Coronary vascular volume remodelling in rainbow trout Oncorhynchus mykiss

The 34% increase in relative ventricular mass (M_rv) resulting from chronic anaemia (induced by an intraperitoneal injection of phenylhydrazine hydrochloride) was accompanied by a 117% increase in coronary vascular volume of diploid rainbow trout Oncorhynchus mykiss. Coronary vascular volume of normocythemic triploid fish was similar to that of normocythemic diploid fish despite a larger M_rv. These observations, in combination with previous studies, suggest that the vascularity of compact myocardium in O. mykiss can vary independently of M_rv.     

Mechanism of sodium uptake in PNA negative MR cells from rainbow trout, Oncorhynchus mykiss as revealed by silver and copper inhibition

The rate of acid-stimulated and phenamil-sensitive sodium (Na(+)) uptake was measured in three different cell lineages: pavement cells (PVC), total mitochondrion-rich (MR) cell populations, and peanut lectin agglutinin-negative mitochondrion-rich cells (PNA(-) MR) isolated from the rainbow trout gill epithelium. Despite the presence of basal levels of Na(+) uptake in PVC, this transport was not enhanced by acidification, nor was it inhibited by independent treatment with bafilomycin (i.e., a V-type H(+)-ATPase inhibitor), phenamil (i.e., a specific inhibitor of ENaC), or Ag (a specific inhibitor of active Na(+) transport in fish). In contrast, Na(+) uptake in PNA(-) MR cells was increased by ~220% above basal levels following acidification of near 0.4 pH units in the presence of 1.0 mM external Na(+). Acid-stimulated Na(+) transport was entirely inhibited by both phenamil and bafilomycin. Silver (Ag) and copper (Cu), which are known to interfere with active Na(+) transport in fish, were also responsible for inhibiting acid stimulated Na(+) uptake in PNA(-) MR cells, but by themselves had no effect on basal Na(+) transport. Thus, we demonstrate that Ag specifically prevented acid-stimulated Na(+) uptake in PNA(-) MR cells in a dose-dependent manner. We also demonstrate rapid (<1 min) and significant inhibition of carbonic anhydrase (CA) by Ag in PNA(-) MR cells, but not in PVC. These data lend further support to the idea of a PNA(-) MR cell type as the primary site for Na(+) uptake in the freshwater (FW) gill phenotype of rainbow trout. Moreover, these findings provide support for the importance of intracellular protons in regulating the movement of Na(+) across the apical surface of the fish gill.     

Factors affecting male potency in pooled gamete crosses of rainbow trout,Oncorhynchus mykiss

Synopsis Numbers of offspring produced by genetically marked male and female rainbow trout,Oncorhynchus mykiss were determined by an electrophoretic analysis of 1713 progeny from crosses produced by premixing gametes from different parents prior to insemination and associated full- and half-sib controls. We examined whether male potency, defined as the ability of males to sire progeny when their semen is pooled with that of other males, is affected by interaction with female gametes, concentration of sperm cells and the timing of semen application. Male potency differed significantly and was not affected by which of two females was involved in either of two sets of experiments. The differential between males decreased significantly when sperm concentrations were equalized. The first male sired over 75% of progeny when semen from four males was added sequentially to ova at 30 s intervals in each of two experiments. The lack of significant deviations of marker genotypes from expected Mendelian segregation in full-sib families suggested that there was no differential survival of progeny with specific marker genotypes. Furthermore, there was no correspondence between the survival of progeny of specific males in control crosses and the number of progeny sired in matings where sperm was premixed prior to insemination. Thus. variation in male reproductive success appeared the result of differential fertilization rates.     

The effects of diet composition and ration on biotransformation enzymes and stress parameters in rainbow trout, Oncorhynchus mykiss

Juvenile rainbow trout (Oncorhynchus mykiss) were fed one of three isoenergetic diets varying in protein (35-55%) and lipid content (8-18%), at full satiation levels or half rations for 6 weeks in order to investigate the effects of diet on baseline stress parameters and biotransformation enzyme activity. Growth was greatest in fish fed to satiation on a low protein and high lipid diet. Stress parameters, including plasma lactate, glucose and cortisol concentrations were not significantly affected by dietary treatment or ration. Basal biotransformation enzymes, including glutathione S-transferase (GST) and ethoxyresorufin O-deethylase (EROD) activity, were also unaffected by dietary treatment. Fish exposed to the biotransformation enzyme inducer beta-naphthoflavone did not exhibit an alteration in stress indicators or GST activity; however, EROD activity was increased (87- to 210-fold) in fish receiving all diets and rations. The results of the present study indicate that, unlike mammals, fish may be more recalcitrant to different levels of ingestion of isoenergetic diets varying in protein and lipid concentration with respect to stress responses and the maintenance of basal titers of biotransformation enzymes and their induction.     

