Induced androgenesis in fish: obtaining viable nucleocytoplasmic hybrids

A self-review of long-standing studies of induced androgenesis in fish. The data are provided on the induction of diploid androgenesis in teleost fish via blocking the first cleavage division and the development of the method of dispermic androgenesis in sturgeon fish. Androgenetic nucleocytoplasmic hybrids were obtained using the method of dispermic androgenesis and eggs of previously produced true interspecific hybrids. Specific morphological features of androgenetic hybrids were described and dispermic androgenesis was realized using cryopreserved sperm. The mechanism of sex determination in sturgeon fish is discussed with reference to possible us of androgenesis for restoration of rare and disappearing species.     

Dispermic androgenesis in sturgeons with the help of cryopreserved sperm: production of androgenetic hybrids between Siberian and Russian sturgeons

Dispermic androgenesis was used to produce, for the first time, an androgenetic progeny of the Siberian sturgeon (Acipenser baerii) and the androgenetic nuclear cytoplasmic hybrids (Siberian sturgeon, A. baerii x Russian sturgeon, A. gueldenstaedtii) using cryopreserved sperm. Microsatellite DNA analysis confirmed exclusively paternal inheritance in the androgenetic progeny of Siberian sturgeon. Heterozygotes for certain microsatellite loci were detected among the androgenetic hybrids, thereby confirming a dispermic nature of androgenesis. According to the data of comparative morphological analysis, the obtained androgenetic hybrid, by the age of 15 months old, was completely identical to the paternal species. Both a female and a male were detected in the androgenetic sturgeon progenies, which is of interest for producing bisexual progenies via androgenesis. The data of this study confirm the feasibility of dispermic androgenesis using cryopreserved sperm to preserve and recover the gene pools of endangered sturgeon species.     

Dispermic androgenesis in sturgeons with the use of cryopreserved sperm: Production of androgenetic siberian sturgeon and androgenetic hybrids between Siberian and Russian sturgeons

Dispermic androgenesis was used to produce, for the first time, an androgenetic progeny of the Siberian sturgeon (Acipenser baerii) and the androgenetic nucleo-cytoplasmic hybrids (Siberian sturgeon, A. baerii × Russian sturgeon, A. gueldenstaedtii) using cryopreserved sperm. Microsatellite DNA analysis confirmed exclusively paternal inheritance in the androgenetic progeny of Siberian sturgeon. Heterozygotes for certain microsatellite loci were detected among the androgenetic hybrids, thereby confirming a dispermic nature of androgenesis. According to the data of comparative morphological analysis, the obtained androgenetic hybrid, by the age of 15 months old, was completely identical to the paternal species. Both a female and a male were detected in the androgenetic sturgeon progenies, which is of interest for producing bisexual progenies via androgenesis. The data of this study confirm the feasibility of dispermic androgenesis using cryopreserved sperm to preserve and recover the gene pools of endangered sturgeon species.     

Nucleocytoplasmic Incompatibility in Androgenetic Fish Hybrids Can Be Overcome

Many androgenetic fish hybrids are nonviable due to the disturbed interaction between the foreign nucleus and the cytoplasm. It has been proposed that this incompatibility can be overcome if eggs from interspecific hybrids and sperm from one of the parental species are used for diploid androgenesis. Indeed, the androgenetic progeny obtained as a result of the insemination of the enucleated eggs of goldfish and carp hybrids by the carp spermatozoa proved to be viable and fertile. This approach may be used for reconstitution of the genotypes of rare or disappearing fish species with the help of induced androgenesis, if their native or cryoconserved sperm is available.     

