Ichthyophthirius (Ciliophora): population studies suggest reproduction in host epithelium

Evidence that Ichthyophthirius multifiliis trophonts may reproduce within the epithelium of the host was obtained from experimental infections of channel catfish. Mean number of parasites spontaneously leaving the fish increased from 0 on day 3 postexposure (PE) to 66.5 per fish on day 7 PE. Mean population density in fin, however, increased five-fold from day 3 to day 5 PE in the absence of opportunity for reinfection. At day 3 PE, 100% of parasite loci in fin and gill arches contained solitary trophonts. At day 4 PE, 10% of loci in fin contained clusters of two trophonts; at day 7 PE, 69% contained clusters of two or more trophonts. The first clusters of four trophonts in fin were observed day 5 PE and of eight trophonts, day 8 PE. Trophonts in clusters were flattened against one another.     

Ichthyophthirius multifiliis (Ciliophora) Development in Gill Epithelium1

ABSTRACT. The development of Ichthyophthirius multifiliis trophonts in gill epithelium of channel catfish was studied on the first five days post-exposure (PE) by light and electron microscopy. Trophonts increased in average diameter from 48 μm at one day PE to 248 μm at five days PE. Although theronts invaded any part of the gill epithelium, at three days PE most parasites were found adjacent to major blood vessels, particularly the afferent vessel. From one to three days PE, 70–90% of trophonts were immediately adjacent to gill epithelium, but at four and five days PE only 50% of trophonts were closely apposed by host epithelial cells. Notable ultrastructural changes in the trophonts over the five-day period occurred in the mucocysts and lipid inclusions, both of which increased markedly in number. The few crystalline mucocysts present at one day PE were near the cell periphery but rarely attached to the plasma membrane. At three days PE, crystalline mucocysts were significantly more abundant, and at five days PE, they occurred in large numbers throughout the cytoplasm as well as at the periphery. At three days PE, secretory mucocysts were first observed. Contractile vacuoles were more prominent at five days PE than earlier in development. Development of mucocysts and lipid reserves is probably essential to survival and reproduction of the ciliate after it leaves the host.     

Effect of immunization of channel catfish with inactivated trophonts on serum and cutaneous antibody titers and survival against Ichthyophthirius multifiliis

Two trials were conducted to determine the effect of immunization of channel catfish with inactivated trophonts on serum and cutaneous antibody titers and survival against Ichthyophthirius multifiliis Fouquet (Ich). In trial I, catfish were immunized intraperitoneally (IP) with: 1) 1% formalin-inactivated trophonts, 2) 3% formalin-inactivated trophonts and 3) freeze-thawed trophonts. Positive and negative control catfish were immunized with live theronts and 5% bovine serum albumin (BSA), respectively. At day 14, 28 and 50 post-immunizations, no statistical difference was noted in serum or cutaneous anti-Ich antibody titers to formalin-inactivated trophonts or freeze-thawed trophonts. The survival of catfish challenged with live theronts ranged from 33.3% to 43.3% for the formalin-inactivated or freeze-thawed trophonts at 50 d post-immunization. The survival of catfish immunized with live theront and BSA was 93.3 and 0%, respectively. In trial II, catfish were IP immunized with sonicated trophonts at doses of 1) 5 trophonts or 10.2 microg protein g(-1) fish, 2) 10 trophonts or 20.4 microg protein g(-1) fish, 3) 20 trophonts or 40.8 microg protein g(-1) fish, and 4) 5% BSA as the control. Fish immunized with 10 or 20 trophonts g(-1) fish showed highest serum (1/210 to 1/480) and cutaneous antibody titers (1/48 to 1/52), respectively, at 22 d post-immunization and survival (63.3-60.0%). The fish immunized with 5 trophonts g(-1) fish had titers of 1/52 and 1/12 for serum and cutaneous antibody and survival of 23.3%. BSA immunized catfish had background titers and a survival of 6.7%. There was a significant correlation between doses of sonicated trophonts used to immunize and catfish survival (correlation coefficient = 0.859, p < 0.01). These results indicate that doses of sonicated trophonts, but not formalin-inactivated or freeze-thawed trophonts provided both serum and cutaneous antibody responses and survival to live trophont challenge.     

