Haplotype structure,adaptive history and associations with exploratory behaviour of the DRD4 gene region in four great tit (Parus major) populations

The assessment of genetic architecture and selection history in genes for behavioural traits is fundamental to our understanding of how these traits evolve. The dopamine receptor D4 (DRD4) gene is a prime candidate for explaining genetic variation in novelty seeking behaviour, a commonly assayed personality trait in animals. Previously, we showed that a single nucleotide polymorphism in exon 3 of this gene is associated with exploratory behaviour in at least one of four Western European great tit (Parus major) populations. These heterogeneous association results were explained by potential variable linkage disequilibrium (LD) patterns between this marker and the causal variant or by other genetic or environmental differences among the populations. Different adaptive histories are further hypothesized to have contributed to these population differences. Here, we genotyped 98 polymorphisms of the complete DRD4 gene including the flanking regions for 595 individuals of the four populations. We show that the LD structure, specifically around the original exon 3 SNP is conserved across the four populations and does not explain the heterogeneous association results. Study‐wide significant associations with exploratory behaviour were detected in more than one haplotype block around exon 2, 3 and 4 in two of the four tested populations with different allele effect models. This indicates genetic heterogeneity in the association between multiple DRD4 polymorphisms and exploratory behaviour across populations. The association signals were in or close to regions with signatures of positive selection. We therefore hypothesize that variation in exploratory and other dopamine‐related behaviour evolves locally by occasional adaptive shifts in the frequency of underlying genetic variants.     

Comparisons of damaging feather pecking and time budgets in male and female turkeys of a traditional breed and a genetically selected male line

Feather and skin pecking leading to feather loss and tissue damage is a welfare problem in commercial turkeys. A factorial experiment was designed to compare a line of unselected turkeys that previously did not exhibit this behaviour with a genetically selected male line with a propensity for damaging pecking. We also compared the time budgets of male and female turkeys from the two lines at 3, 6 and 9 weeks of age and at different times of the day. Damaging feather pecking occurred in 32% of male and 15% of female male line turkeys but was not observed in the traditional line. Time budgets of males and females were similar. Preening increased and resting, feeding and general pecking declined with age. Resting was higher in the afternoon than in the morning and male line turkeys were observed resting more often than traditional turkeys at 3 and 6 weeks. Traditional turkeys displayed more gentle feather pecks than male line poults at 9 weeks. Male turkeys of both lines showed more strong feather pecks and pulls at 3 weeks whereas female turkeys showed more at 9 weeks of age. The frequency of strong feather pulls recorded electronically increased with age but was not related to mortality. It is suggested that damaging feather pecking in turkeys may be the result of vigorous investigative pecking.     

Characterization of a 320-kb region containing the HEXA gene on bovine chromosome 10 and analysis of its association with BSE susceptibility

Bovine spongiform encephalopathy (BSE) belongs to a group of neurodegenerative diseases known as transmissible prion diseases. Recently, variants in the promoter region of the prion protein ( PRNP ) gene have been shown to have a considerable effect on the susceptibility to BSE. However, a previous genome scan revealed other putative BSE-susceptibility loci. Here, we analysed such a region on BTA10, which contains the functional candidate gene HEXA . Three hundred and twenty kilobases that, besides HEXA , also contain ARIH1 , BRUNOL6 and PARP6 were characterized and screened for polymorphisms. Genotyping of 38 SNPs in Holstein–Friesian animals from the UK (350 diseased and 270 controls) revealed two intronic SNPs that were associated with BSE incidence, with experiment-wise P -values of 3.5 × 10⁻³ and 7.7 × 10⁻³ respectively. Both SNPs were in strong linkage disequilibrium and the rare alleles had a protective effect. These alleles were contained in a haplotype dubbed 'UK-protective' that was significantly overrepresented in the controls with a permuted P -value of 2 × 10⁻³. An association study in German Holstein animals (73 diseased and 627 controls) revealed an opposite effect of the 'UK-protective' haplotype in this population, i.e. it was overrepresented in the diseased animals, although not significant after correction for multiple testing. These findings indicate a causal variant for BSE susceptibility on BTA10 in linkage disequilibrium with the markers studied. Candidate gene analyses of the surrounding region and additional association studies will help to clarify the origin of the protective effects and to identify causal variants for BSE susceptibility on BTA10.     

