Reproduction in the marsupial dibbler, Parantechinus apicalis; differences between island and mainland populations

General and Comparative Endocrinology 178 (2012) 347–354

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Reproduction in the marsupial dibbler, Parantechinus apicalis; differences between island and mainland populations H.R. Mills a, F.J. Bradshaw a,⇑, C. Lambert b, S.D. Bradshaw a, R. Bencini a a b

School of Animal Biology M092, The University of Western Australia, Perth, WA 6009, Australia Research Section, Perth Zoological Gardens, Labouchere Road, South Perth, WA 6151, Australia

a r t i c l e

i n f o

Article history: Received 22 April 2012 Revised 11 June 2012 Accepted 12 June 2012 Available online 27 June 2012 Keywords: Marsupial Dibbler Faecal-steroids Gestation Island-syndrome Semelparity

a b s t r a c t Details of the reproductive endocrinology of the dibbler, Parantechinus apicalis, an endangered member of the Family Dasyuridae, are presented from two geographically-separated populations, living either on the mainland or on islands in Jurien Bay, Western Australia. Plasma free cortisol in males measured in the island population during 1998/9 did not differ between the breeding and non-breeding season, but during the March rut in 2000, when males died after breeding, free cortisol levels were significantly raised. Post-mating mortality in dibbler males is facultative, rather than obligatory and the cortisol data implicate the same physiological sequelae described in other dasyurids. In females, a single annual oestrus was recorded during late summer to autumn in both populations with an onset earlier by 12 days in the mainland animals. Faecal steroids excreted as progesterone metabolites (PM) and oestradiol-17b were measured during the annual oestrous period and showed significantly higher PM concentrations in island animals. Oestradiol, although raised, was not different between the two populations. A profile of PM levels throughout gestation revealed a small peak at the time of ovulation, followed by slowly rising levels to peak 8 days before birth, indicating slow development of the corpora lutea. Using collective data, the presumptive day of ovulation could be identified, allowing the calculation of a presumptive gestation length of 45 days in dibblers from mainland populations. This gestation length compares with that of a related species, Pseudantechinus macdonnellensis, reported at 45–55 days. A surprising finding is the significantly shorter gestation period of approximately 38 days in island animals compared with those from the mainland. This and other differences between reproductive parameters of island and mainland populations are discussed in the context of the ‘island syndrome’. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction The dibbler (Parantechinus apicalis) is a small insectivorous marsupial, belonging to the Family Dasyuridae, which was previously thought to be extinct until rediscovered on the south coast of Western Australia (WA) [40]. Its known distribution today is limited to a few localities along the south coast and two small islands, Boullanger and Whitlock, in Jurien Bay, north of Perth (WA). Because of their severely restricted range, dibblers are now classified as endangered under the Environment Protection and Biodiversity Conservation Act (1999). Detailed information on their reproductive biology is therefore essential, not only for understanding life-history strategies but also to provide the basis for a captive breeding programme and subsequent re-introductions. Earlier observations on the mainland dibblers recorded a single breeding period in the year during autumn (March and April), with males probably surviving to breed in a second year [64]. With the ⇑ Corresponding author. E-mail address: [email protected] (F.J. Bradshaw). 0016-6480/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2012.06.013

discovery of dibblers on the offshore islands [16], trapping details there recorded a post-mating mortality of males [13], similar to another dasyurid species Antechinus stuartii (now agilis) [30]. This was at variance with Woolley’s study of the island animals in which she had suggested that post-mating mortality in males ‘‘may not be an inevitable event’’, as cyclic changes in scrotal development and periods of spermatorrhoea suggested that males may be capable of breeding after April [66]. This has been supported by further studies that indicated a facultative, rather than obligatory, die-off in the males following mating [39,61]. Elevated levels of testosterone in the male dasyurid, A. stuartii (now agilis), during the mating season have been shown to depress synthesis of corticosteroid-binding globulin, CBG, by the liver. The consequent elevation of unbound or ‘free’ levels of cortisol leads to a series of pathological conditions, followed by death [7,8,29]. There is a need for hormonal studies on male dibblers in order to establish whether the male die-off is associated with similar sequelae reported in other semelparous dasyurid species. Woolley [64,66] records ‘‘pseudopregnancy’’ in captive island and mainland females as the period between a mating during