Extracellular Calcium Concentration Affects Infectivity of Ichthyophthirius (Ciliophora) Theronts

Tomonts and their theront offspring of the hymenostomatid fish parasite Ichthyophthirius multifiliis were exposed to calcium levels from 0 to 0.8 mM Ca^2+. The survival and reproductive rates of tomonts in the absence of extracellular calcium were not significantly different from rates of tomonts provided calcium. Theronts that developed in the absence of calcium, however, were not infective for Ictalurus punctatus even when the extracellular magnesium concentration was doubled. Theronts that developed in 0.10 mM Ca²⁺ were infective (0.77 trophonts/mm² of pectoral fin) to essentially the same extent as theronts provided 0.33 mM Ca^2+. Infectivity of those provided 0.8 mM Ca²⁺ was 1.79 trophonts/mm² of fin, similar to that of theront controls. Theronts deprived of extracellular calcium as they developed contained significantly fewer secretory mucocysts than did theronts provided 0.1 to 0.8 mM Ca²⁺ although no significant differences among groups occurred with respect to abundance of crystalline or differentiating mucocysts. Theronts deprived of extracellular calcium also had swollen or enlarged mitochondria and abnormal crystalline mucocysts.     

Quantitative trait loci for resistance to fish pasteurellosis in gilthead sea bream (Sparus aurata)

Fish pasteurellosis is a bacterial disease causing important losses in farmed fish, including gilthead sea bream, a teleost fish of great relevance in marine aquaculture. We report in this study a QTL analysis for resistance to fish pasteurellosis in this species. An experimental population of 500 offspring originating from eight sires and six dams in a single mass‐spawning event was subjected to a disease challenge with Photobacterium damselae subsp. piscicida (Phdp), the causative agent of fish pasteurellosis. A total of 151 microsatellite loci were genotyped in the experimental population, and half‐sib regression QTL analysis was carried out on two continuous traits, body length at time of death and survival, and for two binary traits, survival at day 7 and survival at day 15, when the highest peaks of mortality were observed. Two significant QTLs were detected for disease resistance. The first one was located on linkage group LG3 affecting late survival (survival at day 15). The second one, for overall survival, was located on LG21, which allowed us to highlight a potential marker (Id13) linked to disease resistance. A significant QTL was also found for body length at death on LG6 explaining 5–8% of the phenotypic variation.     

The effect of feeding rate on growth of pectoral fin spine of juvenile great sturgeon Huso huso (Linnaeus, 1758)

The effect of two feeding rates (0.5 and 1.5% of total body weight) was assessed on the growth of pectoral fin spines of captive juvenile great sturgeon, Huso huso, after second year of life. The fish received Oxytetracycline (OTC) twice in the first and second years of their lives under basic diet. During the 5‐month experimental period, juveniles (mean 1,187.4 g, 0.1 standard deviation [SD]), n = 50) were reared with two feeding rates under similar conditions in 10 fiberglass tanks (1.5 m³). The fish were fed manually with a commercial diet twice a day (35% Biomar, Nersac, France) throughout the experiment. The OTC marks were distinguished in all pectoral fin spine sections under ultraviolet light. The means of the first and second annular radii were 806.6 µm (27.2 SD) and 2,246.5 µm (50.2 SD), respectively. The marginal increment analysis beyond the second OTC mark revealed a significantly smaller marginal increment for low feeding rate treatment (143.9, 11.2 SD) as compared to the high feeding rate one (269.0, 14.6 SD). The results indicate the slower growth rate in the fish fed the low feeding treatments seen in the pectoral fin spine formation, which can be used as an indicator of recent feeding history in sturgeon juveniles. The best daily feeding rate for great sturgeon of 2,460 g was determined to be 1.5% body weight/day in this study.     