The effects of hypercapnia on branchial and renal calcium fluxes in the rainbow trout (Oncorhynchus mykiss )

Whole body calcium influx, branchial calcium efflux, and renal Ca²⁺ excretion were measured in rainbow trout (Oncorhynchus mykiss) exposed to hypercapnia. These experiments were performed to assess the potential impact on Ca²⁺ balance of the changes in gill morphology known to accompany respiratory acidosis in this species. After 48 h of hypercapnia, gill filamental chloride cell fractional area was significantly reduced. Despite this reduction and the presumed involvement of the chloride cell in calcium influx, whole body calcium influx was increased after 12 h of hypercapnia and remained elevated for 48 h. Branchial calcium efflux was unaltered during hypercapnia exposure, whereas renal Ca²⁺ excretion was elevated over preflux values only at 6 h of hypercapnia. Measurement of the kinetics of whole body calcium influx after 48 h of hypercapnia revealed a significant increase in the maximal uptake rate of Ca²⁺, yet the affinity constant of Ca²⁺ uptake was unaffected. Measurements of high-affinity Ca²⁺ -ATPase activities and ATP-dependent Ca²⁺ transport of gill basolateral membrane vesicles revealed that the ATP-dependent Ca²⁺ extrusion mechanism of the gills was not affected by hypercapnia. The results of the present study clearly show that the reduced chloride cell surface area that accompanies hypercapnia in trout does not impair calcium homeostasis. Although adjustments to the basolateral membrane high affinity Ca²⁺ transporter do not appear to play a role, the mechanism(s) underlying the maintenance of calcium homeostasis under hypercapnic conditions are unresolved. Accepted: 1 July 1996     

The influence of chronic anaemia on catecholamine secretion in the rainbow trout (Oncorhynchus mykiss)

The effect of long-term (7 day) anaemia on catecholamine release was examined in rainbow trout (Oncorhynchus mykiss) in vivo during acute exposure to hypoxia and in situ using a perfused post-cardinal vein preparation. The first goal was to distinguish among reductions in blood O₂ partial pressure, O₂ concentration and haemoglobin percentage saturation as potential stimuli for, or correlates of, catecholamine secretion during hypoxia. The second goal was to elucidate the role of these factors in promoting enhanced chromaffin cell responsiveness in trout subjected to chronic hypoxia (Montpetit and Perry 1998). Anaemic fish (haematocrit lowered from 28.4±2.4% to 11.9±1.6%) displayed a marked reduction in haemoglobin-O₂ binding affinity [P ₅₀ (P _aO₂ at 50% Hb-O₂ saturation) was increased from 14.7 mm Hg to 24.3 mm Hg]. Upon exposure to hypoxia, the anaemic fish released catecholamines into their circulation at higher values of arterial O₂ partial pressure (∼52 mm Hg versus ∼18 mm Hg) and haemoglobin O₂ saturation (<70% versus <55%) than did control fish. In addition, anaemic fish achieved significantly greater circulating levels of total catecholamines (noradrenaline plus adrenaline) during acute hypoxia (294.8±67.3 versus 107.0±35.6 nmol l⁻¹). These results do not support the view that catecholamine release is triggered by a reduction in haemoglobin O₂ saturation or arterial PO₂, per se. Nor are they consistent with the idea that catecholamine release occurs at a threshold value of arterial PO₂ corresponding to a critical reduction in blood O₂ concentration. The effects of the non-selective cholinergic receptor carbachol on catecholamine secretion from chromaffin tissue were assessed using perfused posterior cardinal vein preparations derived from control or anaemic fish. For adrenaline secretion, there was no statistically significant change in the ED₅₀ (dose eliciting 50% response). For noradrenaline secretion however, preparations originating from anaemic fish displayed an enhanced responsiveness to carbachol as indicated by a significant 4.5-fold reduction in the carbachol ED₅₀ value from 2.53 × 10⁻⁶ mol kg⁻¹ to 5.67 × 10⁻⁷ mol kg⁻¹. These results demonstrate that anaemia-induced hypoxaemia, in the absence of any lowering of PO₂, is able to modulate the responsiveness of chromaffin cells to cholinergic stimulation. Accepted: 21 April 1999     

