Genetic analysis of the mating system of the common brushtail possum (Trichosurus vulpecula) in New Zealand farmland

We examined male reproductive success in a common brushtail possum population in New Zealand farmland. Paternity was assigned to 66 of 91 pouch young (maternity known), using a likelihood approach applied to genotypes at six microsatellite loci having an overall average exclusion probability of around 99%. The distribution of number of offspring per male was L-shaped with a standardized variance of 1.52. At least 46% of the 76 sampled reproductively mature males, bred, siring between one and four offspring each. Although breeding males were on average older and larger than nonbreeding males, the small differences did not result in a significant overall difference between the two groups in a multivariate permutation test analagous to a t-test. Paternity analysis of 22 sibling pairs (resulting from experimental removal of pouch young early in the breeding season, inducing a second oestrous) suggested that sequential mating of females with the same male was uncommon ( approximately 16-27%). Whilst there was a tendency for female possums to mate with nearby males, consistent with previous observations of territorial mating behaviour in Australian populations, some interhabitat matings were also inferred. The study population displayed only a low degree of polygyny, which may in part reflect population and habitat characteristics of the study site. A comprehensive understanding of the mating system of Trichosurus vulpecula awaits genetic paternity analysis in additional populations from both Australia and New Zealand, using quantitative approaches undertaken in this study.     

Social interactions among captive brushtail possums (Trichosurus vulpecula)

A biological control strategy relying on a self-disseminating agent may provide the only affordable long-term technique for reducing brushtail possum (Trichosurus vulpecula) numbers throughout New Zealand. The objective of this study was to determine the frequency and patterns of social interactions in single and mixed-sex groups of possums, in order to identify interactions that may assist in the dissemination of biocontrol agents. Thirty-two sexually mature wild-caught possums (16 female, 16 male) were housed in captive groups (of four) in enclosures during the breeding and non-breeding seasons. Groups consisted of either all females, females and males or all males. Four types of social interactions were defined: threats, fights, affiliative and sexual interactions. Interactions only occurred between two animals at one time and were most frequent in the non-breeding season. Within all female groups, possums readily engaged in interactions that had either an agonistic or affiliative nature. Mixed-sex groups interacted less frequently than females, but also engaged in both agonistic and affiliative interactions. In contrast, possums in all male groups rarely interacted, with only a few fights and no affiliative interactions observed. Some mixed-sex dyads appeared to 'associate' during the breeding period. Young were produced by three females that regularly associated with a male and one female that showed little associative behaviour. Given the different interaction patterns observed in each group type, biological control agents that rely on specific interaction patterns for dissemination, are likely to spread at different rates among different possum groups and in different seasons.     

Den sharing behaviour of captive brushtail possums (Trichosurus vulpecula)

Abstract

Den sharing among wild brushtail possums (Trichosurus vulpecula) has important implications for disease transmission. This study investigated den sharing in captive possums, and measured interactions between possums sharing dens. Thirty‐four sexually mature possums (16 female, 18 male) were housed in single‐sex or mixed‐sex pairs in large enclosures that contained two dens. Daily patterns of den sharing were recorded for each pair over a 69 day period in the breeding or non‐breeding season. Social behaviour within shared dens was sampled using miniature infrared cameras. Male pairs rarely shared dens in the breeding or non‐breeding seasons (4% and 1% of days respectively) and usually engaged in ‘threats’ and ‘fights’ associated with den defence. Pairs of female possums (in both seasons) and mixed‐sex pairs housed together in the breeding season shared dens most frequently (between 84% and 91% of days), and also spent the most time together in dens each night. While sharing dens, affiliative interactions were frequent, including long periods of ‘touching’, and also ‘food sharing’ and ‘allogrooming’. The preference for den sharing and close contact shown by captive possums highlights the importance of den sharing as a potential route for disease transmission.     

