvae were observed. Their number decreased significantly to 30.8 and 6.2% on average during 2-5 and 10-15months of life, respectively. The karyotype of tetraploid progeny (4n=98) included 3n=74 chromosomes of the parental female and n=24 of C. taenia male. The number of tetraploid progeny indicated indirectly that about 66% of eggs from 3n females were fertilized with C. taenia. The rest of the eggs developed clonally via gynogenesis or hemiclonally via hybridogenesis into triploids of the same karyotype structure as parental females. We have documented for the first time that (at least under experimental conditions) tetraploids are commonly formed, but are less viable than triploids, and a ratio similar to what is found under natural conditions is finally attained. The current explanation concerning the ploidy and karyotype structure of the progeny confirms that the eggs of 3n Cobitis females are not only capable of maintaining all chromosomes but are also capable of incorporating the sperm genome, thus creating the potential to produce tetraploids.

Effects of feeding level and sexual maturation on fatty acid composition of energy stores in diploid and triploid rainbow trout (Oncorhynchus mykiss)

Sexual maturation is an energy demanding, physiological process that alters growth efficiency and compromises muscle quality in many food-fish species. Lipid mobilization supplies energy required for this process. To study the effect of ration level on fatty acid composition, diploid (2N) rainbow trout, approaching ovulation, were fed at 0.25 and 0.50% of tank biomass/day and to apparent satiation (~0.75% of tank biomass/day). In addition, triploid (3N) female trout, which exhibit only minimal ovarian development, were fed at 0.50% of tank biomass/day. The primary objective of this study was to determine effects of ration level on fatty acid composition in different lipid compartments (muscle, visceral adipose tissue, liver, and gonad) during sexual maturation. Lower feeding levels produced smaller fish, but did not affect the onset of sexual maturation. Higher feeding levels resulted in fish muscle with higher relative amounts of saturated fatty acids (SFAs), but monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) were not affected by ration level. While ration level affected the fatty acid profile of each of the four tissues analyzed, the number of fatty acids affected was greatest in white muscle. An additional objective was to determine differences in the fatty acid composition of energy stores during maturation in female rainbow trout that were fed at a moderately restricted feeding level (0.50% of tank biomass/day). These differences were determined by comparing mature 2N to sterile 3N females of the same age. Diploid muscle contained higher amounts of PUFAs (44.4±1.0%) than 3N muscle (39.7±0.8%). Saturated fatty acids were in the highest concentrations in muscle and visceral adipose tissue, and 2N liver contained more PUFAs and fewer MUFAs than 3N liver; however these values are relative values. In general, fatty acids affected by ration level were not the same as fatty acids affected by ploidy. Triploid fatty acid profiles did not mimic those of the satiation fed group; which was expected if 3N fish were simply consuming excess energy. Both 2N and 3N muscle fatty acid profiles were similar to that of the diet, except muscle had lower amounts of PUFA precursors (18:3n-3 and 20:5n-3) and higher relative amounts of their product (22:6n-3). Also, 2N muscle had higher 16:1 and 3N muscle had higher 16:0 compared to the diet. It is unclear if these differences are hormonally driven or if there are other physiological dissimilarities between 2N and 3N trout causing these differences. Overall, our data suggest that 2N and 3N fatty acid metabolism is regulated differently.

Evaluation of long‐chain n3 fatty acid content in diploid and triploid rainbow trout

Intake of long chain n3 fatty acids (LCn3), eicosapentaenoic acid (EPA; 20:5 n3) and docosahexaenoic acid (DHA; 22:6 n3), is associated with reduced cardiovascular disease. There is growing interest in farmed fish like rainbow trout, Oncorhynchus mykiss, as sources of LCn3. The trout industry raises diploid (2N) and triploid (3N) fish. 3N females fail to sexually mature and avoid the negative effects of maturation on fillet quality. In this work we examined the LCn3 content of 2N and 3N trout. Female rainbow trout were selected from independent populations of 1 yr and 2 yr old 2N and 2 yr old 3N fish. Although each group consumed the same diet formulation, rearing conditions and feeding rates were different and only the 2 yr 2N trout were sexually mature. Boneless, skinless fillets were removed from 4 fish per group and the total fatty acid content of the fillets was determined by FAME analysis. The contents of LCn3 in the 3N 2yr trout (EPA: 1.9 ± 0.5 mg/g; DHA 7.6 ± 1.7 mg/g) were almost 2‐fold higher than in the 2N 2yr trout (EPA:0.8 ± 0. mg/g; DHA 3.9 ± 0.5 mg/g) and the 2N 1yr trout (EPA:1.0 ± 0.3 mg/g; DHA 4.0 ± 0.7 mg/g). The higher content of LCn3 was in part the result of total higher content of fatty acid in the 3N trout. The percent content of LCn3 and ratios of short chain to long chain n3 were similar in all groups. These data indicate that farm‐raised rainbow trout are a rich source of EPA and DHA. This work was supported by the USDA‐ARS.