Dietary Na does not reduce dietary Cu uptake by juvenile rainbow trout

Rainbow trout Oncorhynchus mykiss fry in moderately hard water were exposed to control or high levels of dietary Cu (c. 6 and 580 ug Cu g food^?1) at one of three levels of Na (1·5, 3·0 or 4·5%) in the diet, i.e. six experimental groups. Fish were fed a 4% body mass ration daily for 28 days and 10 individuals from each group were sampled every 7 days. Concentrations of Cu and Na were measured in the gills, liver, gut and remaining carcass of sampled fish. Growth was not affected and no consistent differences were found in mass, total lengths (L_T) or indices of body condition among any of the groups on any sampling day. Copper concentration was significantly higher in tissues of Cu‐exposed groups, although within treatment types (control Cu v. high Cu diet), it did not differ consistently among groups that received different levels of dietary Na. Tissue Na concentration did not differ among any of the groups and did not show any marked changes over time. In Cu‐exposed groups, the proportion of total body Cu burden contained in the liver approximately doubled over time, from c. 30% on day 7 to c. 60% on day 28. In unexposed fish, the liver maintained c. 25% of the total Cu burden throughout the experiment. In contrast, the proportion of the total body Cu burden contained in the gut decreased somewhat over time in Cu‐exposed fish, from c. 40% on day 7 to c. 30% on day 28, and remained fairly stable at c. 25–30% in control groups, i.e. approximately equal to liver values. In all groups, the carcass contained by far the largest portion of the total Na content (>80%). Measurements made 36 h post‐feeding indicated that all six groups had much higher Na efflux relative to influx, suggesting that the fish were eliminating excess Na taken up from the diet, and differences in Na influx rates were small. Na efflux rate was significantly higher in the high Cu and high Na group than in the high Cu and low Na group. The results indicate that at the concentrations used in this experiment, dietary Na has little effect on dietary Cu uptake by juvenile rainbow trout, and dietary Cu has little effect on Na homeostasis.     

Genetics and expression of two pectinesterase genes in Valencia orange

The genetics and expression of pectinesterase (PE) genes were examined in Valencia orange. Degenerate primers based on partial amino acid sequence of a 36 kDa PE protein isolated from juice vesicles were used to amplify a 350 bp DNA fragment from cDNA prepared from juice vesicle total RNA. Two groups of 350 bp PE clones with 66% sequence identity were isolated. A clone from each group was used to screen a Valencia orange genomic DNA λ library. Two different lambda clones that contained complete PE coding sequence (CsPME1 and CsPME3) and a third lambda clone that contained partial PE sequence (CsPME2) were characterized. The CsPME1 gene contained two exons (1063 and 689 bp) interrupted by a 1452 bp intron, whereas the CsPME3 gene had two exons (844 and 686 bp) interrupted by a 771 bp intron. CsPME1 shared significant sequence similarity with the partial clone CsPME2, including the entire cloned region of the first exon, a large region in the 5′ portion of the intron and the 3′ portion of the second exon, but the 3′ portion of the intron and the 5′ portion of the second exon were dissimilar. Southern blots suggested that Valencia orange has two genes within each PE group. Full-length cDNA clones that shared 99% sequence identity with CsPME1 and CsPME3 were isolated. Both groups of PE genes were differentially expressed in tissues of Valencia orange, and in addition CsPME3 appeared to be ethylene-regulated. The deduced proteins of PE cDNA clones CsPME1 (63.5 kDa) and CsPME3 (56.3 kDa) were considerably larger than the PE protein we isolated from Valencia orange juice vesicles and also other mature plant PE proteins. The estimated size of group I (2.2 kb) and group II (2.0 kb) PE mRNAs also predicted a larger protein than was isolated from juice vesicles. Alignment of the mature tomato and mung bean PE proteins, the most N-terminal sequence we obtained from polypeptides derived from the 36 kDa PE isolated from juice vesicles and the deduced amino acid sequences of plant PE cDNA clones suggest that a post-translational cleavage event separates the variable N-terminus from the more conserved C-terminal domain of the mature PE protein.     