Follicle development in cyclic, anoestrous and FSH-treated brushtail possums (Trichosurus vulpecula)

The common brushtail possum (Trichosurus vulpecula) is a pest of considerable economic importance in New Zealand. Attempts to develop methods of suppressing reproduction in this species are currently hampered by the lack of reliable methods to synchronise oestrus and ovulation in this species. The objective of this study was to compare antral follicle populations in anoestrous and cyclic brushtail possums and to assess the efficacy of exogenous FSH to induce follicle development in anoestrous animals. Ovaries were recovered from anoestrous possums after administration of either exogenous FSH (1.0 mg/injection) or the saline vehicle alone (0.5 ml/injection) at 12-h intervals for 3 days (n = 6/group), and from cyclic animals (n = 6) that were euthanised in mid-follicular phase (5 days after removal of their pouch young). All antral follicles > or =1.0 mm in diameter were dissected free of extraneous tissue, incubated in vitro to measure oestradiol production, and then processed for histological assessment of health status. Mean weight of ovaries and vaginal cul-de-sac tissues were both significantly greater (P<0.001) in FSH-treated anoestrous females (24.2+/-5.1 mg and 6.50+/-1.34 g, respectively), but did not differ significantly between saline-treated anoestrous possums (12.4+/-3.0 mg and 1.31+/-0.27 g) and cyclic animals (13.5+/-1.6 mg and 2.62+/-0.95 g). Mean uterine weights in both cyclic (889+/-161 mg) and FSH-treated (1098+/-184 mg) animals were significantly heavier(P<0.001) than those of anoestrous possums (414+/-61 mg). The mean number of follicles (> or =1.0-mm diameter) present was significantly greater (P<0.001) in FSH-treated, than in cyclic and anoestrous possums (38.0+/-4.4, 23.2+/-3.2 and 10.7+/-3.4 follicles/animal, respectively). Cyclic animals had significantly more (P<0.01) follicles than anoestrous possums. The proportion of follicles that were classified as healthy, was significantly lower (P<0.01) in cyclic possums(38%) than in anoestrous (69%) and FSH-treated (88%) animals. The mean diameter of the largest healthy follicle present was 2.5+/-0.41, 2.1+/-0.08, and 3.1+/-0.15 mm for cyclic, anoestrous and FSH-treated animals, respectively. None of the follicles harvested from saline-treated anoestrous possums produced measurable levels of oestradiol in vitro, whereas 7% and 59% of those from cyclic and FSH-treated animals did so. In summary, cyclic possums had more antral follicles present than anoestrous animals, but a lower percentage of these follicles were healthy. Less than 10% of healthy follicles from cyclic possums, and none of those from anoestrous animals, were capable of producing oestradiol when incubated in vitro. Treatment with ovine FSH promoted follicle development in anoestrous possums, to significantly increase the number of follicles present, the proportion that were healthy and the percentage capable of producing oestradiol.     

Sexual dimorphism and synchrony of breeding: variation in polygyny potential among populations in the common brushtail possum, Trichosurus vulpecula

Male-biased sexual dimorphism in both size and body mass isa common characteristic of many polygynous mammals and is oftenattributed to sexual selection favoring large males. The degreeof dimorphism is thought to be related to the potential forsome males to monopolize access to estrous females, which isin turn related to the distribution of receptive females inspace and time. In the present study, we investigated the relationshipbetween the temporal distribution of breeding females and thedegree of mass dimorphism among 11 populations of the commonbrushtail possum from northern Australia. Breeding patternsvaried from complete aseasonality in some populations to a seasonalconcentration of births within 2 or 3 months in others. We predictedthat in populations in which mating opportunities were distributedthroughout the year, dominant males would be able to monopolizeaccess to larger numbers of estrous females than in populationsin which matings were more seasonal, and in such populations,large body size in males would be favored. We found that dimorphismwas related to seasonality of breeding, being greatest in populationswith a more aseasonal pattern. Mean body mass of male possumsalso decreased with increasing population density. Populationdensity may influence the degree of breeding synchrony withinpopulations, particularly in locations with a more seasonalclimate. The present study is the first to demonstrate plasticityin mass dimorphism in response to local variation in the synchronyof breeding in a mammal species.     