Reaction to frustration in high and low feather pecking lines of laying hens from commercial or semi-natural rearing conditions

The effect of rearing conditions on feather pecking and reaction to frustration was studied in two lines of laying hens. From commercial rearing conditions (large group, no mother hen), seven birds from a high feather pecking line (HC birds) and eight birds from a low feather pecking line (LC birds) were used. From semi-natural rearing conditions (small group, mother hen present) seven birds from the high feather pecking line (HN birds) were used. Feather pecking behaviour of HC, LC, and HN groups was recorded for 30 min. After that, each bird was food deprived and trained to peck a key for a food reward in a Skinnerbox. After training, each bird was subjected to a frustration session in a Skinnerbox, where the feeder was covered with Perspex. Three HC birds showed severe feather pecking, compared with one HN bird and zero LC birds. Differences in reaction to frustration were found between birds from different lines, but not in birds from different rearing conditions. LC birds tended to put their head in the feeder more frequently than HC birds over all sessions. Although limited, this study indicates that rearing conditions influence feather pecking, but not reaction to frustration.     

Selection method and early-life history affect behavioural development, feather pecking and cannibalism in laying hens: A review

The aim of this review is to discuss the effects of selection method and early-life history on the behavioural development of laying hens. Especially in larger groups, laying hens often develop damaging behaviours, such as feather pecking and cannibalism, leading to impaired animal welfare. We hypothesise that the propensity to develop feather pecking and cannibalism is affected by a bird's genetic background and by its early-life history. The genetic background can be influenced by genetic selection. Laying hens are traditionally selected on individual performance, which may lead to co-selection of feather pecking and cannibalism. For hens kept in small groups, it has recently been demonstrated that a novel group selection method, focusing on group performance, can help to reduce cannibalism. However, the biological background behind the success of group selection is unknown. It is also not known whether these results from small groups can be translated to larger groups of laying hens. Regarding early-life history, laying, brooding and rearing conditions have been shown to have major effects on behavioural development and on feather pecking and cannibalism. The presence of a hen during rearing has been shown to improve foraging- and social behaviour, to decrease feather pecking and to decrease fearfulness in chicks. Applying group selection and rearing laying hens in a more natural environment may be key factors in solving the problems caused by feather pecking and cannibalism, especially if the promising results of group selection from small groups in experimental settings can be translated to large-group housing systems.     

Association between DRD4 gene polymorphism and personality variation in great tits: a test across four wild populations

Polymorphisms in the dopamine receptor D4 gene (DRD4) have been related to individual variation in novelty‐seeking or exploratory behaviour in a variety of animals, including humans. Recently, the human DRD4 orthologue was sequenced in a wild bird, the great tit (Parus major) and a single nucleotide polymorphism in exon 3 of this gene (SNP830) was shown to be associated with variation in exploratory behaviour of lab‐raised individuals originating from a single wild population. Here we test the generality of this finding in a large sample of free‐living individuals from four European great tit populations, including the originally sampled population. We demonstrate that the association between SNP830 genotype and exploratory behaviour also exists in free‐living birds from the original population. However, in the other three populations we found only limited evidence for an association: in two populations the association appeared absent; while in one there was a nonsignificant tendency. We could not confirm a previously demonstrated interaction with another DRD4 polymorphism, a 15 bp indel in the promoter region (ID15). As yet unknown differences in genetic or environmental background could explain why the same genetic polymorphism (SNP830) has a substantial effect on exploratory behaviour in one population, explaining 4.5–5.8% of the total variance—a large effect for a single gene influencing a complex behavioural trait—but not in three others. The confirmation of an association between SNP830 genotype and personality‐related behaviour in a wild bird population warrants further research into potential fitness effects of the polymorphism, while also the population differences in the strength of the association deserve further investigation. Another important future challenge is the identification of additional loci influencing avian personality traits in the wild.     

Induction of a wheat Em promoter by ABA and optically pure ABA analogs in white spruce (Picea glauca) protoplasts

