MicroRNA Alternations in the Testes Related to the Sterility of Triploid Fish

The knowledge of understanding the molecular traits of the sterile triploid fish is sparse. Herein, we analyzed the microRNA (miRNA) alternations in the testes of the sterile triploid fish produced by crossing the tetraploid fish with the diploid fish, compared with those of tetraploids and diploids used as the controls. A total of 136, 134, and 142 conserved miRNAs and 105, 112, and 119 novel miRNAs were identified in the diploid, triploid, and tetraploid fish, respectively. The genes targeted by the differentially expressed miRNAs were identified and were enriched in the GO term cell surface receptor signaling pathway, cellular process, G-protein coupled receptor signaling pathway, and metabolic process. KEGG pathway enrichment was also assessed to evaluate the target genes with differentially expressed miRNAs and these genes were enriched in four pathways (synthesis and degradation of ketone bodies, pentose and glucuronate interconversions, cyanoamino acid metabolic process, and ascorbate and aldarate metabolism). Nine differentially expressed miRNAs were verified by quantitative real-time PCR analysis (qPCR). The upregulated miRNAs in triploids, including miR-101a, miR-199-5p, miR-214, miR-222, and miR-193a, showed the same results with high-throughput sequencing. Among the selected downregulated miRNAs, miR-7b and miR-153b had significantly lower expression levels in triploids. Dnah3 and Tekt1 genes targeted by miR-199-5p showed lower expression in triploids by qPCR. These verified differentially expressed miRNAs may participate in testicular development and sperm activity by targeting functional genes, which were identified with differential expression in the triploid. This evidence provides insights into the epigenetic regulatory mechanisms of sterility in triploid cyprinids.     

Global analysis of the small RNA transcriptome in different ploidies and genomic combinations of a vertebrate complex--the Squalius alburnoides

The Squalius alburnoides complex (Steindachner) is one of the most intricate hybrid polyploid systems known in vertebrates. In this complex, the constant switch of the genome composition in consecutive generations, very frequently involving a change on the ploidy level, promotes repetitive situations of potential genomic shock. Previously in this complex, it was showed that in response to the increase in genome dosage, triploids hybrids could regulate gene expression to a diploid state. In this work we compared the small RNA profiles in the different genomic compositions interacting in the complex in order to explore the miRNA involvement in gene expression regulation of triploids. Using high-throughput arrays and sequencing technologies we were able to verify that diploid and triploid hybrids shared most of their sequences and their miRNA expression profiles were high correlated. However, an overall view indicates an up-regulation of several miRNAs in triploids and a global miRNA expression in triploids higher than the predicted from an additive model. Those results point to a participation of miRNAs in the cellular functional stability needed when the ploidy change.     

The role of tetraploids in the sexual-asexual cycle in dandelions (Taraxacum)

Apomictic plants often produce pollen that can function in crosses with related sexuals. Moreover, facultative apomicts can produce some sexual offspring. In dandelions, Taraxacum, a sexual-asexual cycle between diploid sexuals and triploid apomicts, has been described, based on experimental crosses and population genetic studies. Little is known about the actual hybridization processes in nature. We therefore studied the sexual-asexual cycle in a mixed dandelion population in the Netherlands. In this population, the frequencies of sexual diploids and triploids were 0.31 and 0.68, respectively. In addition, less than 1% tetraploids were detected. Diploids were strict sexuals, triploids were obligate apomicts, but tetraploids were most often only partly apomictic, lacking certain elements of apomixis. Tetraploid seed fertility in the field was significantly lower than that of apomictic triploids. Field-pollinated sexual diploids produced on average less than 2% polyploid offspring, implying that the effect of hybridization in the 2x-3x cycle in Taraxacum will be low. Until now, 2x-3x crosses were assumed to be the main pathway of new formation of triploid apomicts in the sexual-asexual cycle in Taraxacum. However, tetraploid pollen donors produced 28 times more triploid offspring in experimental crosses with diploid sexuals than triploid pollen donors. Rare tetraploids may therefore act as an important bridge in the formation of new triploid apomicts.     