Understanding home range behaviour and resource selection of invasive common brushtail possums (Trichosurus vulpecula) in urban environments

Because invasive species are often opportunistic and behaviourally flexible, they tend to be successful in urban landscapes, where they may use space differently than in their more traditional habitats. Consequently, control strategies developed for invasive pest species in non-urban areas may not succeed in an urban context. Using GPS tracking, we examined habitat use by an invasive pest, the brushtail possum (Trichosurus vulpecula) in residential New Zealand habitats; these habitats comprise a large proportion of the urban landscape and are currently not targeted for pest control. We predicted that home ranges of possums in residential areas would be larger than those in non-urban habitats due to lower densities of their primary food resource, plant material; that possums would prefer forest fragment habitat; and that they would avoid roads due to the mortality risk. Home range sizes estimated using mechanistic Brownian bridges and 100 % minimum convex polygons were smaller or similar in size to those recorded in rural and forest habitats. Resource utilization functions and the regression of utilization distribution probabilities against environmental variables showed that possums selected forest fragments primarily, followed by residential habitat characterised by large gardens with a degree of vegetative structural complexity. Roads were not avoided. Control operations should expand their scope to include residential areas containing mature, diverse vegetation rather than only focussing on forest fragments, a strategy that ignores biodiversity values in residential habitats, and is likely to be unsuccessful due to reinvasion potential from neighbouring residential areas.     

Maternal influence on philopatry and space use by juvenile brushtail possums (Trichosurus vulpecula)

1. The causes of juvenile sex-biased philopatry and space use in mammals remain poorly understood, and results of previous research have been conflicting. Experimental interventions and manipulations on wild populations are rare, but can play an important role in establishing the factors governing offspring space use. 2. We experimentally removed mothers of independent juvenile brushtail possums from the maternal home range and examined changes in offspring space use with global positioning system collars. We examined the influence of mother absence on philopatric behaviour, and determined whether or not maternal presence affected offspring space use. 3. We fitted a longitudinal linear mixed effects model to demonstrate a change over time in the home range size of juveniles following experimental treatment by the removal of their mothers. When mothers were removed from the natal range, juveniles occupied significantly larger home range areas, with average increases of 175% in 95% kernel density estimates and 289% in minimum convex polygon estimates. This increase occurred within the first month following mother absence and was independent of juvenile sex. Home ranges of control juveniles did not change during the same time period. 4. Changes in the spatial structure of mammalian populations in response to removal of individuals have important implications for pest management. The impacts of management strategies which target particular individuals in a population may counteract conservation benefits through their effect on the space use of survivors. Studies involving experimental removals provide important information on consequences of control and also yield insights into the causes of mammalian space use, philopatric behaviours and ultimately dispersal.     

Long-term paternity skew and the opportunity for selection in a mammal with reversed sexual size dimorphism

Most mammalian groups are characterized by male-biased sexual size dimorphism, in which size-dependent male-male competition and reproductive skew are tightly linked. By comparison, little is known about the opportunity for sexual selection in mammalian systems without male-biased dimorphism, where the traits under sexual selection might be less obvious. We examined 10 years of parentage data in a colony of greater horseshoe bats (Rhinolophus ferrumequinum) to determine the magnitude of male reproductive skew and the opportunity for sexual selection in a mammal in which females are the larger sex. Annual paternity success was weakly skewed but consistent patterns led to strong longitudinal paternity skew among breeders. Just three males accounted for a third of all paternity assignments, representing at least a fifth of all colony offspring born in a decade. Paternity success was in part determined by age but was not influenced by dispersal status. Our results show that paternity skew and the opportunity for sexual selection in a species with reversed sexual size dimorphism can approach levels reported for classical examples of species with polygyny and male-biased dimorphism, even where the traits under sexual selection are not known.     