Reproductive biology of the endangered Oxleyan pygmy perch Nannoperca oxleyana Whitley

The reproductive biology of the Oxleyan pygmy perch Nannoperca oxleyana is described from simultaneous studies of wild populations in north-eastern New South Wales and mature fish held in aquaria. In the wild, 50% of males and females matured at total lengths of 24·0–25·9 and 28·0–29·9 mm, respectively. The species displays sexual dichromatism during the spawning season, with males developing more intense red and brown fin and body colouration, and black pelvic fins. Captive male broodfish displayed territoriality during the breeding season, closely guarding sites within artificial, plant-like substrata in which pairs of fish spawned adhesive eggs. Protracted serial spawning of wild and captive fish occurred from September to April and May at mean water temperatures ≥16·6° C and day length ≥10·7 h. Captive broodfish spawned on an average of 57% of days during the 256 day spawning period. Gonado-somatic indices averaged 0·7% for all ripe males and 4·1–4·2% for all ripe females collected. Mean total and batch fecundities of captive females were 1323 eggs per fish and 7·8 eggs per fish per day, respectively, and relative fecundity was 587 eggs g⁻¹ of body mass. Batch fecundity of wild females was estimated at 7·8 eggs per fish. The adaptive significance of this reproductive strategy in a harsh, variable environment is discussed.     

In vitro culture technique for Cryptocaryon irritans, a parasitic ciliate of marine teleosts

A medium for the in vitro culture of Cryptocaryon irritans, which is an obligatorily parasitic ciliate of marine teleosts and causes 'white spot disease', was developed. The medium consisted of a layer of cultured fish cells (FHM), with an agarose gel layer covering the cell layer. The agarose gel contained 0.22% agarose, 10% fetal calf serum, 100 I.U. ml(-1) Penicillin G potassium and 100 microg ml(-1) streptomycin sulphate. Theronts of C. irritans transformed to trophonts and grew to 180 microm in mean length in the medium, although they gradually decreased in number. When trophonts fully developed in medium were transferred into seawater 4 d after inoculation, approximately 70% of them transformed to encysted tomonts and released theronts. When fish were challenged with theronts obtained from in vitro-raised parasites, approximately 40% of the theronts were recovered from fish, indicating comparative infectivity of in vitro-raised theronts to those of in vivo-raised theronts. This is the first report that C. irritans fully developed in vitro and its entire life cycle was completed without a host fish.     

Age‐dependent decline in fin regenerative capacity in the short‐lived fish Nothobranchius furzeri

The potential to regenerate declines with age in a wide range of organisms. A popular model system to study the mechanisms of regeneration is the fin of teleost fish, which has the ability to fully regrow upon amputation. Here, we used the short‐lived killifish Nothobranchius furzeri to analyse the impact of aging on fin regeneration in more detail. We observed that young fish were able to nearly completely (98%) regenerate their amputated caudal fins within 4 weeks, whereas middle‐aged fish reached 78%, old fish 57% and very old fish 46% of their original fin size. The difference in growth rate between young and old fish was already significant at 3 days post amputation (dpa) and increased with time. We therefore hypothesized that early events are crucial for the age‐related differences in regenerative capacity. Indeed, we could observe a higher percentage of proliferating cells in early regenerating fin tissue of young fish compared with aged fish and larger fractions of apoptotic cells in aged fish. Furthermore, young fish showed peak upregulation of several genes involved in fgf and wnt/β‐catenin signalling at an earlier time point than old fish. Our findings suggest that regenerative processes are initiated earlier and that regeneration overall is more efficient in younger fish.     