Hematologic and enzymatic analysis of Ctenopharyngodon idella x Hypophthalmichthys nobilis F1 hybrids

1. Erythrocyte counts, hemoglobin concentrations and hematocrit values were determined for diploid and triploid Ctenopharyngodon idella X Hypophthalmichthys nobilis hybrids and the parental species. 2. Comparisons of diploid and triploid hybrids with the parental species revealed low erythrocyte counts for triploids, high mean corpuscular hemoglobin values for triploids, elevated hematocrits for diploids and triploids and similar hemoglobin concentrations for all fish. 3. Alkaline phosphatase, aldolase, and lactate dehydrogenase specific activities were determined spectrophotometrically. Levels of specific activity of these enzymes in the hybrids were consistently elevated above that of the parental species. These higher levels of enzyme activities in hybrids were probably the result of a breakdown in gene regulation.     

Genome relationships in the diploid oats

Genome relationships between the three diploid oats, Avena strigosa (S.), A. longiglumis (L.) and A. prostrata (P.) were studied by chromosome pairing in diploid hybrids and in synthetic triploids and tetraploids combining these genomes. Fairly regular pairing in the diploid hybrid and typical autopolyploid behavior in the triploids and in the amphidiploid suggest small differentiation in the chromosome architecture of A. longiglumis and A. prostrata. A. strigosa diverges from the other two oats by complex chromosome rearrangements. Conspicuous preferential pairing took place in triploids with SSL, SSP and SPP genomic constitution. The low bivalent frequency in the SLL triploid suggests that preferential pairing in triploids with two S genomes is not a consequence of chromosome rearrangement but is rather of genetic origin. The presence of the three genomes in a triploid or a tetraploid caused considerable meiotic irregularities suggesting a better pairing competition of the S genome.     

MicroRNA expression changes following synthesis of three full-sib <Emphasis Type="Italic">Populus</Emphasis> triploid populations with different heterozygosities

Key message

Through high-throughput sequencing, we compared the relative expression levels of miRNA in three full-sib Populus triploid populations with that in their parents and one diploid hybrid population. We found similar numbers of miRNAs differentially expressed between the parents and the four progeny hybrid populations. In addition, unbalanced parental expression level dominance of miRNAs were found in the three allotriploid and interspecific hybrid populations, which may reprogram gene expression networks and contribute to the growth of Populus hybrids. These results indicated that hybridization has a great impact on the miRNA expression variation in the newly synthesized Populus triploid and diploid hybrid populations. However, we also found no significant differences in miRNA expression among one diploid and three triploid hybrid populations, hinting that miRNA abundances do not increase with the genome content. No dosage effect of miRNA expression could lead to dosage-dependent negative effects on target genes and their downstream pathway in polyploids. We speculate that polyploids may gain advantages from the slight decrease in miRNA regulation, suggesting an important molecular mechanism of polyploid advantage.

Abstract

Hybridization with three types of induced 2n gametes transmitted different parental heterozygosities has been proven as an efficient method for Populus triploid production. Several researches have shown that miRNA could be non-additively expressed in allopolyploids. However, it is still unclear whether the non-additively expressed miRNAs result from the effect of hybridization or polyploidization, and whether a dose response to the additional genomic content exists for the expression of miRNA. Toward this end, through high-throughput sequencing, we compared the expression levels of miRNA in three full-sib Populus triploid populations with that in their parents and one interspecific hybrid population. We found similar numbers of miRNAs differentially expressed between the parents and the four progeny hybrid populations. Unbalanced parental expression level dominance of miRNAs were found in the three triploid and diploid hybrid populations, which may reprogram gene expression networks and affect the growth of Populus hybrids. These results indicated that hybridization has a great impact on the miRNA expression variation in the newly synthesized Populus triploid and diploid hybrid populations. However, we also found no significant differences in miRNA expression among the three triploid populations and the diploid hybrid population. No dosage effect of miRNA expression could lead to dosage-dependent negative effects on target genes and their downstream pathway in polyploids. We speculate that polyploids may gain advantages from the decrease in miRNA negative regulation, suggesting an important molecular mechanism of polyploid advantage.