In white spruce ( Picea glauca ) protoplasts, abscisic acid (ABA) and optically pure ABA analogs induced expression of a reporter gene under regulation of a wheat ABA-responsive promoter. A fusion of a 650 bp promoter fragment from the wheat Em gene promoter and the Escherichia coli uidA sequence encoding β -glucuronidase (GUS) was linked in the plasmid pBM 113Kp. Expression of the Em-uidA fusion varied among 6 white spruce genotypes. Protoplasts from 4-day-old embryogenic suspension cultures gave the highest GUS activity relative 10 other stages in the 7-day growth cycle of suspension cultures. Racemic ABA [R.S-(±)-ABA] induced a significant increase of protoplast GUS activity over background at a concentration of 1 × 10⁻⁵ M , but maximum GUS activity was found at 1 × 10⁻³ M , ABA stereochemistry had a significant effect on gene expression. The natural isomer of ABA [S-(+)-ABA] was an effective inducer at a concentration as low as 1 × 10⁻⁷ M , but a concentration of greater than 1 × 10⁻⁴ M was required for induction by [R-(—)-ABA]. Moreover, analogs with the same configuration at C-l¹ as that of natural ABA were more effective for induction of expression from the Em-uidA . insert at 1 × 10⁻⁴ M than were their enamiomers. Plasnud pBI511. carrying the chloramphenicol acety] transferase (CAT) gene driven by the constitutively expressed, tandemly duplicated cauliflower mosaic virus 35S promoter, was co-electroporated with pBM113Kp for monitoring Ihe influence of addition of exogenous ABA or ABA analogs on heterologous gene expression in protoplasts. CAT activity was not significantly affected by the presence or absence of ABA or the analogs used.     

Are genetic differences in foraging behaviour of laying hen chicks paralleled by hybrid-specific differences in feather pecking?

Feather pecking is a behavioural disorder in laying hens which consists of pecking the feathers of conspecifics, causing feather damage or even injuries to the skin. Its development can be explained by redirection of foraging behaviour. While the occurrence of feather pecking strongly depends on the kind of housing condition, it is also known that there are strain differences in the tendency to feather peck. From the inverse relation between feather pecking and foraging behaviour found earlier, we hypothesised that genetically determined differences in foraging behaviour could be responsible for the observed differences in feather pecking between strains.In a first experiment we tested whether there are differences in the foraging behaviour of two hybrids. As hybrids, we used Lohman selected leghorn (LSL) and Dekalb; eight groups of 20 1-day old chicks each. They were kept in enriched pens (265cmx90cm) with a litter area (200cmx90cm) consisting of wood-shavings, chaff, straw, polystyrene blocks, sand area (65cmx90cm) and elevated perches. Behavioural observations were carried out in week 4. In a subsequent experiment with the same birds we tested how the foraging behaviour of the two hybrids differed when housing conditions were changed from enriched to restricted and to what extent they developed feather pecking. A 2x2 factorial design with hybrid (LSL, Dekalb) and housing condition (restricted, enriched) as factors and with four replicates of each factor combination was used. Half of the pens of each hybrid were changed from enriched to restricted housing conditions by covering the litter area with slats. Behavioural observations were carried out in weeks 5 and 6.In experiment 1, LSL and Dekalb spent the same amount of time foraging, but Dekalb spent significantly more of that time with pecking and hacking at the polystyrene blocks. On the other hand, LSL spent significantly more time at the feeding troughs and rested significantly less than Dekalb. In the restricted environment of experiment 2, again, the total foraging time did not differ between hybrids, but LSL chicks spent significantly less time scratching, while Dekalb spent significantly more time moving. Both hybrids developed feather pecking but LSL showed significantly higher rates than Dekalb.Our results demonstrate genetic differences in the foraging behaviour and in the way hybrids cope with the change in housing condition from enriched to an environment that is restricted in relation to foraging possibilities. We conclude that the results support the hypothesis put forward that genetic differences in foraging behaviour could be the basis for the genetic influence in the development of feather pecking.     

Divergent selection on feather pecking behaviour in laying hens (Gallus gallus domesticus)

A selection experiment was initiated in 1996 in which selection for (HP line) and against (LP line) feather pecking was performed. The foundation stock was a White Leghorn layer strain established in 1970 and maintained since then as a random bred control line at the Institute. Six hatches were produced over three generations. At the age of 68 weeks (generation 0, 1996), 35 weeks (generation 1, 1997), 30 weeks (generation 2, 1998), and 27 weeks (generation 3, 1999) female birds were transferred to observation pens and their feather pecking behaviour was recorded. In each generation, 30 females and 8 males were selected from approximately 200 females and 60 males. The selection criterion was breeding value estimated by animal model on the trait 'number of bouts of feather pecking per bird per hour'.Feather pecking behaviour in adult hens was significantly higher in HP than in LP. In generation 2 the following was recorded: 3.10 versus 1.37 bouts per bird per hour (P<0.01), 7.04 versus 3.58 pecks per bird per hour (P<0.05) and the proportion of hens recorded feather pecking in the 180min observation period was 67 versus 56% (P<0.05). In generation 3 the following was recorded: 4.56 versus 0.63 bouts per bird per hour (P<0.001), 13.9 versus 2.51 pecks per bird per hour (P<0.001) and the proportion of hens recorded feather pecking in the 180min observation period was 75 versus 49% (P<0.001).In generation 3, plumage condition was better in LP on neck, breast, back, wings and tail, as well as overall (P<0.001). Body weight did not differ between lines in generation 2, but in generation 3, HP hens were on average heavier than LP hens at the age of 27 weeks (1435g versus 1371g, P<0.001).     