Comparison of fin ray sampling methods on white sturgeon Acipenser transmontanus growth and swimming performance

Effects of two fin‐ray sampling methods on swimming performance, growth and survival were evaluated for hatchery‐reared sub‐adult white sturgeon Acipenser transmontanus. Fish were subjected to either a notch removal treatment in which a small section was removed from an anterior marginal pectoral‐fin ray, or a full removal treatment in which an entire marginal pectoral‐fin ray was removed. Control fish did not have fin rays removed, but they were subjected to a sham operation. A modified 3230 l Brett‐type swim tunnel was used to evaluate 10 min critical station‐holding speeds (S_CSH) of A. transmontanus, immediately after the fin ray biopsies were obtained with each method. Survival and growth were evaluated over a 6 month period for a separate group of fish subjected to the same biopsy methods. Mean ± s.e . 10 min S_CSH were 108·0 ± 2·3, 110·0 ± 2·6 and 115·0 ± 3·5 cm s^?1 for the notch removal group, full removal group and control group, respectively, and were not significantly different among treatments. Behavioural characteristics including tail‐beat frequency and time spent hunkering were also not significantly different among treatment groups swimming at the same speeds. There were no mortalities and relative growth was similar among treatment groups. Average biopsy time for the notch removal method was lower and the wounds appeared to heal more quickly compared with the full removal method.     

Protective immunity of Nile tilapia against Ichthyophthirius multifiliis post-immunization with live theronts and sonicated trophonts

Two immunization trials were conducted to evaluate host protection of Nile tilapia, Oreochromis niloticus against Ichthyophthirius multifiliis (Ich). Immunizations were done with live theronts or sonicated trophonts by bath immersion and intraperitoneal (IP) injection. The immunized fish were challenged with theronts 21 days post-immunization in trial I and 180 days post-immunization in trial II. The serum anti-Ich antibody and cumulative mortalities of tilapia were determined after theront challenge. Serum anti-Ich antibody was significantly higher (P<0.05) in tilapia immunized with live theronts by immersion or IP injection or with sonicated trophonts administered by IP injection than tilapia immunized with sonicated trophonts by immersion, with bovine serum albumin by IP injection, or non-immunized controls. Host protection was acquired in fish immunized with live theronts by immersion or IP injection. Tilapia immunized with sonicated trophonts by IP injection were partially protected with a 57-77% survival in both trials. At 180 days post-immunization, serum antibody titers had declined in immunized fish yet they were still able to survive challenge. The protection appears not to be solely depending on serum antibody response against Ich.     

COLONY-FORMING UNITS IN DIFFUSION CHAMBERS (CFU-d) AND COLONY-FORMING UNITS IN AGAR CULTURE (CFU-c) OBTAINED FROM NORMAL HUMAN BONE MARROW: A POSSIBLE PARENT–PROGENY RELATIONSHIP

A series of experiments was performed to elucidate the relationship between cells that form granulocytic colonies in fibrin clot diffusion chambers implanted into the peritoneum (i.p.) of irradiated mice (CFU-d) and day 7 and day 14 CFU-c which give rise to colonies after 7 and 14 days in agar cultures in vitro, respectively. Normal human bone marrow cells were cultured in suspension in vitro or in diffusion chambers implanted into irradiated or non-irradiated mice. During these culture conditions there was an initial decrease in the number of CFU-c per culture. This was followed by an increase between day 2 and day 7 of culture. No similar increase of neutrophilic CFU-d was observed. When CFU-d, day 14 and day 7 CFU-c in normal marrow were separated by velocity sedimentation and cultured in suspension culture or in diffusion chambers for 7 days, the maximum increase of day 7 and day 14 CFU-c was observed in slowly sedimenting cell fractions which contained the majority of CFU-d. After 3 days in suspension culture, the maximum increase of day 14 CFU-c was found in fractions which also gave rise to maximum numbers of CFU-c after 7 days. However, day 7 CFU-c were found in fractions which initially contained the majority of day 14 CFU-c. No increase in CFU-d was found in fractions initially containing peak numbers of CFU-c. Between 53 and 71% of CFU-c harvested from diffusion chambers in irradiated mice or from suspension cultures were sensitive to pulse incubation with tritiated thymidine, suggesting that the cells were proliferating during these culture conditions. In diffusion chambers implanted into non-irradiated mice, however, CFU-c were found to be relatively resistant to this treatment (3–11% sensitive to tritiated thymidine). Thus marked increases in CFU-c were also observed during experimental conditions, where no significant DNA synthesis was detected. A reproducible time sequence of increase in CFU-c populations in culture was observed. Day 14 CFU-c and cells that gave rise to clusters on day 7 in agar increased between day 2 and day 4, whereas day 7 CFU-c increased between day 4 and day 7. The results suggested that CFU-d gave rise to CFU-c in culture and that day 14 CFU-c were precursors of day 7 CFU-c.     