     

Influence of environmental parameters on growth pattern and population structure of Carassius auratus gibelio in Eastern Ukraine

Population structure and growth parameters of Prussian carp (Carassius auratus gibelio) were studied in 12 freshwater ecosystems of the Donbass region (Eastern Ukraine). These ecosystems differed significantly with respect to their surface area, water transparency and annual concentrations of phosphorus and nitrogen. Amongst the studied ecosystems, diploid males and females as well as a smaller percentage of triploid females were found. The population structure of C. a. gibelio varied significantly in terms of the percentage of triploids and sex ratio amongst diploids. A considerable proportion of triploid females (>10%) was found in four ecosystems with intermediate surface area (38–50 ha) and relatively high growth rate of specimens. The sex ratio amongst diploids was significantly female-biased in seven of 12 ecosystems, including those where triploids were present in considerable numbers. The growth parameters of Prussian carp were significantly correlated with ecosystem characteristics, but the growth patterns of diploids and triploids were not significantly different from each other. The restricted distribution of triploid specimens suggests that the triploid form might counter diverse environmental challenges, whereas the diploid form of Prussian carp seems to be efficiently adapted to a wide range of ecosystem conditions.     

黄姑鱼三倍体的诱导、鉴定及其性腺发育特征

研究采用冷休克方法诱导黄姑鱼(Nibea albiflora)三倍体并进行鉴定,进一步采用组织学和生殖相关基因表达分析比较了三倍体黄姑鱼的性腺发育特征。研究结果表明:(1)在受精后2.5min以3℃进行10min的冷休克处理,冷休克处理组的受精率和孵化率分别为(70.31±4.49)%和(21.5±6.63)%,其受精率和孵化率显著低于二倍体对照组;(2)经流式细胞仪倍性检测和染色体核型分析发现,三倍体的DNA含量为二倍体的1.5倍,染色体数目为72条,而二倍体的染色体数目为48条,三倍体的比例为100%;(3)三倍体的生殖腺指数显著低于二倍体,进一步通过组织切片观察发现三倍体的精巢和卵巢发育较二倍体滞后,在12月龄时,二倍体精巢和卵巢处于Ⅴ期,而三倍体精巢和卵巢分别处于Ⅲ期和Ⅰ期;(4)三倍体精巢的dmrt1和vasa基因及三倍体卵巢的cyp19a基因表达量均显著低于二倍体(P<0.05);三倍体卵巢的vasa基因表达量也比二倍体低(P>0.05)。综上结果表明:研究通过冷休克处理成功诱导了黄姑鱼三倍体;三倍体的性腺发育较二倍体滞后,育性明显降低。     

A hypothesis on the origin of genetic heterozygosity in diploids and triploids in Japanese Cayratia japonica species complex (Vitaceae)

We previously reported the occurrence of triploid strains in Japanese populations of Cayratia japonica (Thunb.) Gagnep. Interestingly, the triploid and most diploid strains had variably reduced pollen fertility. Two questions emerged from this earlier work: (1) How do triploids arise, and are they allotriploids or autotriploids? and (2) Why is there low pollen fertility in some diploid plants? We used a molecular genetic approach to determine the phylogenetic origins of triploids in C. japonica and the closely related species Cayratia tenuifolia (Wight & Arn.) Gagnep. In our analysis, we compared the sequences of the nuclear single-copy genes LEAFY and ASYMMETRIC LEAVES1. As a result, most triploids and diploids were heterozygous for the loci examined; the triploid genome shared an allele with the diploid genome, but other alleles differed between the ploidies. Therefore, Japanese populations of C. japonica and C. tenuifolia almost certainly arose from repeated hybridization events among genetically differentiated strains. Using our sequence data, we discuss possible scenarios accounting for the occurrence of triploids in the two species of Cayratia.     