Distant hybridization leads to different ploidy fishes

Distant hybridization makes it possible to transfer the genome of one species to another, which results in changes in phenotypes and genotypes of the progenies. This study shows that distant hybridization or the combination of this method with gynogenesis or androgenesis lead to different ploidy fishes with genetic variation, including fertile tetraploid hybrids, sterile triploid hybrids, fertile diploid hybrids, fertile diploid gynogenetic fish, and their derived progenies. The formations of the different ploidy fishes depend on the genetic relationship between the parents. In this study, several types of distant hybridization, including red crucian carp (Carassius auratus red var.) (2n=100, abbreviated as RCC) (♀)×common carp (Cyprinus carpio L.) (2n=100, abbreviated as CC) (♂), and RCC (2n=100) (♀)×blunt snout bream (Megalobrama amblycephala) (2n=48, abbreviated as BSB) (♂) are described. In the distant hybridization of RCC (♀)×CC (♂), bisexual fertile F3–F18 allotetraploid hybrids (4n=200, abbreviated as 4nAT) were formed. The diploid hybrid eggs and diploid sperm generated by the females and males of 4nAT developed into diploid gynogenetic hybrids and diploid androgenetic hybrids, respectively, by gynogenesis and androgenesis, without treatment for doubling the chromosome. Improved tetraploid hybrids and improved diploid fishes with genetic variation were derived from the gynogenetic hybrid line. The improved diploid fishes included the high-body RCC and high-body goldfish. The formation of the tetraploid hybrids was related to the occurrence of unreduced gametes generated from the diploid hybrids, which involved in premeiotic endoreduplication, endomitosis, or fusion of germ cells. The sterile triploid hybrids (3n=150) were produced on a large scale by crossing the males of tetraploid hybrids with females of diploid fish (2n=100). In another distant hybridization of RCC (♀)×BSB (♂), different ploidy fishes were obtained, including diploid bisexual fertile natural gynogenetic fish (2n=100), sterile triploid hybrids (3n=124), and bisexual fertile tetraploid hybrids (4n=148). Furthermore, two kinds of pentaploid hybrids (5n=172 and 5n=198) were formed. The biological characteristics and the mechanisms of formation of the different ploidy fish were compared and discussed at the cellular and molecular level. The results indicated distant hybridization or the combination of this method with gynogenesis or androgenesis affects the formation of different ploidy fish with genetic variation.     

鱼类雄核发育的研究进展

雄核发育是在雄核控制下发育的一种特殊的有性生殖方式,它是鱼类单性发育的重要组成部分。本文全面综述了国内外鱼类雄核发育研究的进展状况。主要包括雌核染色体灭活和雄核二倍化诱导技术。雌核染色体灭活主要通过γ、X和紫外线(UV)等辐射处理完成的,其辐射剂量和方法因研究者不同而不同。雄核的二倍化诱导则主要是采用静水压和温度休克阻止第一次有丝分裂来实现的,其二倍化诱导条件与研究的种类不同而有所差异。同时,本文对鱼类雄核发育在遗传育种中的应用前景作了评价。 Abstract：Fish andtogenesis is a kind of secial sexual development which is controlled by male nucleus and it is an important part of fish unisexual development. This paper summarizes the progress of fish androgenesis comprehensively in the world.It includes the techniques of inactive female nucleus of eggs and diploidization of male nucleus. The methods of inactive female nucleus are carride out by using γ,X and ultraviolet (UV)rays to irradiate the eggs,the irradiation dosages and methods are different based on the different researchers. The techniques of diploidization of male nucleus are carried out by using hydrostatic pressure, heat and cold shock to restrain the fish mitosis,the diploidization condition are different from species to species.Meanwhile,the paper evaluates the application perspectives of fish androgenesis on the genetics and breeding.     

Failure of interspecies androgenesis in salmonids

Androgenetic development of salmonid embryos was induced in recipient oocytes from the same or other species (intra- or interspecies androgenesis). Parameters for induced androgenesis were investigated in brown trout Salmo trutta and brook trout Salvelinus fontinalis . Reciprocal androgenetic and control crosses were conducted among fishes from three genera: Oncorhynchus (rainbow trout, O. mykiss ), Salmo (brown trout) and Salvelinus (brook trout), and within two genera: Salmo (brown trout and Atlantic salmon, S. salar ) and Salvelinus (brook trout and Arctic charr, S. alpinus ). Live hatched androgenetic progenies were obtained in all intraspecies variants, where oocytes and sperm originated from the same species. Interspecies androgenesis resulted in no viable larvae, despite the fact that most hybrid controls and intraspecies androgenetic individuals were viable. When recipient oocytes originated from other genera (interspecific intergeneric androgenesis), embryonic development ceased in early developmental stages, except for haploid controls of brook trout produced in eggs of brown trout. Survival of embryos to the eyed-egg stage was relatively high in the intrageneric androgenesis experiment. Nevertheless, none of these embryos survived to hatching. Some of the presumed Atlantic salmon individuals developing in brown trout eggs contained maternal DNA, questioning the accuracy of enucleation using irradiation. The inability to induce interspecific androgenesis among the examined salmonid species may have been the result of substantial kariotypical and developmental differences between spermatozoal donors and oocyte recipients, causing an incompatibility between maternal cytoplasmic regulatory factors and the paternal nuclear genome.     