Branchial and renal ion fluxes and transepithelial electrical potential differences in rainbow trout,Oncorhynchus mykiss: effects of aluminium at low pH

Synopsis Adult rainbow trout, Oncorhynchus mykiss, were acutely exposed for 4 hours to low pH (4.4) and elevated Al-concentrations (300 µgI^–1) in soft water (Ca²⁺ + Mg²⁺ = 25 µmolI^–1). Comparison of branchial and renal ion fluxes (Na⁺, Cl^–, Mg²⁺, Ca²⁺ and NH₄ ⁺) gave evidence that pH and Al effects were primarily localized at the gill site. The negative whole body ion balance seemed to be caused by stimulatory effects on Na and Cl efflux especially under Al stress and to a lesser extent by inhibition of influx. Measurements of gill potentials indicated positive shifts, which were similar in response to increasing levels of H⁺ ions and Al. It is suggested that Al-induced changes of branchial potentials causes high diffusable loss of ions through interference with membrane-bound Ca²⁺ at the gill site.     

Production of recombinant leptin and its effects on food intake in rainbow trout (Oncorhynchus mykiss)

Leptin is a key factor for the regulation of food intake and energy homeostasis in mammals, but information regarding its role in teleosts is still limited. There are large differences between mammalian and teleost leptin at both gene and protein levels, and in order to characterize the function of leptin in fish, preparation of species-specific leptin is therefore a key step. In this study, full-length cDNA coding for rainbow trout leptin was identified. In spite of low amino acid sequence similarity with other animals, leptin is highly conserved between trout and salmon (98.7%). Based on the cDNA, we produced pure recombinant trout leptin (rt-leptin) in E. coli, with a final yield of 20 mg/L culture medium. We then examined the effects of intraperitoneal (IP) injection of rt-leptin on feeding behavior and gene expression of hypothalamic NPY and POMCs (POMC A1, A2 and B) in a short-term (8 h) experiment. The rt-leptin suppressed food intake and led to transient reduction of NPY mRNA levels, while the expression of POMCs A1 and A2, was elevated compared with vehicle-injected controls. These results for rainbow trout are the first that describe a physiological role of leptin using a species-specific orthologue in teleosts, and they suggest that leptin suppresses food intake mediated by hypothalamic regulation. This anorexic effect is similar to that observed in mammals and frogs and supports that the neuroendocrine pathways that control feeding by leptin are ancient and have been conserved through evolution.     

Changes in tissue and mitochondrial membrane composition during rapid growth, maturation and aging in rainbow trout, Oncorhynchus mykiss

Membrane compositions, particularly of mitochondria, could be critical factors in the mechanisms of growth and aging processes, especially during phases of high oxidative stress that result in molecular damage. In the present study, liver and mitochondrial membrane phospholipid (PL) compositions were analyzed in rainbow trout during its four first years of life, a period characterized by rapid growth and high oxidative stress. Specifically, farmed fish of three ages (1-, 2- and 4-years) were studied, and PL compositions of whole liver and liver mitochondria, and fatty acid compositions of individual PL classes were determined. Liver mitochondrial membranes showed a PL composition different to that of the whole tissue suggesting adaptation of cell and subcellular membranes to specific functions. Individual PL had characteristic fatty acid compositions that were similar in whole liver and mitochondrial membranes. Whole liver and mitochondria showed increased lipid peroxidation with age along with changes in membrane PL fatty acid compositions. Most PL classes showed similar changes in fatty acid composition among the age groups, with reduced proportions of docosahexaenoic acid (DHA) and, generally, concomitantly increased levels of monounsaturated fatty acids, which together resulted in reduced peroxidation index (PIn). However, total polyunsaturated fatty acid (PUFA) content did not change significantly with age due to increased eicosapentaenoic acid, docosapentaenoic acid and, in most PL, increased n−6 PUFA. These results suggest there may be oxidation of PL DHA with compensatory mechanisms to maintain membrane fluidity and function. However, modification of fatty acid composition of specific PLs, such as cardiolipin, could affect the electron transport chain efficiency and propagate the oxidative reaction throughout the cell. In addition, both the content and fatty acid composition of sphingomyelin, which has been suggested as a possible mediator of cell dysfunction and apoptosis, changed with age differently to the other PL classes. Moreover, these changes showed different trends between mitochondria and whole liver. These data suggest there is marked oxidative stress associated with rapid growth and maturation in rainbow trout. Changes observed in membrane lipids point to their possible participation in the processes involved in this species response to oxidative stress and damage accumulation rate.     