Effect of sexual maturation and triploidy on chemical composition and fatty acid content of energy stores in female rainbow trout, Oncorhynchus mykiss

Sexual maturation is an energy and nutrient demanding physiological process that alters growth efficiency and compromises muscle quality in many food fish species. Lipid mobilization supplies energy required for this process. To study the effect of sexual maturation on lipid mobilization in female rainbow trout on a high nutritional plane, fish growth, egg development, proximate composition, and fatty acid composition of muscle, liver, visceral fat, and gonads were determined for diploid (2N; fertile) and triploid (3N; sterile) females from July 2008 thru March 2009, when most 2N females ovulated. Previously, we reported there were minimal differences in most growth measurements between the 2N and 3N fish in this study, but there were changes in muscle proximate composition and visceral fat stores. Here, we report changes in proximate composition of liver, gonads, visceral adipose tissue, and fatty acid composition of these tissues and muscle during sexual maturation of 2N females with 3N females serving as the control. Similar and distinct patterns of change in fatty acids were observed between tissues of 2N and 3N fish, and among different tissues as the 2N fish were maturing. Highly unsaturated, long chain fatty acids appear to play an important role in gonadogenesis, either as a source of energy or for deposition in eggs to support offspring development and survival. Differences in growth and preferential mobilization of fatty acids are observed when fish are on a high nutritional plane compared to more typical, lower nutritional regimens.

Effect of Sexual Maturation on Thermal Stability, Viscoelastic Properties, and Texture of Female Rainbow Trout, Oncorhynchus mykiss, Fillets

The nutrient and energy demand of sexual maturation in many fish cultivars causes structural change to key contractile proteins and thereby, affects fillet firmness. Thermal denaturation and viscoelastic properties of white muscle from diploid (2N; fertile) and triploid (3N; sterile) female rainbow trout were investigated at 6 age endpoints from July 2008 through spawning in March 2009. Differential scanning calorimetry showed, in March, that the actin denaturation temperature (T(max,actin)) of 2N females was higher than that observed in 3N females (78.17 versus 77.27 °C). From 35 to 45 °C, viscoelastic measurement revealed that muscle from 2N females and younger fish (July, 16 mo) had greater elasticity (lower tan δ) than muscle from 3N females and older fish (November to March; 20 to 24 mo), respectively. The highest elastic response and the firmest fillets were observed in July. Raw fillets were softer (Allo-Kramer shear; P < 0.05) from September to January (288.77 g/g on average) than those collected in July (475.15 g/g) and March (366.79 g/g). Soft fillets became firmer after cooking except for January samples. Greater cook yield and softer fillets were observed in January compared to December. Lipid accumulation in 3N females may lubricate muscle fibers and protect them from losing functionality during the spawning season for animals on a high plane of nutrition. The relationship between fish maturation, measured as egg development, and chemical characteristics of fillets from fertile and sterile fish was evaluated. Thermal denaturation and viscoelastic characterization revealed changes in stability and gelling properties of muscle proteins that were related to changes in fillet texture.

Effect of sexual maturation on growth, fillet composition, and texture of female rainbow trout ( Oncorhynchus mykiss) on a high nutritional plane

Nutrient and energy demands of sexual maturation influence fillet composition and texture in many fish cultivars. Effects of sexual maturation on growth and on fillet composition and texture were investigated in rainbow trout on a high nutritional plane. Diploid (2N; fertile) and triploid (3N; sterile) female rainbow trout were reared from July 2008 through spawning in March 2009. Growth rates were greater in the 2N females from August through October; whereas, the reverse response was true in February and March, resulting in similar overall growth rates and body weight (3140 ± 76 g). Raw fillets were softer, regardless of ploidy, between September and January (Allo-Kramer shear) compared to July, but increased again in March (366.79 g/g). By November, 2N females had lower fillet fat content and higher moisture content than 3N females. Fat content negatively correlated with shear force (r = − 0.35, R 2 = 0.12). Alkaline-insoluble (a-i) hydroxyproline (HYP) concentration was similar for 2N and 3N muscle through January but increased 2 and 4 folds for 3N and 2N muscles, respectively, by March. A-i HYP positively correlated with energy of shear (r = 0.41, R 2 = 0.17). In summary, during the five months prior to spawning and in females on a high nutritional plane, increased accumulation of fat in 3N females resulted in decreased firmness of raw fillets; whereas, increased insoluble collagen content, primarily in 2N females, increased fillet firmness in the month of spawning.