Comparative characterization of the ultrastructure of intestinal epithelium of various sturgeon species

The ultrastructure of the mid-intestine of four sturgeon species and their hybrids was studied. Apart from enterocytes and glandular goblet cells, the cells with microvilli, endocrine cells, and immunocompetent elements were revealed. Specific cells responsible for the primary immune response, analogous to mammalian M-cells, were discovered in sturgeon fishes. The ultrastructural characteristics common for the mid-intestinal epithelium of all the sturgeon fishes studied were determined. Species-specific features of intestinal epithelium of the parent species, which were characteristic of the hybrids, were revealed.     

Use of heterologous sperm for the dispermic induction of androgenesis in barbs

Dispermic activation of genome‐inactivated eggs of the grey tiger barb Puntius tetrazona , facilitated by 2·5% polyethylene glycol (PEG)‐incubated sperm of the golden rosy barb Puntius conchonius , resulted in the generation of interspecific androgenetic clones of the golden rosy barb. A 10 min incubation of the golden rosy barb sperm in increasing concentrations (1·0, 1·5, 2·0, 2·5 and 3·0%) reduced the frequency of motile sperm (to 70%), motility duration (110–50 s) and fertilizability (to 80%) of the sperm; however, the frequency of sperm with double head size increased. At 3% PEG, motility pattern of the sperm completely changed from 'zig‐zag' to 'circular'. Incubation in 2·5% PEG facilitated the dispermic entry and production of diploid androgenetic female and male progenies at the ratio of 0·27 : 0·73. Polymerase chain reaction (PCR) analysis using Tc1 transposon specific primers confirmed the purity of paternal inheritance of P. conchonius through the surrogate eggs of P. tetrazona . Survival and breeding, body colour, diploidy (karyotyping and erythrocyte measurements) and 0·27F : 0·73M sex ratio of the androgenotes provided evidence for the successful induction of dispermic androgenesis. Despite increased heterozygosity and reduced 'damage cost' on restoration of diploidy, survival of dispermic androgenotes induced in heterologous eggs was lower (1·7%) than those reported for androgenetic golden rosy barbs induced using homologous sperm (14·0%) and heterologous eggs (7·0%), i.e . tiger barb eggs.     

The formation of improved tetraploid population of red crucian carp × common carp hybrids by androgenesis

Bisexual fertile diploid androgenetic individuals (A₀) (2n=100) were formed by androgenesis. In this way, the diploid spermatozoa from male allotetraploid hybrids (AT) (4n=200) of red crucian carp (Carassius auratus red var.) (♀) × common carp (Cyprinus carpio L.) (♂) were used to fertilize the UV-treated haploid eggs of goldfish (Carassius auratus), and living androgenetic diploid fish were developed. The A₀ became sexually mature at the age of 2 years, and they fertilized with each other to form their offspring (A₁). In this study, we observed the chromosomal number, gonadal structure and appearance of A₁ fish. The results are as follows: (1) In A₁, there were 85% tetraploids (A₁-4n), 10% triploids (A₁-3n) and 5% diploids (A₁-2n), suggesting that diploid A₀ could produce diploid gametes. It was concluded that the formation of diploid gametes generated from diploid A₀ was probably related to the mechanism of pre-meiotic endoreduplication. (2) Among A₁, only A₁-4n possessed normal ovaries and testes. The mature males of A1-4n produced white semen. Under the electron microscope, the head of diploid sperm generated by A₁-4n was bigger than that of haploid sperm generated by red crucian carp. In the testes of the A₁-4n, there were many mature normal spermatozoa with a head bearing plasma membrane and a tail having the typical structure of “9+2” microtubules. Between the head and the tail, there were some mitochondria. The ovaries of A₁-4n developed well and mainly contained II, III and IV-stage oocytes. The IV-stage oocytes were surrounded by inner and outer follicular cells. The micropyle was observed on the oolemma of follicular cells. There were abundant yolks and plenty of endoplasmic reticulum in the cytoplasm of IV-stage oocytes. Because A₁-2n and A₁-3n were distant crossing diploid hybrids and triploid hybrids respectively, they possessed abnormal gonads, and no mature semen and eggs were observed. (3) Compared with allotetraploids, the A₁-4n fish not only had advantages such as fast growth rate and strong resistibility but also showed some new good performances such as high ratio of body width to body length, smaller heads and shorter tails. These results indicated that androgenesis could produce bisexual fertile tetraploids and improve the shape of allotetraploid hybrids as well, which will be of great significance in both the cell genetics research and fish breeding.     