Relationship between feather pecking and ground pecking in laying hens and the effect of group size

The aim of this experiment was to study the relationship between feather pecking and ground pecking in laying hens and the effect of group size on feather pecking behaviour. Hisex White hens were kept in floor pens in group sizes of 15, 30, 60 and 120 birds, each with four replicates. Behavioural observations were performed at four different ages and focused on the number of feather pecks and aggressive pecks, both given and received. The part of the body pecked and the location of the bird was recorded as well as the number of pecks made to the floor, feeder and drinker.The results showed that most feather pecking activity occurred in the largest group size (120 birds) and there was some evidence of an increasing frequency of aggressive pecks with increasing group size. The parts of the body which were targets for feather pecking varied depending on the location of the bird giving the peck and the bird receiving it. When looking at the behaviour of individuals, birds doing a lot of feather pecking also showed more ground pecking.     

Evaluation of insecticidal and genotoxic effects of imidacloprid and acetochlor in Drosophila melanogaster

Two pesticides, the insecticide imidacloprid and the herbicide acetochlor, were evaluated for their insecticidal and genotoxic effects in Drosophila melanogaster . Their insecticidal effects were assessed by calculating LC₅₀ values after acute or chronic exposure of larvae and adults to different concentrations of the test compounds. After acute exposure, the LC₅₀ of imidacloprid was 7.59 × 10⁻⁵ M for larvae and 1.43 × 10⁻⁴ M for adults, and after chronic exposure, it was 2.67 × 10⁻⁵ M and 6.09 × 10⁻⁵ M for larvae and adults, respectively. On the contrary, the herbicide acetochlor showed no acute or chronic insecticidal effect against either larvae or adults, even at very high concentrations (8 × 10⁻³ M). For the evaluation of genotoxic properties of the two pesticides, the Somatic Mutation and Recombination Test in D. melanogaster was used. Our results suggest that neither imidacloprid nor acetochlor exhibits mutagenic or recombinogenic activity at applicable concentrations.     

Emission rate of amino acids and ammonia and their role in olfactory preference behaviour of juvenile Arctic charr, Salvelinus alpinus (L.)

The behaviour of juvenile Arctic charr, Salvelinus alpinus (L.) , to an abrupt concentration step of L-amino acids, L-alanine and ammonium chloride was studied by fluviarium technique. The emission rates of these substances were studied. Juvenile Arctic charr emit 8.0 × 10⁻⁴ mol total ammonia-N kg⁻¹ h⁻¹ and 3.3 × I0⁻⁵ mol amino acids kg⁻¹ h⁻¹. In behaviour tests the charr avoided 5.6x 10⁻⁶and 5.6 × 10⁻⁷ M ammonium chloride. The 17 L-amino acid mixture, ranked as observed in the analysis of emission, was avoided at 4.6 × 10⁻⁷ M, while 100 times dilution of this value gave neither avoidance nor attraction. The charr avoided L-alanine tested alone at the concentration of 4.6 × 10 ⁻⁷ M. Anosmic charr showed neither avoidance nor attraction to the mixture of 17 amino acids tested at 4.6 × 10⁻⁷ M. The results indicate that ammonia as well as emitted amino acids are not responsible for the olfactory mediated attraction to conspecific odour shown earlier in Arctic charr. On the contrary, these substances may have a negative effect by reducing the strength of attraction.     

Is there social transmission of feather pecking in groups of laying hen chicks?