Older triploid fish retain impaired reproductive endocrinology in the European sea bass Dicentrarchus labrax

This paper reports on an evaluation of growth, gonadal development and reproductive endocrinology of older triploid (3n) European sea bass Dicentrarchus labrax in comparison with their diploid (2n) counterparts throughout their fifth and seventh annual cycle of life. While older triploids retained impaired reproductive endocrinology, a sexually related dimorphic growth was observed with 3n females attaining the largest sizes. Comparisons of some body indexes showed that 3n females had a significantly lower hepato-somatic index (I(H)) than 2n females but a significantly higher viscero-somatic index (I(F)). In contrast, both male and female triploids showed significantly lower gonado-somatic index (I(G)) than diploids. Accordingly, diploids produced mature gametes but triploids did not, demonstrating that despite the longer time given to triploids for gonadal development, they could not reproduce. Furthermore, older triploids had lower levels of plasma sex steroids (testosterone, T; 11-ketotestosterone, 11-KT and oestradiol-17beta, E(2)) and luteinizing hormone (LH) than their 2n counterparts with 3n females showing drastic effects of triploidization on their reproductive endocrinology. Vitellogenin (VTG) was undetectable in 3n females. Gonadal content of steroid hormones and Sparus aurata-type gonadotropin-releasing hormone (sbGnRH) in the brain and pituitary were also lower in triploids compared with diploids. These results suggest that older 3n D. labrax retain functional sterility in both sexes, and 3n females might reach larger sizes than 3n males and their 2n counterparts in this species.

A VERTEBRATE REPRODUCTIVE SYSTEM INVOLVING THREE PLOIDY LEVELS: HYBRID ORIGIN OF TRIPLOIDS IN A CONTACT ZONE OF DIPLOID AND TETRAPLOID PALEARCTIC GREEN TOADS (BUFO VIRIDIS SUBGROUP)*

The rise and consequences of polyploidy in vertebrates, whose origin was associated with genome duplications, may be best studied in natural diploid and polyploid populations. In a diploid/tetraploid (2n/4n) geographic contact zone of Palearctic green toads in northern Kyrgyzstan, we examine 4ns and triploids (3n) of unknown genetic composition and origins. Using mitochondrial and nuclear sequence, and nuclear microsatellite markers in 84 individuals, we show that 4n (Bufo pewzowi) are allopolyploids, with a geographically proximate 2n species (B. turanensis) being their maternal ancestor and their paternal ancestor as yet unidentified. Local 3n forms arise through hybridization. Adult 3n mature males (B. turanensis mtDNA) have 2n mothers and 4n fathers, but seem distinguishable by nuclear profiles from partly aneuploid 3n tadpoles (with B. pewzowi mtDNA). These observations suggest multiple pathways to the formation of triploids in the contact zone, involving both reciprocal origins. To explain the phenomena in the system, we favor a hypothesis where 3n males (with B. turanensis mtDNA) backcross with 4n and 2n females. Together with previous studies of a separately evolved, sexually reproducing 3n lineage, these observations reveal complex reproductive interactions among toads of different ploidy levels and multiple pathways to the evolution of polyploid lineages.

Chemical composition of flesh of diploid and triploid population of tench (Tinca tinca, Linnaeus 1758)

The objective of this study was to determine differences in relevant parameters of the basic chemical composition of flesh (descriptors: dry matter &amp;ndash; DM, crude proteins &amp;ndash; CP, net proteins &amp;ndash; NP and net muscle proteins &amp;ndash; NMP, fat &amp;ndash; F, ash &amp;ndash; A) of communal stock of diploid (2n) and triploid (3n) tench (Tinca tinca L.) of the same origin, in relation to sex (F: female vs. M: male) and age (T&lt;sub&gt;3&lt;/sub&gt;: 36 months, after overwintering vs. T&lt;sub&gt;3+&lt;/sub&gt;: 42&amp;nbsp;months, after one summer in the pond). Altogether 137 siblings of tench were studied. The basic chemical composition of flesh of 2n and 3n tench of T&lt;sub&gt;3&lt;/sub&gt; age was found to be nearly the same. The only significant difference (P &amp;lt; 0.05) was found in DM content in diploids (5.7% higher DM content in females). Marked changes in the basic chemical composition were related to the increasing age of tench: after one summer in the pond (from T&lt;sub&gt;3&lt;/sub&gt; to T&lt;sub&gt;3+&lt;/sub&gt;) the DM content increased significantly in all groups of tench (by 5.4% in 2n females, P &amp;lt; 0.05; by 6.5% in 2n males, 9.9% in 3n females and by 7.4% in 3n males, all at P &amp;lt; 0.01). The content of nitrogenous substances was found to increase significantly (P &amp;lt; 0.01) in flesh of 2n males: CP by 3.2%, NP by 4.2% and NMP by 3.3% and in flesh of 3n males: CP by 7.0%, NP by 6.6% and NMP by 7.3%. Muscles of 3n females contained 107.3% higher fat content (P &amp;lt; 0.05). The proportion of ash decreased in 2n and 3n by 2.5% and 1.7%, respectively (P &amp;lt; 0.05). The flesh quality of T&lt;sub&gt;3+ &lt;/sub&gt;tench after one summer in the pond was higher if compared with that of T&lt;sub&gt;3 &lt;/sub&gt;tench after overwintering. Related to the ploidy level, more favourable chemical composition of flesh and more favourable flesh quality were found for triploids of T&lt;sub&gt;3+&lt;/sub&gt; age. &amp;nbsp;