The formation of improved tetraploid population of red crucian carp × common carp hybrids by androgenesis

Bisexual fertile diploid androgenetic individuals (A0) (2n=100) were formed by androgenesis. In this way, the diploid spermatozoa from male allotetraploid hybrids (AT) (4n=200) of red crucian carp (Carassius auratus red var.) (♀) × common carp (Cyprinus carpio L.) (♂) were used to fertilize the UV-treated hap- loid eggs of goldfish (Carassius auratus), and living androgenetic diploid fish were developed. The A0 became sexually mature at the age of 2 years, and they fertilized with each other to form their offspring (A1). In this study, we observed the chromosomal number, gonadal structure and appearance of A1 fish. The results are as follows: (1) In A1, there were 85% tetraploids (A1-4n), 10% triploids (A1-3n) and 5% diploids (A1-2n), suggesting that diploid A0 could produce diploid gametes. It was concluded that the formation of diploid gametes generated from diploid A0 was probably related to the mechanism of pre-meiotic endoreduplication. (2) Among A1, only A1-4n possessed normal ovaries and testes. The mature males of A1-4n produced white semen. Under the electron microscope, the head of diploid sperm generated by A1-4n was bigger than that of haploid sperm generated by red crucian carp. In the testes of the A1-4n, there were many mature normal spermatozoa with a head bearing plasma mem- brane and a tail having the typical structure of "9 2" microtubules. Between the head and the tail, there were some mitochondria. The ovaries of A1-4n developed well and mainly contained II, III and IV-stage oocytes. The IV-stage oocytes were surrounded by inner and outer follicular cells. The micropyle was observed on the oolemma of follicular cells. There were abundant yolks and plenty of endoplasmic reticulum in the cytoplasm of IV-stage oocytes. Because A1-2n and A1-3n were distant crossing diploid hybrids and triploid hybrids respectively, they possessed abnormal gonads, and no mature semen and eggs were observed. (3) Compared with allotetraploids, the A1-4n fish not only had advantages such as fast growth rate and strong resistibility but also showed some new good performances such as high ratio of body width to body length, smaller heads and shorter tails. These results indicated that an- drogenesis could produce bisexual fertile tetraploids and improve the shape of allotetraploid hybrids as well, which will be of great significance in both the cell genetics research and fish breeding.     

Induced androgenesis in acipenserids may be obtained by ultraviolet radiation

The results are presented on haploid androgenesis in Siberian sturgeon and sterlet induced by UV irradiation of ovicells. During irradiation, the cells in Ringer solution were rotated around a UV lamp. The efficiency of genetic inactivation of ovicells was estimated by the following parameters: manifestation of Hertwig effect, the fraction of embryos demonstrating haploid syndrome at final developmental stages, by arrest of embryonic development in hybrids Siberian sturgeon × great sturgeon, and by absence of maternal alleles of microsatellite loci in embryos. The dose-effect curve suggests that, during UV irradiation of ovicells of Siberian sturgeon, the complete genetic inactivation is attained at exposition of 120 s, while that in sterlet is 90 or 105 s. The results show a principal possibility of inactivation of ovicells by UV irradiation and use of such cells for producing androgenetic progeny of acipenserids.     