Sequence and analysis of zona pellucida 2 cDNA (ZP2) from a marsupial,the brushtail possum,Trichosurus vulpecula

All mammalian eggs are surrounded by the zona pellucida, an extracellular coat involved in vital functions during fertilization and early development. The zona pellucida glycoproteins are promising antigenic targets for development of contraceptive vaccines to control pest populations of marsupials in Australia and New Zealand. Our current understanding of the function of the zona pellucida glycoproteins is based almost entirely on the mouse and may not be representative of gamete interactions in all eutherian or marsupial mammals. This study reports the isolation and characterization of the ZP2 gene from the brushtail possum (Trichosurus vulpecula). The brushtail possum ZP2 mRNA is 2,182 nucleotides long with an open reading frame coding for a polypeptide chain of 712 amino acids with a molecular mass of 79,542 d. The deduced amino acid sequence of possum ZP2 is 48 to 55% identical to that of eutherian mammals. It shares several structural characteristics including N-linked glycosylation sites, location and number of cysteine residues, and hydropathy profile. The brushtail possum ZP2gene is expressed exclusively in the ovary. Further studies are planned to elucidate the specific site of ZP2 expression within the ovary and its function during fertilization in marsupials. Mol. Reprod. Dev. 51:322–329, 1998. © 1998 Wiley-Liss, Inc.     

VAP1, with cystatin C motif, an oocyte protein encoded by a novel ovarian-specific gene during oogenesis in the common brushtail possum (Trichosurus vulpecula)

In the brushtail possum oocyte, vesicles accumulate in a polarized fashion at the vegetal pole and cytoplasm rich in mitochondria and containing the germinal vesicle comprise the animal pole. During cleavage to early blastocyst stages, animal pole cytoplasm locates to the cells of the embryonic hemisphere (pluriblast) and vegetal pole vesicular cytoplasm to cells of the abembryonic hemisphere (trophoblast). Previously identified 16 amino acid residues, associated with the vesicle-rich cytoplasm were used for molecular cloning and characterization of a vesicle associated protein, VAP1. The degenerate primer was used in a 3'RACE for vap1 gene cloning. The cDNA encoding VAP1 was 516 bp in length with no significant homologies and coded for 172 amino acid residues for the mature protein. The N-terminal domain of VAP1 showed a structural homology to the cysteine protease inhibitor, Cystatin. Gene expression studies during oogenesis revealed that vap1 had an ovary-specific, possibly oocyte-specific expression, which occurs during follicle formation and growth and in adult ovaries. Recombinant VAP1 fusion protein generated polyclonal antibodies in the mouse and in the brushtail possum.     

Acid loads induced by the detoxification of plant secondary metabolites do not limit feeding by common brushtail possums (Trichosurus vulpecula)

We fed common brushtail possums artificial diets containing a buffer and the plant secondary metabolite (PSM), orcinol, to test the hypothesis that organic acids, common products of PSM metabolism, limit feeding by common brushtail possums (Trichosurus vulpecula). We introduced several diets containing orcinol and a buffer (urinary alkalising agent) over a course of three experiments. A diet containing 2% orcinol (wet matter) caused possums to reduce their food intake immediately, but feeding returned to normal 1–2 days later. Even though possums excreted strongly acidic urine (pH 5.1) and had perturbed nitrogen metabolism, they maintained their food intake and body mass until the experiment terminated 9 days after the introduction of orcinol. Possums ate 52% less when the basal diet contained 4% orcinol. As expected, the acid loads caused a change in the composition of urinary nitrogen with possums excreting more ammonium than urea and a large amount of unidentified nitrogenous material. Supplementing the diet containing orcinol with buffer neutralised the metabolic acid load and partly restored normal nitrogen metabolism, but did not restore feeding. Also, animals eating orcinol excreted normal amounts of 3-methylhistidine, indicating no increase in muscle protein catabolism. This suggests that a limitation to the rate of detoxification or toxicosis, rather than acid loads, limits the ingestion of acid-inducing PSMs.     