四类不同倍性鲫鱼在胚胎发育期间同工酶基因表达的比较分析

Isozyme zymograms of esterase (EST), lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and superoxide dismutase (SOD) were analysed by polyacrylamide gradient gel electrophoresis at different developmental stages of embryogenesis in 4 types of various ploidy crucian carp embryos, including haploids, diploids, natural triploids, and multiple tetraploids, and 2 types of haploid and diploid common carp embryos. Haploid embryos of crucian carp (Carassius auratus) and common carp (Cyprinus carpio) were produced by treating eggs with UV-irradiated milt from blunt snout bream (Megalobrama amblycephala). Natural triploid embryos were obtained from the eggs of gynogenetic silver crucian carp (Carassius auratus gibelio) inseminated with milt from red common carp. Multiple tetraploid embryos were also produced by gynogenesis from eggs of the newly discovered multiple tetraploid females inseminated with milt from red common carp. Gradient gel electrophoresis indicated that the band types and staining intensity of 4 isozymes expressed in haploid embryos of crucian carp and red common carp were similar to that in the correlative diploid embryos. In natural triploid silver crucian carp embryos, the zymograms of MDH and SOD isozymes were identical with that of diploid crucian carp embryos, but the EST and LDH isozymes manifested more new enzyme bands in comparison with diploid embryos. The corresponding expressed products of some bands in the triploid embryos, such as EST5 and EST6, could be observed also in red common carp embryos, which provided evidence for hybrid origin about the gynogenetic fish. The multiple tetraploids incorporated one foreign genome of red common carp, therefore, the effects of genes from the foreign genome could be observed in the multiple tetraploid embryos. Gene expression of the isozymes in the tetraploid embryos was somewhat similar to that in hybrids. Owing to interaction of triploid silver crucian carp genomes and common carp haploid genome, some isozyme bands, such as EST5 and EST6, changed in quantity, and some bands increased, such as s-SOD1, s-SOD2, s-SOD3 and s-SOD4 in the tetraploid embryos. Moreover, the heterogeneity was revealed among embryos developed from gynogenetic eggs of 3 different multiple tetraploid individuals.     

Why are triploid zebrafish all male?

Adult triploid zebrafish Danio rerio has previously been reported to be all male. This phenomenon has only been reported in one other gonochoristic fish species, the rosy bitterling Rhodeus ocellatus, despite the fact that triploidy is induced in numerous species. To investigate the mechanism responsible, we first produced triploid zebrafish and observed gonad development. Histological sections of juvenile triploid gonads showed that primary growth oocytes were able to develop in the juvenile ovary, but no cortical alveolus or more advanced oocytes were found. All adult triploids examined were male (n = 160). Male triploids were able to induce oviposition by diploid females during natural spawning trials, but fertilization rates were low (1.0 ± 3.1%) compared with diploid male siblings (67.4 ± 16.6%). The embryos produced by triploid sires were aneuploid with a mean ploidy of 2.4 ± 0.1n, demonstrating that triploid males produce aneuploid spermatozoa. After confirming that adult triploids are all male, we produced an additional batch of triploid zebrafish and exposed them (and a group of diploid siblings) to 100 ng/L estradiol (E2) from 5 to 28 dpf. The E2 treated triploids and nontreated triploids were all male. The nontreated diploids were also all male, but the E2 treated diploids were 89% female. This demonstrates that triploidy acts downstream of estrogen synthesis in the sex differentiation pathway to induce male development. Based on this and the observations of juvenile gonad development in triploids, we suggest that triploidy inhibits development of oocytes past the primary growth stage, and this causes female to male sex reversal.     

Surface feeding and aggressive behaviour of diploid and triploid brown trout Salmo trutta during allopatric pair‐wise matchings

Diploid and triploid brown trout Salmo trutta were acclimated for 6 weeks on two feeding regimes (floating and sinking). Thereafter, aggression and surface feeding response were compared between pairs of all diploid, all triploid and diploid and triploid S. trutta in an experimental stream. In each pair‐wise matching, fish of similar size were placed in allopatry and rank was determined by the total number of aggressive interactions recorded. Dominant individuals initiated more aggression than subordinates, spent more time defending a territory and positioned themselves closer to the surface food source (Gammarus pulex), whereas subordinates occupied the peripheries. In cross ploidy trials, diploid S. trutta were more aggressive than triploid, and dominated their sibling when placed in pair‐wise matchings. Surface feeding, however, did not differ statistically between ploidy irrespective of feeding regime. Triploids adopted a sneak feeding strategy while diploids expended more time defending a territory. In addition, we also tested whether triploids exhibit a similar social dominance to diploids when placed in allopatry. Although aggression was lower in triploid pairs than in the diploid and triploid pairs, a dominance hierarchy was also observed between individuals of the same ploidy. Dominant triploid fish were more aggressive and consumed more feed items than subordinate individuals. Subordinate fish displayed a darker colour index than dominant fish suggesting increased stress levels. Dominant triploid fish, however, appeared to be more tolerant of subordinate individuals and did not display the same degree of invasive aggression as seen in the diploid and diploid or diploid and triploid matchings. These novel findings suggest that sterile triploid S. trutta feed similarly but are less aggressive than diploid trout. Future studies should determine the habitat choice of triploid S. trutta after release and the interaction between wild fish and triploids during the breeding season prior to utilization of triploids as an alternative management strategy within freshwater fisheries.     