Ontogenetic patterns in prey use by pallid sturgeon in the Missouri River, South Dakota and Nebraska

The pallid sturgeon ( Scaphirhynchus albus ) is an endangered species native to the Missouri and Mississippi rivers. To date, recovery efforts have focused on stocking juvenile fish, but little is known about ontogenetic changes in diet composition. Although diet composition for pallid sturgeon is believed to change from macroinvertebrates to fish, it is unclear at what size and/or age these ontogenetic diet shifts occur. To evaluate diet composition, 29 hatchery-stocked pallid sturgeon (range 356–720 mm fork length [FL]; mean = 549; SE = 23) were collected from the Missouri River downstream of Fort Randall Dam, South Dakota and Nebraska during summer 2006. The majority of pallid sturgeon (72%) were captured within a large delta region formed by the Niobrara River in the headwaters of Lewis and Clark Lake. Predominant prey of pallid sturgeon based on percent occurrence was Ceratopogonidae (81%), Isonychiidae (67%), Chironomidae (52%), and fishes (24%). Percent composition by wet weight showed that diets were composed of fishes (68%), Ephemeroptera (23%), Decapoda (6%), and Diptera (3%). Graphical analysis of combined data showed that mayflies, particularly Isonychiidae, were an important component of pallid sturgeon diets. Nonetheless, the percent composition of fishes in the diet increased with pallid sturgeon body size; for fish > 600 mm FL (5–7 years of age) diets were composed primarily of fish prey (66%, mostly johnny darters Etheostoma nigrum ). These findings highlight the importance of ontogenetic changes in diet composition for pallid sturgeon. Moreover, the unique habitat formed in the delta region is characterized by higher fish and invertebrate densities that may enhance foraging opportunities and thus improve recovery efforts for stocked pallid sturgeon.     

Discrimination of interspecific hybrids in natural populations of Amur sturgeon fish by means of multilocus RAPD-PCR markers

A RAPD-PCR analysis of a sample of Amur sturgeon fish (46 individuals) is performed. An estimate of the genetic state of native populations of Amur sturgeon Acipenser schrenckii (Brandt, 1869) and kaluga fish Huso dauricus (Georgi, 1775) is given. Genetic evidence for the hybrid origin of two phenotypical hybrids is obtained. Estimates of the genetic distances between the species and the hybrids proved to be at the level of interspecific distances. It is shown that an Exact Test for differentiation of the populations is the most effective means of discriminating the two species, while multidimensional scaling (MDS) is the most effective means of discriminating the hybrids. It is concluded that the populations of sturgeon fish in the Amur River have maintained a substantial level of genetic diversity; the presence of hybrids in these populations is judged to be one of the risk factors. Multilocus RAPD-PCR markers are acknowledged as a convenient and reliable tool for conducting genetic monitoring of populations of Amur sturgeon fish for the purpose of preserving the gene pool of the fish.     

Nucleocytoplasmic incompatibility in androgenetic fish hybrids can be overcome

Many androgenetic fish hybrids are nonviable due to the disturbed interaction between the foreign nucleus and the cytoplasm. It has been proposed that this incompatibility can be overcome if eggs from interspecific hybrids and sperm from one of the parental species are used for diploid androgenesis. Indeed, the androgenetic progeny obtained as a result of the insemination of the enucleated eggs of goldfish and carp hybrids by the carp spermatozoa proved to be viable and fertile. This approach may be used for reconstitution of the genotypes of rare or disappearing fish species with the help of induced androgenesis, if their native or cryoconserved sperm is available.     

Diet of farm‐escaped sturgeon in the Yangtze River

The diet of farm‐escaped sturgeon accidentally introduced into the Yangtze River ecosystem was investigated from samples collected over 10 days. The fish had consumed adequate quantities of natural food, with prey type related to fish body size. The fish were categorized according to their primary dietary component: demersal fish were most common prey of kaluga Huso dauricus and H. dauricus × Acipenser schrenckii hybrids of total length 120.3–188.7 cm. Shrimp Macrobrachium nipponensis made up the major portion of the diet of H. dauricus × A. schrenckii and Acipenser baerii × A. schrenckii of total length 91.1–106.8 cm. Gammarid amphipods were consumed by A. baerii × A. schrenckii of total length 35.0–81.2 cm. The body length of prey was proportional to the fork length of the sturgeon, Y = 0.19X‐9.46, R² = 0.997. Escaped sturgeon had travelled at least 898 km downstream from the point of escape in the Yangtze River and were feeding on native macrobenthos and demersal fish.     