Endogenous type D retrovirus in a marsupial, the common brushtail possum (Trichosurus vulpecula)

We have sequenced and characterized an endogenous type D retrovirus, which we have named TvERV(D), from the genome of an Australian marsupial, the common brushtail possum (Trichosurus vulpecula). Intact TvERV(D) gag, pro, pol, and env open reading frames were detected in the possum genome. TvERV(D) was classified as a type D retrovirus, most closely related to those of Old World monkeys, New World monkeys, and mice, based on phylogenetic analyses and genetic organization. Approximately 30 TvERV(D) proviruses are present in the genomes of possums, as detected by Southern hybridization. However, variability in fragment patterns between possums was observed and suggests recent (or ongoing) retrotranspositional activity.     

Leptospirosis serology in the common brushtail possum (Trichosurus vulpecula) from urban Sydney, Australia

The common brushtail possum (Trichosurus vulpecula) is indeed a common marsupial in major cities of Australia. This species is known to be susceptible to leptospirosis and often lives in close contact with humans, raising concerns about the potential for transmission of this disease in urban areas. A total of 192 brushtail possum blood samples were collected from 136 individuals in suburban areas of metropolitan Sydney from November 2002 to November 2004. Sera were screened against a reference panel of 21 Leptospira spp. using the microscopic agglutination test. Leptospiral antibodies were detected in 9.6% (13/136) of tested brushtail possums and represented two serovars; antibodies to Leptospira interrogans serovar Hardjo were most frequently identified (11/136). A representative of the exotic sero-group Ballum, most likely serovar Arborea, was found in two of 136 brushtail possums. Exposure to leptospirosis seemed to be associated with age, as older animals had a higher incidence, but there was no distinction in relation to gender. Antibody prevalence varied between the different sampling sites and seropositive animals were clustered and restricted to a few sites. These data support the possible role of brushtail possums as a maintenance host for Leptospira spp. in urban environments and also identified them as a previously unknown and potential source of serovar Arborea.     

Chemical characterization of milk oligosaccharides of the common brushtail possum (Trichosurus vulpecula)

Structural characterizations of marsupial milk oligosaccharides have been performed in only three species: the tammar wallaby, the red kangaroo and the koala. To clarify the homology and heterogeneity of milk oligosaccharides among marsupials, 21 oligosaccharides of the milk carbohydrate fraction of the common brushtail possum were characterized in this study. Neutral and acidic oligosaccharides were separated from the carbohydrate fraction of mid-lactation milk and characterized by ¹H-nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The structures of the 7 neutral oligosaccharides were Gal(β1-3)Gal(β1-4)Glc (3’-galactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3”, 3’-digalactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I), Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl lacto-N-novopentaose I), Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)Gal(β1-4)Glc (galactosyl lacto-N-novopentaose II). The structures of the 14 acidic oligosaccharides detected were Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Glc (sialyl 3’-galactosyllactose), Gal(β1-3)(O-3-sulfate)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate a) Gal(β1-3)[Gal(β1-4)(O-3-sulfate)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)(?3-O-sulfate)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)[Gal(β1-4)(?3-O-sulfate)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(?3-O-sulphate)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)(?3-O-sulphate)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)(?3-O-sulphate)GlcNAc(β1-6)]Gal(β1-4)Glc and Gal(β1-3)Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl sialyl lacto-N-novopentaose b). No fucosyl oligosaccharides were detected. Galactosyl lacto-N-novopentaose II, lacto-N-novopentaose I sulfate a, lacto-N-novopentaose I sulfate b and galactosyl sialyl lacto-N-novopentaose b are novel oligosaccharides. The results are compared with those of previous studies on marsupial milk oligosaccharides.     