Chryseobacterium oncorhynchi sp. nov., isolated from rainbow trout (Oncorhynchus mykiss)

Genotypic and phenotypic analyses were performed on five Gram-negative, catalase and oxidase-positive, rod-shaped bacteria isolated from the gill and liver of four rainbow trout. Studies based on comparative 16S rRNA gene sequence analysis showed that the five new isolates shared 99.8-100% sequence similarity and that they belong to the genus Chryseobacterium. The nearest phylogenetic neighbours of the strain 701B-08(T) were Chryseobacterium ureilyticum F-Fue-04IIIaaaa(T) (99.1% 16S rRNA gene sequence similarity) and Chryseobacterium joosteii LMG 18212(T) (98.6%). DNA-DNA hybridization values between the five isolates were 91-99% and ranged from 2 to 53% between strain 701B-08(T) and the type strains of phylogenetically closely related species of Chryseobacterium. Strain 701B-08(T) had a DNA G+C content of 36.3 mol%, the major fatty acids were iso-C(15:0), iso-C(17:1)ω9c, C(16:1)ω6c and iso-C(17:0) 3-OH and the predominant respiratory quinone was MK-6. The novel isolates were distinguished from related Chryseobacterium species by physiological and biochemical tests. The genotypic and phenotypic properties of the isolates from rainbow trout suggest their classification as representatives of a novel species of the genus Chryseobacterium, for which the name Chryseobacterium oncorhynchi sp. nov. is proposed. The type strain is 701B-08(T) (=CECT 7794(T)=CCUG 60105(T)).     

Immunostimulant effects of Cotinus coggyria on rainbow trout (Oncorhynchus mykiss)

In this study, non-specific immune effects of tetra (Cotinus coggyria) on rainbow trout (Oncorhynchus mykiss) by dietary intake were investigated. Fish were fed daily ad libitum with diets containing 0.5% and 1.0% tetra for 3 weeks. After this period, fish were switched back to the basal diet for additional 6 weeks. Extracellular and intracellular respiratory burst activities, phagocytosis in blood leukocytes, lysozyme activities, and total plasma protein levels were evaluated at the end of the tetra feeding period and every 3 weeks during the basal diet period. Extracellular and intracellular respiratory burst activities, phagocytic activity, lysozyme activity and total protein level parameters of the groups containing 0.5% and 1.0% tetra were higher than the control group at the end of the 3rd, 6th and 9th weeks, respectively (P < 0.05). The highest values of the non-specific immune parameters were observed in the group fed with 1.0% tetra. Tetra groups did not show any significant difference (P > 0.05) in terms of specific growth rate and average weight of the fish.     

Compliance and smooth muscle reactivity of rainbow trout (Oncorhynchus mykiss) vessels in vitro