三种鲟科鱼类和两种鲑科鱼类卵黄免疫球蛋白性质的比较

从施氏鲟(Acipenser schrencki)、小体鲟(Acipenser ruthenus)、西伯利亚鲟(Acipenser baeri)、哲罗鲑(Hucho taimen)和金鳟(Oncorhynchus mykiss)五种鱼类卵黄中分离、纯化Ig,并对其分子结构进行了初步研究。结果表明:五种鱼类卵中Ig的分子量分别为施氏鲟524kD、小体鲟468kD、西伯利亚鲟475kD、哲罗鲑为490kD,金鳟498kD,其Ig重链的相对分子量相同,约为97kD。轻链的相对分子量各不相同,施氏鲟为27kD,小体鲟28.5kD,西伯利亚鲟30kD,哲罗鲑28.5kD和金鳟16kD。其中西伯利亚鲟、施氏鲟和小体鲟Ig等电点约为5.85,哲罗鲑和金鳟的Ig等电点约为6.55,其蛋白质结构上具有一定的相似性,且均为糖蛋白     

Endogenous polyamine content during in vivo maturation and in vitro culture of maize pollen

Changes in polyamine content during in vivo maturation and in vitro culture of maize (Zea mays L.) pollen were studied. The endogenous content of free, conjugated and bound polyamines was analyzed during 30 days of pollen evolution, in both developmental pathways (microsporogenesis and androgenesis). The induction of androgenesis from cold-pretreated uninucleate pollen results, in most of cases, in a lower total polyamine content than that of the in vivo uninucleate pollen. These differences indicate that polyamine metabolism is altered during the induction of androgenesis, and this could be a consequence of increased polyamine assimilation. In general, pollen stages that involve cell division (tetrades, pre-anthesis pollen and four-day cultured pollen) are characterized by a predominance of free Spd. The increase of Spd and Spm in 15-day cultured pollen, when the first embryoids are formed, outline the possible implication of these polyamines in embryogenetic processes. Furthermore, these findings may contribute to the improvement of maize androgenesis yield, especially in recalcitrant genotypes, by the exogenous application of polyamines or polyamine-inhibitors to the culture medium.Abbreviations PAs polyamines - Put putrescine - Spd spermidine - Spm spermine - S free polyamine fraction - SH conjugated polyamine fraction - PH bound polyamine fraction     

Important considerations for methaemoglobin measurement in fish blood: assay choice and storage conditions

Spectrophotometric assays of methaemoglobin (metHb) in rainbow trout Oncorhynchus mykiss , channel catfish Ictalurus punctatus , tilapias Tilapia niloticus and Tilapia zillii and white sturgeon Acipenser transmontanus , under baseline conditions, were low (<4%) for each species, and yet higher than human values (<1%). MetHb results for a given fish species varied significantly between assays and two assays were deemed unacceptable for particular animals. For rainbow trout, white sturgeon, and the two species of tilapia, the Dubowski method gave uncharacteristically high estimates of metHb. MetHb could not measured in tilapia blood using the Evelyn & Malloy method due to spectral interference. Only the Horecker & Brackett assay worked well for all species. Storage conditions were extremely important in the quantification of metHb in rainbow trout blood. For consistent values, samples can be stored up to 4 h on ice (0° C) or at least 20 days under liquid nitrogen (− 196° C). Auto-oxidation, however, elevates rainbow trout metHb at − 20 and − 80° C. It should not be assumed that the blood of fishes and humans perform similarly during assays of metHb.     

Induction of Haploid Androgenesis in Pacific Oyster by UV Irradiation

Androgenesis, development from paternal but not maternal chromosomes, can be induced in some organisms including fish, but has not been induced previously in mollusk. In this study we investigated the induction of haploid androgenesis in the Pacific oyster by ultraviolet irradiation and observed nuclear behavior in the androgenetic eggs. Irradiation for 90 seconds at a UV intensity of 72 erg/mm² per second (6480 erg/mm²) was the optimal dose to achieve haploid androgenesis. The fertilization and development rates of D-shaped larvae decreased with increasing exposure time, and the development of the genetically inactivated eggs terminated before reaching the D-shaped stage. Cytologic observations showed that UV irradiation did not affect germinal vesicle breakdown or chromosomal condensation but caused various nuclear behavioral patterns during meiosis and first mitosis: 21.7% of eggs extruded all maternal chromosomes as 2 or 3 polar bodies, and 59.1% of eggs formed one female pronucleus. The maternally derived nucleus did not participate, or partially participated, in the first karyokinesis. The cytologic evidence demonstrates that the male genome is directing development in haploids produced by UV irradiation.