Description of Triplophysa luochengensis sp. nov. (Teleostei: Nemacheilidae) from a karst cave in Guangxi,China

A new cave‐dwelling fish Triplophysa luochengensis is described based on specimens collected from a karst cave in Guangxi Zhuang Autonomous Region, China, that is interconnected to the Hongshui River drainage. The species can be distinguished from its congeners by a combination of characters: eyes degenerated, anal fin with six branched rays, caudal fin with 16–17 branched rays, pectoral‐fin length 72·4–95·8% of the distance between pectoral‐fin origin and pelvic‐fin origin, lateral head length 26·2–28·2% of standard length (L_S), eye diameter 7·5–8·6 of L_S, body covered by sparse scales, lateral line complete and 7–8 pre‐operculo‐mandibular pores. Dark pigments irregularly present on dorsum of head, dorsum and flank.     

Relatedness of Indian Flax Genotypes (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.): An Inter-Simple Sequence Repeat (ISSR) Primer Assay

The objective of this study was to analyze the genetic relationships, using PCR-based ISSR markers, among 70 Indian flax (Linum usitatissimum L.) genotypes actively utilized in flax breeding programs. Twelve ISSR primers were used for the analysis yielding 136 loci, of which 87 were polymorphic. The average number of amplified loci and the average number of polymorphic loci per primer were 11.3 and 7.25, respectively, while the percent loci polymorphism ranged from 11.1 to 81.8 with an average of 63.9 across all the genotypes. The range of polymorphism information content scores was 0.03–0.49, with an average of 0.18. A dendrogram was generated based on the similarity matrix by the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), wherein the flax genotypes were grouped in five clusters. The Jaccard’s similarity coefficient among the genotypes ranged from 0.60 to 0.97. When the omega-3 alpha linolenic acid (ALA) contents of the individual genotypes were correlated with the clusters in the dendrogram, the high ALA containing genotypes were grouped in two clusters. This study identified SLS 50, Ayogi, and Sheetal to be the most diverse genotypes and suggested their use in breeding programs and for developing mapping populations.     

Morphological variation in diadromous and landlocked populations of the spotted galaxias,Galaxias truttaceus,in Tasmania,south-eastern Australia

Synopsis A comparison of a suite of morphometric measurements and meristic counts of individuals of two landlocked lacustrine and two diadromous riverine populations of Galaxias truttaceus was carried out utilising both univariate and canonical variate analyses. Lacustrine fish had fewer dorsal and anal fin rays than did riverine fish. Differences were not as clear for gill rakers and vertebrae. Comparisons of serial counts were made with two derived lacustrine species, G. auratus and G. tanycephalus, also from Tasmania. Lacustrine G. truttaceus varied in the same direction as the derived species, relative to riverine G. truttaceus. From an analysis of 12 body measurements, the first canonical variate clearly separated lacustrine fish from riverine fish largely based on measurements associated with fins (pre-anal fin length, length of anal base, pre-dorsal fin length, maximum length of dorsal fin and inter-orbital width). An overall value for the correct classification of fish into groups based on locality was 84%. The percentage of fish classified into the wrong habitat (lake or stream) was much less than the percentage classified between localities within habitats. Overall morphological variation was greater between than within habitats. It is suggested that the differences in water movement and food type may in part account for the differences shown and that selective pressures peculiar to the lacustrine environment may be causing the lake populations to diverge from the riverine populations.     