Sexual maturation in triploid rainbow trout, Salmo gairdneri Richardson

This paper compares some morphological and endocrinological characteristics of diploid and triploid rainbow trout.
Significant differences were found between diploid and triploid females in GSI, condition factor, gut weight, liver weight and percentage dress-out, and between diploid and triploid males in GSI, condition factor and gut weight.
Diploid females had large, well-developed ovaries containing yolk-filled secondary oocytes whereas the triploids had only string-like ovaries containing nests of oogonia. No primary oocytes were present.
All the diploid males produced copious quantities of milt but it was possible to express a thin, watery milt containing motile spermatozoa from only two of the 12 triploid males. Testes weights in triploids were similar to those of diploids but, while the diploid testes were packed with spermatozoa, those of the triploids consisted mainly of spermatocytes and spermatids with few spermatozoa present. Measurements of the heads of spermatozoa revealed that those from triploids were larger and had a wider size range than those from diploids.
Levels of testosterone and 11-ketotestosterone in triploid and diploid males were not significantly different. However, levels of testosterone and 17β-oestradiol in diploid females were considerably higher than those of triploid females.     

四类不同倍性鲫鱼在胚胎发育期间同工酶基因表达的比较分析

用聚丙烯酰胺梯度凝胶电泳比较分析了单倍体、二倍体、三倍体和复合四倍体4类不同倍性鲫鱼以及单倍体和二倍体鲤鱼在胚胎发育时期4种同工酶(EST,LDH,MDH,SOD)酶谱。结果表明,单倍体鲫鱼和单倍体鲤鱼胚胎与各自的二倍体胚胎相比,同工酶酶谱看不出差异;天然三倍体银鲫胚胎的MDH和SOD同工酶酶谱与二倍体鲫相似,但EST和LDH同工酶比二倍体增多了酶带,有的酶带如EST5和EST6还可在鲤鱼胚胎中找到相应的表达产物,提供了天然雌核发育三倍体银鲫杂交起源的证据;复合四倍体由于含有鲤鱼的一个外来基因组,其胚胎的基因表达有些与杂种类似,在所分析的4种同工酶酶谱中,都可观察到来自鲤鱼基因的影响。此外,在由源于不同复合四倍体个体的卵子发育形成的胚胎间,还观察到同工酶基因表达的异质性。     

A cryptic clonal line of the loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) evidenced by induced gynogenesis,interspecific hybridization,microsatellite genotyping and multilocus DNA fingerprinting

In Memanbetsu town, Hokkaido island, Japan, a high frequency of natural triploid loaches Misgurnus anguillicaudatus (7.4% on average) was detected by flow cytometry for relative DNA content. Among sympatric diploid females (n=6) from a single population, we found two unique females that laid unreduced diploid eggs. They gave normal diploid progeny even after induction of gynogenesis with genetically inert UV-irradiated sperm. When fertilized with normal loach sperm, some unreduced eggs developed into triploids, but the rest into diploids. Hybridization using goldfish Carassius auratus sperm gave both normal diploid loaches and inviable allotriploid hybrids possessing the diploid loach genome and the haploid goldfish genome. Microsatellite genotyping and DNA fingerprinting demonstrated that the diploid progeny developing from the unreduced eggs were genetically identical to the mother, while the triploids had some of the paternal DNA. These results indicate that the diploid eggs reproduced unisexually as a diploid clone and in other cases developed into triploids after accidental incorporation of sperm nucleus. The presence of at least one clonal line in this area was shown by the identical DNA fingerprint detected in five out of 17 diploid loaches examined.     