Presence of males affects the incidence of ovulation after pouch young removal in brushtail possums (Trichosurus vulpecula)

The traditional method for inducing and synchronising oestrus in the brushtail possum (Trichosurus vulpecula) is by removal of their suckling pouch young (RPY). However, our studies have recently shown that, in addition to wide variation between animals in the time of ovulation after RPY, a proportion of animals failed to ovulate. Evidence from several mammalian species indicates that the presence of males can stimulate ovarian activity and synchronise oestrus in females. The aim of this study was to determine the effect of the male on the oestrous cycle of the female brushtail possum after RPY. A total of 67 adult female brushtail possums were treated as three replicates. In order to observe the day of preovulatory follicle emergence and ovulation, animals underwent laparoscopic examination at 1–4 day intervals over a period from 0–21 days after RPY. The first replicate (N=18, May/June 1995) involved only animals kept in isolation from males, whereas the two remaining replicates compared ovarian responses between animals kept with (N=10, July 1995; N=14, June 1996) or in isolation from (N=10, July 1995; N=15, June 1996) males. The incidence of ovulation after RPY was significantly higher in females that were housed with males than in those kept in isolation from males (100%, 92.8% vs. 50.0%, 66.7%, 14.3%; P<0.001). Every animal that ovulated, had previously had a preovulatory follicle present at the site where the corpus luteum formed. Conversely, none of the animals that failed to ovulate, developed a preovulatory follicle during the period of study. The range of mean day of preovulatory follicle emergence (6.00–6.86 days), of ovulation (11.80–12.20 days) and the synchrony of ovulation between animals (range 8–17 days) after RPY, were not significantly affected by the presence of males. This study demonstrates for the first time, that the presence of males significantly increases the incidence of ovulation after RPY in the brushtail possum. However neither the timing of reproductive events nor the synchrony of ovulation were affected by presence of the male.     

Body size and sexual size dimorphism in calanoid copepods

Patterns of sexual size dimorphism and body size in calanoid copepods are examined. We hypothesize that favorable conditions for development will result in large body size and high sexual size dimorphism among populations of a given species and that differences in this allometric relationship among species is governed by the male's role in insemination. We confirm that there is a greater advantage to large female size, normally the larger sex, when compared to males, hence leading to selection for developmental patterns favoring high size dimorphism. Individuals from populations of four centropagid copepod species were measured; other sizes were obtained from published sources. In the four species we examined, the relationships between prosome length and both clutch size and the ability to produce multiple clutches with one insemination were determined. Results show a trend toward hyperallometry in all centropagid species examined: sexual size dimorphism increases with increasing size. Large females produce larger clutches and more additional clutches on one insemination. That hyperallometry is not observed in diaptomid copepods may result from the greater role the male plays in reproduction. Males are needed for each clutch produced, hence the selective pressure to be larger is greater than that in the centropagidae.     

Isolation and characterisation of a cDNA encoding a zona pellucida protein (ZPB) from the marsupial Trichosurus vulpecula (brushtail possum)

We have cloned a cDNA containing the entire coding sequence of a marsupial (the brushtail possum, Trichosurus vulpecula) zona pellucida protein (ZPB). The open reading frame of 1,581 nt is predicted to encode a ZPB polypeptide of 527 amino acids which contains 20 cysteine residues, 7 potential N‐linked glycosylation sites, a potential N‐terminal signal peptide and a potential C‐terminal trans‐membrane domain, preceded by a furin proteolytic processing signal. Sequence comparisons between possum ZPB and orthologous polypeptides from 7 eutherian species and from Xenopus laevis, reveal the existence of a high degree of sequence similarity, particularly in the central portion of the molecule. Cysteine residues are highly conserved, and all nine species possess potential N‐terminal signal peptide sequences and C‐terminal trans‐membrane domains of approximately the same length. In situ hybridisation revealed that expression of ZPB was restricted to oocytes of primordial and primary follicles of adult possums; no expression was detected in the surrounding granulosa cells. The broad conservation of ZPB sequence, structure and expression over a wide range of mammalian species, revealed by our studies, makes it unlikely that these features account for the different properties of the marsupial and eutherian zona pellucidae. Mol. Reprod. Dev. 52:174–182, 1999. © 1999 Wiley‐Liss, Inc.