Systemic veins have a profound influence on cardiac output in mammals. Venoregulatory mechanisms have not been adequately studied in fish and their existence has been questioned. In the present study, two characteristics of vascular mechanics, compliance and agonist-induced tension development, were investigated in rainbow trout vessels in vitro. Rapid compliance in the anterior cardinal vein and efferent branchial artery was calculated from step-wise changes in the volume-pressure curve of isolated vessel segments. Agonist-induced tension development was examined in four veins; anterior and posterior cardinals, intestinal and duct of Cuvier. Venous compliance was not altered in response to epinephrine, norepinephrine or angiotensin II, while efferent branchial artery compliance was decreased by 10^-6 mol·l^-1 epinephrine and norepinephrine but not angiotensin II. The ratios of venous to arterial compliance in vessels from two rainbow trout strains were similar (21:1 and 32:1) and consistent with the ratio reported for mammalian viens (24:1). Trout veins contracted in response to agonists in both an, agonist- and vesselspecific manner. The greatest tension per vessel wet weight was produced in anterior cardinal vein. The response pattern of anterior cardinal vein and duct of Cuvier were similar; acetylcholine, arginine vasotocin, epinephrine and norepinephrine, and the thromboxane A₂ agonist, U-44,069, produced approximately identical contractions, whereas angiotensin II was virtually ineffective. Conversely, angiotensin II was more potent than epinephrine in posterior cardinal vein. In cumulative dose-response experiments, epinephrine was equipotent in anterior cardinal vein and duct of Cuvier, whereas the latter was less sensitive to acetylcholine. Both atrial natriuretic peptide and sodium nitroprusside relaxed precontracted veins. This is the first study to determine compliance in fish vessels and the contractile nature of different rainbow trout veins. These findings suggest that venous tone and therefore cardiac output in fish may be regulated by neural or humoral mechanisms.Abbreviations ACH acetylcholine - ACV anterior cardinal vein - ANG II salmon asn¹-val⁵ angiotensin II - ANP rat atrial natriuretic peptide - AVT arginine vasotocin - DNR Department of Natural Resources - DOC duct of Cuvier - EBA efferent branchial artery - EC₅ threshold dose producing 5% maximal contraction - EC₅₀ dose producing 50% maximal contraction - EPI epinephrine - HI K⁺ 80 mmol·l^-1 - KCl IV, intestinal vein - NEPI norepinephrine - PBS phosphate buffered saline - PCV posterior cardinal vein - SNP sodium nitroprusside - U-44,069 thromboxane A₂ agonist     

Analysis of chorion hardening of eggs of rainbow trout, Oncorhynchus mykiss

We estimated changes of chorion hardness of rainbow trout (Oncorhynchus mykiss) egg by the use of three parameters, namely increase of resistance of an egg to rupture by extraneously applied pressure, decrease of solubility of chorion proteins in 8 mol/L urea and a change in the content of γ-glutamyl-ε-lysine crosslink. Unfertilized egg chorions became hardened after egg activation. During chorion hardening, 49, 56 and 65 kDa protein components of the chorion gradually disappeared, high molecular weight intermediates (113,160–170 and higher than 250 kDa) were newly formed and, finally, all components became undetectable by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The content of γ-glutamyl-ε-lysine (γ-Glu-ε-Lys) crosslink in the chorion increased after hardening. Chorion hardening was inhibited by the incorporation of monodansyl-cadaverine, a competitive inhibitor for transglutaminase (TGase), into the chorions. TGase activity was detected in unfertilized eggs and localized in the chorion fraction rather than in the ooplasmic fraction. The findings suggest that chorion hardening depends upon polymerization of the chorion components by TGase-dependent γ-Glu-ε-Lys crosslink formation.     

Formation of the vitelline envelope precedes the active uptake of vitellogenin during oocyte development in the rainbow trout,Oncorhynchus mykiss

In order to study the initial formation of the vitelline envelope and the appearance of vitellogenin in oocytes of rainbow trout, females were sampled monthly from 19 to 5 mo before ovulation. lmmunohistochemistry revealed that the formation of the vitelline envelope starts when the oocytes reach a diameter of about 450 μm. Oocytes of this size were first found in females sampled a year before ovulation at the time when plasma levels of estradiol-17β increased from 0.2 to 0.6 ng/ml. An antiserum directed against vitellogenin crossreacted with small vesicles (around 2 μm) present just inside the oolemma, when the oocytes reached a diameter of 600 μm. This was interpreted as an active uptake of vitellogenin. Oocytes of this size were first found in females sampled 9 mo before ovulation at the time when estradiol 17β levels increased from 0.6 to 1.0 ng/ml and the gonadal somatic index was doubled. Oocytes with a diameter of 600 μm had an immunoreactive vitelline envelope with a thickness of about 3 μm. It is apparent that the initial formation of the vitelline envelope starts before the active uptake of vitellogenin and that the low previtellogenic plasma levels of estradiol-17β observed in females are of physiological significance. © 1994 Wiley-Liss, Inc.     