Gonad development, growth, survival and quality traits in triploids of the protandrous hermaphrodyte gilthead seabream Sparus aurata (L.)

The effect of triploidy on the protandrous hermaphrodite gilthead seabream, Sparus aurata, was evaluated from 2 g to 1347 g (42 months old). No difference of growth was recorded until 484 g (17 months old), 2n and 3n being all males. From 20 to 42 months old, the 3n exhibited a lower growth, the difference increasing to 15.3% at the end of the experiment. At the two reproductive seasons studied, 3n matured as males. The 2n and 3n males had the same weight throughout the experiment. It is postulated that the 2n's advantage is mainly due to the occurrence of their feminisation, the 2n females presenting a significant better growth. Out of the breeding seasons, the 3n presented higher gutted yields (from 0.5% to 2%) and a lower level of lipid concentration in the muscle (from − 7.5% to − 17.5%). The fillet of the 2n females is more fatty than the fillets of the 2n and 3n males. This study demonstrated that until the common commercial weight (480 g), 3n present the same growth as 2n and do not exhibit a difference in survival, processing yields and quality traits. For bigger sizes, 3n are lower performers in growth but are leaner and have a better gutted yield. As no spawning behaviour was observed in this land based farm, these results need to be evaluated in sea cages in which breeders spend 3–4 months a year in spawning.

The First Results on Relationships Among Amphimictic Diploid, Diploid Gynogenic and Triploid Tench, Tinca tinca L. under Communal Testing

Amphimictic diploid (2n), induced meiotic gynogenic (MeiG) and induced triploid (3n) siblings of tench, Tinca tinca L., were separately nursed, marked and kept in long-term communal testing in pond until sex maturation and natural spawning. Survival rates during the initial periods were lesser in 2n and 3n compared to MeiG population (65%). The first overwintering period showed decreased survival of 3n (62.67%) compared to MeiG (90.0%) and 2n populations (91.67%). Survival rates during the next seasons ranged in 70.50–90.97% similarly in both 3n and 2n while in MeiG population it dropped to 38%. Weight and length growth of all populations studied showed significantly higher growth of 3n females and of 3n in general, compared to 2n and MeiG populations (P < 0.001). Health examinations found metacercaria of Diplosthomus spathaceum in eyes of 2-year-old 3n specimens, weak polyparasitic infections of skin and gills by Dactylogyrus, Trichodina, Thrichophrya, Apiosoma and Chilodonella spp. in 3n and 2n specimens but medium strong to strong infections of 100% of MeiG fish which might contribute to their enhanced mortality. Ploidy analysis of progeny obtained from spontaneous spawning of the broodstock showed 100% diploidy, indicating the following possibilities: 2n population might spawn itself, 2n males might also mate to gynogens, or 3n males might also mate to 2n and/or MeiG females causing spontaneous gynogenesis. Further pedigree analyses will be focused to highlight these questions.

The egg size determining gene,Esd, is a unique morphological marker on the W chromosome ofBombyx mori

Triploid females of the silkworm,Bombyx mori, were produced by crossing a tetraploid female with a male carrying asch (sex-linked chocolate) gene on its Z chromosomes. Since thesch gene expresses the color of reddish brown (chocolate) in newly hatched larvae, the females with ZWW chromosomes become chocolate, while those with ZZW have normal color (black). The effects of the W chromosome on oogenesis were studied by comparing these two types of 3n females. Despite the fact that the theoretical value of the ratio of chocolate larvae to black larvae should be 1:5, the actual ratio was found to be 1:80. The chocolate larvae revealed very low hatchability. Thirteen adult ZWW female moths were obtained, all laying normal-shaped eggs and even larger than those of 4n females which also have two W chromosomes. Eighty percent of the eggs of ZZW 3n females were irregular-shaped, and the size of the normal-shaped eggs was as large as that of 2n females which possess one W chromosome. It is hypothesized that egg size is determined by a gene on the W chromosome. The higher frequency of normal-shaped eggs in the ZWW female group than the ZZW female group suggests the presence of another kind of gene on the W chromosome, which leads to normal oogenesis.