Feather pecking is an abnormal behaviour where laying hens peck the feathers of conspecifics, damaging the plumage or even injuring the skin. If it occurs in a flock, more and more birds show it within a short period of time. A possible mechanism is social transmission. Several studies have shown that laying hen chicks, Gallus gallus domesticus, are able to modify their own behaviour when observing the behaviour of other chicks, for example, when feeding and foraging. As there is good experimental evidence that feather pecking originates from foraging behaviour, we hypothesized that feather pecking could also be socially transmitted. To test this, we reared 16 groups of 30 chicks. After week 4, the birds were regrouped into 16 groups of 20 chicks into each of which we introduced either five chicks that showed high frequencies of feather pecking or, as controls, five chicks that had not developed feather pecking. We then determined the feather-pecking rate and the frequency of foraging, dustbathing, feeding, drinking, preening, moving, standing and resting of all birds in a group. Data from the introduced birds were analysed separately and excluded from the group data. Chicks in groups with introduced feather-pecking chicks had a significantly higher feather-pecking rate than chicks in the control groups. In addition, birds in groups with introduced feather peckers showed significantly lower foraging frequencies than those in the control groups, although the housing conditions were identical and there were no differences in either the number or the quality of the stimuli relevant to foraging behaviour. The study therefore suggests that feather pecking is socially transmitted in groups of laying hen chicks. Copyright 2000 The Association for the Study of Animal Behaviour.     

Heart rate variability in domestic chicken lines genetically selected on feather pecking behavior

Domestic chicken lines of the White Leghorn type differing in their level of feather pecking were developed by divergent genetic selection specifically on feather pecking behavior. We determined parameters of heart rate variability to elucidate the relative activation of the sympathetic and parasympathetic nervous systems during rest and stressful situations. A total of 48 hens were tested in 8 batches. Segments of 2 min were extracted from electrocardiograms recorded by radio-transmitter implants, before (basal undisturbed conditions) and during physical restraint and a social test. Under basal conditions mean distance between R-waves were shorter in the low and high lines compared to the control. During physical restraint, stress reactions [reduced root of the mean squares of successive differences (RMSSD), reduced high frequency (HF), high low frequency (LF/HF) and low vagal-sympathetic effect (VSE) compared to basal levels] were significant in all lines. During the physical restraint the high feather pecking (HFP) line reacted significantly stronger than control (CON) and low feather pecking (LFP) line. During social test the LFP line reacted different than the other two lines. Seemingly birds from LFP conceived the social test as less stressful than birds from the CON and HFP lines. From this it follows that (1) physical restraint generally induced higher stress reactions than the social test and (2) genetic selection for higher levels of feather pecking increased the autonomic nervous system reaction to physical restraint whereas selection against feather pecking has reduced the response to increased social contact.     

The presence of extreme feather peckers in groups of laying hens

Feather pecking is a serious economic and welfare problem in laying hens. Feather damage occurs mainly through severe feather pecking (SFP). Selection experiments have proved that this behavior is heritable and lines have been divergently selected for high (HFP) and low feather pecking (LFP). The number of bouts of SFP per hen follows a Poisson distribution with a maximum nearby 0. A few studies indicate that the distribution within flocks is not homogenous but contains sub-groups of birds showing extremely high levels of feather pecking (EFP). It was the aim of the current study to re-analyze data on SFP of lines selected for HFP/LFP and their F2 cross so as to uncover hidden sub-populations of EFP birds. Data of seven selection generations of HFP and LFP selection lines as well as their F2 cross have been used. We fitted a two-component mixture of Poisson distributions in order to separate the sub-group of EFP from the remaining birds. HFP and LFP lines differed mainly in mean bouts per bird. The proportion of EFP was only marginal in the LFP as compared with the HFP and the F2 population. Selection for LFP did not result in total elimination of EFP. The presence of even small proportions of EFP may play an important role in initiating outbreaks of feather pecking in large flocks. Further studies on feather pecking should pay special attention to the occurrence of EFP sub-groups.     

No evidence for strong recent positive selection favoring the 7 repeat allele of VNTR in the DRD4 gene

The human dopamine receptor D4 (DRD4) gene contains a 48-bp variable number of tandem repeat (VNTR) in exon 3, encoding the third intracellular loop of this dopamine receptor. The DRD4 7R allele, which seems to have a single origin, is commonly observed in various human populations and the nucleotide diversity of the DRD4 7R haplotype at the DRD4 locus is reduced compared to the most common DRD4 4R haplotype. Based on these observations, previous studies have hypothesized that positive selection has acted on the DRD4 7R allele. However, the degrees of linkage disequilibrium (LD) of the DRD4 7R allele with single nucleotide polymorphisms (SNPs) outside the DRD4 locus have not been evaluated. In this study, to re-examine the possibility of recent positive selection favoring the DRD4 7R allele, we genotyped HapMap subjects for DRD4 VNTR, and conducted several neutrality tests including long range haplotype test and iHS test based on the extended haplotype homozygosity. Our results indicated that LD of the DRD4 7R allele was not extended compared to SNP alleles with the similar frequency. Thus, we conclude that the DRD4 7R allele has not been subjected to strong recent positive selection.     