Sublethal ammonia and urea concentrations inhibit rainbow trout (Oncorhynchus mykiss) erythrocyte glucose-6-phosphate dehydrogenase

In vitro and in vivo effects of sublethal ammonia and urea concentrations were assayed on glucose-6-phosphate dehydrogenase (G6PD) of rainbow trout (Oncorhynchus mykiss) erythrocyte. G6PD was purified from erythrocytes with a specific activity of 16.7 EU (mmol NADP+/min)/mg protein and approximately 1600-fold in a yield of approximately 60% by ammonium sulphate precipitation and 2',5'-ADP Sepharose 4B affinity chromatography. The purity of the enzyme was confirmed using SDS polyacrylamide gel electrophoresis. Experiments with ammonia (2.2-5.5 microM) and urea (20-50 microM) showed the inhibitory effects on the enzyme, in vitro. Inhibition effects were determined in vitro by Lineweaver-Burk and regression graphs. The dissociation constant of the enzyme inhibitor complex (Ki) and 50% inhibitory values were 2.26+/-1.21 and 2.86+/-3.51 microM for ammonia and 18.69+/-6.75 and 23.77+/-4.58 microM for urea, respectively. In vivo studies in rainbow trout erythrocytes showed significant (p < 0.01) inhibition of G6PD by ammonia and urea. However, ammonia inhibited more than urea since there were significant differences between the final values of erythrocyte G6PD activities.     

Passive and active transport properties of a gill model, the cultured branchial epithelium of the freshwater rainbow trout (Oncorhynchus mykiss)

Branchial epithelia of freshwater rainbow trout were cultured on permeable supports, polyethylene terephthalate membranes ("filter inserts"), starting from dispersed gill epithelial cells in primary culture. Leibowitz L-15 media plus foetal bovine serum and glutamine, with an ionic composition similar to trout extracellular fluid, was used. After 6 days of growth on the filter insert with L-15 present on both apical and basolateral surfaces, the cultured preparations exhibited stable transepithelial resistances (generally 1000-5000 ohms cm2) typical of an electrically tight epithelium. Under these symmetrical conditions, transepithelial potential was zero, and unidirectional fluxes of Na+ and Cl- across the epithelium and permeability to the paracellular marker polyethylene glycol-4000 (PEG) were equal in both directions. Na+ and Cl- fluxes were similar to one another and linearly related to conductance (inversely related to resistance) in a manner indicative of fully conductive passive transport. Upon exposure to apical fresh water, transepithelial resistance increased greatly and a basolateral-negative transepithelial potential developed. At the same time, however, PEG permeability and unidirectional effluxes of Na+ and Cl- increased. Thus, total conductance fell, and ionic fluxes and paracellular permeability per unit conductance all increased greatly, consistent with a scenario whereby transcellular conductance decreases but paracellular permeability increases upon dilution of the apical medium. In apical fresh water, there was a net loss of ions from the basolateral to apical surfaces as effluxes greatly exceeded influxes. However, application of the Ussing flux ratio criterion, in two separate series involving different methods for measuring unidirectional fluxes, revealed active influx of Cl- against the electrochemical gradient but passive movement of Na+. The finding is surprising because the cultured epithelium appears to consist entirely of pavement-type cells.     

Marker-assisted estimation of quantitative genetic parameters in rainbow trout,Oncorhynchus mykiss

Estimation of quantitative genetic parameters conventionally requires known pedigree structure. However, several methods have recently been developed to circumvent this requirement by inferring relationship structure from molecular marker data. Here, two such marker-assisted methodologies were used and compared in an aquaculture population of rainbow trout (Oncorhynchus mykiss). Firstly a regression-based model employing estimates of pairwise relatedness was applied, and secondly a Markov Chain Monte Carlo (MCMC) procedure was employed to reconstruct full-sibships and hence an explicit pedigree. While both methods were effective in detecting significant components of genetic variance and covariance for size and spawning time traits, the regression model resulted in estimates that were quantitatively unreliable, having both significant bias and low precision. This result can be largely attributed to poor performance of the pairwise relatedness estimator. In contrast, genetic parameters estimated from the reconstructed pedigree showed close agreement with ideal values obtained from the true pedigree. Although not significantly biased, parameters based on the reconstructed pedigree were underestimated relative to ideal values. This was due to the complex structure of the true pedigree in which high numbers of half-sibling relationships resulted in inaccurate partitioning of full-sibships, and additional unrecognized relatedness between families.