Identification of triploid individuals and clonal lines in Carassius auratus complex using microsatellites

The Carassius auratus complex in natural populations includes diploid triploid and polyploidy individuals. Diploid individuals belong to the species Carassius auratus whereas triploid and polyploidy individuals are from the subspecies Carassius auratus gibelio. Triploid individuals are all female and reproduce clonally by gynogenesis. Therefore the Carassius auratus complex is an ideal system for studying evolution of unisexual reproduction. Identification of triploid individuals and clonal lines is the first step towards understanding of the evolution of unisexual clonal lines. We examined the ability of 10 microsatellites in identifying triploid individuals in 94 individuals from Japan and China. In 40 confirmed triploid individuals and eight confirmed diploid individuals, all triploid and diploid individuals can be identified by genotyping 10 microsatellite, and four triploid clonal lines were identified. Using the 10 microsatellites we genotyped 46 adult individuals (40 females and six males) from a natural population in China and found that all six males were diploid whereas the majority of females (36 of 40) were triploid and three triploid clonal lines were detected. In 18 diploid individuals from China, all individuals showed different genotypes, suggesting there is no diploid clonal line in diploid crucian carp. A phylogenetic analysis of 94 individuals from China and Japan showed that triploid individuals and clonal lines have originated recurrently.     

二倍体和三倍体太平洋牡蛎鳃扫描电镜的比较

利用扫描电子显微镜技术对二倍体和三倍体太平洋牡蛎(Crassostrea gigas)鳃的表面结构进行了观察和比较。结果显示：三倍体牡蛎鳃丝的宽度、鳃丝间的距离较二倍体大;鳃丝的微细结构比二倍体更致密;鳃丝间通过丝问连接形成的孔洞大于二倍体。这些不同表明二倍体和三倍体呼吸及摄食可能存在差异。     

Comparative studies on histological and ultra-structure of the pituitary of different ploidy level fishes

The histological and ultra-structure of the pituitary in diploid red crucian carp(Carassius auratus red var.),triploid crucian carp and allotetraploid hybrids within and after the breeding season were comparatively studied.The result showed that there were six endocrine cell types in the pituitary of these three kinds of fishes,and there was an obvious difference in cell size among different ploidy level fishes.As for the same type of pituitary cells,the cell size was increased gradually with the in- creasing ploidy level.In the breeding season,the allotetraploid hybrids had higher proportion of go- nadotropin cells(GTH)than triploids,and the triploids had higher proportion of GTH than diploids.The results were related to the earlier sexual maturity of allotetraploid hybrids and sterility of triploid cru- cian carp.On the other hand,among the three kinds of fishes,the proportion of somatotropin(STH) cells in triploids crucian carp was the highest,whereas that in allotetraploid hybrids was the lowest. The results might be connected with the faster growth rate of triploids and slower growth rate of al- lotetraploid hybrids.In addition,in GTH cells of meso-adenohypophysis after the breeding season, there were many endocrine particles in triploids,while those endocrine particles were released from the cells in allotetraploids and diploids.This result showed that the sterility of triploid crucian carp might be related to the hormone which was not released from the GTH cells.In a word,the present study indicated that the differences in the structure of pituitary among different ploidy level fishes contributed to their difference in the growth rate and gonadal development.     

Delayed meiosis and polar body release in eggs of triploid Pacific oysters, Crassostrea gigas, in relation to tetraploid production

The dynamics of polar body release are important for creating polyploid shellfish. For producing triploids, these dynamics concern meiosis in diploid eggs and are well understood. For creating tetraploids, eggs from triploids are employed and the dynamics, variation, and environmental influences upon polar body release are less studied. We investigated the effects of several agents on the timing of 50% first polar body (PB1) release in eggs of triploids. PB1 release is generally slower in triploid eggs than diploid ones at 26 degrees C. Lowering the temperature (from 26 to 19 degrees C) had a marked effect on timing of 50% PB1 in both diploid and triploid eggs. While lower temperature merely slowed development in diploid eggs, it nearly halted it in triploid eggs. At any temperature, the variability in 50% PB1 release was much higher in triploid eggs than diploid ones; this variation occurred both within eggs from individual females and among eggs from different females. The amount of time eggs remain in seawater between the time they are stripped and fertilized (or time of hydration) also affected rate of meiosis. In triploid eggs, the average time necessary for the expulsion of 50% PB1 was 23 min post-fertilization (PF) for 75 min of hydration versus 29 min PF for 35 min. However, increasing the time of hydration had no effect on the variability in the timing among females. Serotonin also had no effect on the dynamics of polar body release in triploids. Variability among triploid females in timing of meiosis cannot be improved with any treatments we tried. Consequently we recommend that treatments of triploid eggs to produce tetraploids incorporate a single female at a time.