Reducing feather pecking when raising laying hen chicks in aviary systems

Aviary systems for laying hens offer several advantages over battery cages. However, pecking the feathers of conspecifics remains a serious problem that negatively affects the welfare of the birds as well as the economy of a farm. From experimental studies with small groups, it has been shown that feather pecking and foraging behaviour are related and that both behaviour are influenced by early access to litter substrate. We, therefore, hypothesised, that feather pecking in aviaries can be reduced with an adequate management in the first 2 weeks of life.Each of seven pens on six commercial poultry farms, was divided into two identical compartments (matched pair design). In one of the compartments (experimental compartment) chicks were reared for the first 2 weeks of life with access to litter (wood shavings, in one case with additional straw), while the chicks in the other compartment (control) were kept on a plastic grid. Thereafter, all chicks had unrestricted access to litter and there were no differences between the two compartments neither in housing conditions nor in management procedures.Chicks in the experimental compartments spent significantly more time foraging (week 5), showed significantly less feather pecking (weeks 5 and 14) and significantly fewer birds had damaged tail feathers (weeks 5 and 14).The study demonstrates that in aviaries, under commercial conditions, early access to litter substrate has a significant effect on the development of feather pecking. In order to reduce feather pecking and to increase foraging behaviour, it is recommended that laying hen chicks raised in aviary systems do get access to litter from day 1 on.     

Dopamine system genes associated with parenting in the context of daily hassles

The current study examined the molecular genetic foundations of sensitive parenting in humans and is the first to test the interaction between genes and environment in modulating parental sensitive responses to children. In a community sample of 176 Caucasian, middle class mothers with their 23-month-old toddlers at risk for externalizing behavior problems, the association between daily hassles and sensitive parenting was investigated. We tested whether two dopamine-related genes, dopamine D4 receptor ( DRD4 ) and catechol-O-methyltransferase ( COMT ) gene polymorphisms, modulate parents' vulnerability to the negative influence of daily hassles on sensitive parenting behavior to their offspring. Sensitive parenting was observed in structured settings, and parents reported on their daily hassles through a standard questionnaire. In parents with the combination of genes leading to the least efficient dopaminergic system functioning ( COMT val/val or val/met, DRD4 -7Repeat), more daily hassles were associated with less sensitive parenting, and lower levels of daily hassles were associated with more sensitive parenting d  = 1.12. The other combinations of COMT and DRD4 polymorphisms did not show significant associations between daily hassles and maternal sensitivity, suggesting differential susceptibility to hassles depending on parents' dopaminergic system genes. It is concluded that the study of (multiple) gene–environment interactions (in the current case: gene by gene by environment interaction, G × G × E) may explain why some parents are more and others less impacted by daily stresses in responding sensitively to their offspring's signals.     

Analysis of parentage determination in Atlantic salmon (Salmo salar) using microsatellites

Microsatellite genetic markers are becoming increasing important tools in the investigation of alternate reproductive strategies in wild plants and animals, and in the implementation of optimal breeding programs for endangered species, and managed cultured populations. Overall, little attention is paid to the frequency and impact of scoring errors and mutations on the resolution and accuracy of such analyses. Here, parentage of 792 Atlantic salmon ( Salmo salar ) reared communally were determined using di- and tetranucleotide microsatellites. Over 99·5% of the offspring could be unambiguously matched to one set of parents in the original 12 (1 × 1) experimental cross (each of 12 males uniquely crossed to one of 12 females) and in a simulated 36 (1 × 1) cross (involving additional parents), and over 80% in a 12 × 12 cross (all 12 males crossed to all 12 females). Mutations were rare (≈3·4 × 10⁻⁻⁴), though scoring errors were relatively common (2–3% per allele scored), with the rate of error varying among loci. Approximately 90% of scoring errors (or mutations) are expected to be detected in this analysis, and of those that are not, fewer than 0·5% should lead to a false or incorrect determinations of parentage. Based on several indices, we expect that greater than 99·7% of offspring assayed were matched to their true parents.