Genetic differences between wild and hatchery‐bred brown trout (Salmo trutta L.) in single nucleotide polymorphisms linked to selective traits

To study effects from natural selection acting on brown trout in a natural stream habitat compared with a hatchery environment, 3,781 single nucleotide polymorphism (SNP) markers were analyzed in three closely related groups of brown trout (Salmo trutta L.). Autumn (W/0+, n = 48) and consecutive spring (W/1+, n = 47) samples of brown trout individuals belonging to the same cohort and stream were retrieved using electrofishing. A third group (H/1+, n = 48) comprised hatchery‐reared individuals, bred from a mixture of wild parents of the strain of the two former groups and from a neighboring stream. Pairwise analysis of F_ST outliers and analysis under a hierarchical model by means of ARLEQUIN software detected 421 (10.8%) candidates of selection, before multitest correction. BAYESCAN software detected 10 candidate loci, all of which were included among the ARLEQUIN candidate loci. Body length was significantly different across genotypes at 10 candidate loci in the W/0+, at 34 candidate loci in the W/1+ and at 21 candidate loci in the H/1+ group. The W/1+ sample was tested for genotype‐specific body length at all loci, and significant differences were found in 10.6% of all loci, and of these, 14.2% had higher frequency of the largest genotype in the W/1+ sample than in W/0+. The corresponding proportion among the candidate loci of W/1+ was 22.7% with genotype‐specific body length, and 88.2% of these had increased frequency of the largest genotype from W/0+ to W/1+, indicating a linkage between these loci and traits affecting growth and survival under this stream's environmental conditions. Bayesian structuring of all loci, and of the noncandidate loci suggested two (K = 2), alternatively four clusters (K = 4). This differed from the candidate SNPs, which suggested only two clusters. In both cases, the hatchery fish dominated one cluster, and body length of W/1+ fish was positively correlated with membership of one cluster both from the K = 2 and the K = 4 structure. Our analysis demonstrates profound genetic differentiation that can be linked to differential selection on a fitness‐related trait (individual growth) in brown trout living under natural vs. hatchery conditions. Candidate SNP loci linked to genes affecting individual growth were identified and provide important inputs into future mapping of the genetic basis of brown trout body size selection.     

Polymorphic microsatellite loci for assigning parentage in least flycatchers (Empidonax minimus)

Least flycatchers (Empidonax minimus) are socially monogamous birds that form tight territorial aggregations on the breeding grounds. We designed five polymorphic microsatellite loci for assigning parentage to offspring within least flycatcher clusters. The number of alleles per locus ranged from 7 to 18. Mean polymorphic information content was 83.8%; the probability of exclusion with known maternal genotype was 0.999. These microsatellites are powerful DNA markers for identifying extra‐pair paternity in this species. Preliminary data also suggest that these loci may be useful for other members of this genus.     

In vitro Maintenance of the Trophonts and Sporozoites of Pyxinia crystalligera*

SYNOPSIS. In a study of in vitro maintenance of the trophonts and sporozoites of the cephaline gregarine, Pyxinia crystalligera, a basal medium consisting of salts, reducing agents, and antibiotics was supplemented by fetal bovine serum, serum protein fraction IV-4, or protein hydrolysates. Motility was used as the viability criterion. The tests were terminated when viability was ～30% of the original inoculum. Two-week survival of trophonts was observed with 0.75% protein fraction, 80% of organisms remaining viable at the end of 1 week. Similar results were noted with 2% casein hydrolysate. With lactalbumin hydrolysate and Casamino acids trophonts remained viable for 6 days, and with fetal bovine serum the results were similar to those obtained with lower concentrations of protein fraction IV-4. Sporozoites remained motile for up to 5 days in the basal medium supplemented by both protein fraction IV-4 and diluted haemocoelic contents of the host, Dermestes vulpinus, but only for 3 days or less in that supplemented by the protein fraction alone. The characteristic crystals of trophonts were dissolved or possibly utilized during in vitro maintenance. A new type of crystal, however, was observed in forms kept in the presence of higher protein hydrolysate concentrations. Smaller trophonts were more adaptable to maintenance than larger ones, for rapid depletion of paraglycogen and resorption of the deutomerite often was observed in the latter. Results of cytochemical tests for carbohydrates, neutral lipids, and proteins indicated that the trophonts were capable of paraglycogen synthesis in vitro.