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oestrus, and the appearance of a glandular pouch, ranging from 43 to 53 days. Although she noted that mating was attempted, no young were born, but ovulation most probably occurred, as progesterone (P4) from the corpus luteum plays a principal role in developing the mammary ducts for lactation in marsupials [55,57]. This period may have represented a single oestrous cycle, but, as dibblers appear to be monoestrous, the term ‘pseudopregnancy’ may be more apposite. If the period represents a failed pregnancy, then the female dibbler has the longest reproductive cycle of all dasyurids studied to date. A mating, however, may not signal the beginning of a pregnancy, as dasyurid reproduction features frequent mating during a lengthy period of oestrous behaviour that is characterised by raised P4 and oestrogen (E2) levels [15,22,23,24,37,53,63]. The time of ovulation and subsequent fertilisation is thus concealed, making the actual length of gestation difficult to determine. A reliable indicator, however, is the conjunction between three parameters, a decrease in the prevalence of cornified epithelial cells (CEC), the appearance of polymorphonuclear leucocytes in urinogenital smears and a temporary fall in body mass [67]. A recent review of P4 throughout pregnancy in marsupials documents the occurrence of raised levels, both before and during oestrus in most dasyurid marsupials, which decline concurrently with the parameters described above [9]. Although largely unexplored in dasyurids, in the female grey short-tailed opossum (Monodelphis domestica), P4, in the presence of E2, plays the dual roles of inducing oestrous behaviour in the female and stimulating the cytological development of the reproductive tract [14,20,27]. The aims of the present study were (a) to document changes in plasma cortisol levels in male dibblers in order to establish whether high levels of unbound hormone are implicated in postmating mortality, (b) to gain insight into P4 and E2 levels associated with oestrus, (c) to document the parameters associated with ovulation, and thereby estimate the length of gestation in island and mainland populations of female dibblers and (d) to establish the secretory profile of P4 throughout a reproductive period. The endangered status of the dibbler has necessitated the use of faecal analysis of P4, excreted as P4 metabolites, or progestagens (PM), and E2. These methods are now widely used to detect periods of oestrus and subsequent luteal phases during reproduction, both in eutherian [46] and marsupial mammals [10,53,62] and should provide insights into the hormonal control of reproduction in the dibbler. Over and above this, the comparison of life-history traits of island and mainland populations of dibblers contributes to the wider study of island biogeography and the nature of adaptive traits in island animals – known as the ‘island syndrome’ [1]. Gigantism, dwarfism, delayed sexual maturation and reduced reproductive output have all been described in a range of vertebrate species confined to islands and the present study is the first to focus and provide quantitative data on a marsupial.

2. Materials and methods 2.1. Animals Dibblers were captured from two sites in WA; a mainland population from the Fitzgerald River National Park on the south coast (lat. 33°580 S; long. 119°320 E) and two islands, Boullanger and Whitlock Islands (lat. 30°180 5800 S long. 115°000 E) and maintained separately at the Perth Zoological Gardens as part of the Native Species Breeding Programme [28]. Animals were housed in airconditioned buildings where the temperature was controlled to match that in the environment, although moderated between 10 and 30 °C. Lighting conditions represented natural daylight as far as possible with exposure to natural light through windows and

skylights and extra fluorescent lighting between 0730 and 1600 h. The diet consisted of live invertebrates, kangaroo meat, neonatal rats, cooked chicken, sultanas, fig, Rhagodia baccata berries, ‘small carnivore mix’ (Wombaroo Food Products, Glen Osmond, South Australia) and vitamin supplements (SP40, Glen Forest Stockfeeds, Glen Forrest, WA) and water ad libitum. Inflorescences of Grevillea, Banksia and Callistemon were occasionally provided. Males were trapped on both Boullanger and Whitlock islands over the period 1998–2000 and bled soon after capture. Adults and sub-adults were distinguished by body mass and scrotal measurements. Blood samples (<200 lL) were collected either from the infra-orbital sinus or the lateral tail vein and transported on ice to the mainland where they were centrifuged for 2 min at 5000 rpm. Plasma fractions were stored frozen at 20 °C until assayed for cortisol levels. All procedures were approved by the University of Western Australia’s Animal Ethics Committee and complied with the National Health and Medical Research Council’s Australian code of practice for the care and use of animals for scientific purposes. Mainland females entered the captive study in 2001, within the year of their capture in 2000. The island females, monitored in 1999, were from a captive population that was established at Perth Zoological Gardens in March 1997 from eight founder animals, two pairs (male and female) removed from each island. All females entering the study were housed separately and care was taken in the pairing procedure to avoid the odour of the male reaching the female before their placement together. Urinary cellular analysis and pouch inspection were first carried out in island females during 1998 in 14 females paired with males and in nine that were maintained in isolation, in order to detect any difference in the length between pregnancy and pseudo-pregnancy. Between 1998 and 2001, eight females from the mainland and a further nine from island localities were monitored during February to early June for steroid hormone analysis throughout the pregnant cycle. Urine and faeces were collected three times a week and the pouch was inspected every second day. Faeces were stored at 20 °C until assayed and urine was examined on the day of collection for presence of spermatozoa or cornified epithelial cells (CEC) from the reproductive tract. Pairing of the animals occurred when spermatozoa were present in the urine of the male and the number of CECs increased in the female urine from more than one cell per grid. An ‘observed’ mating was recorded if males were mounted on the female for longer than one hour. Pregnancy was confirmed by identifying new-born young in the pouch. 2.2. Urinary cellular analysis Microscopic examination revealed the relative proportions of nucleated and anuclear superficial (cornified) cells (CEC), parabasal and intermediate cells and neutrophils (leucocytes) that were counted to <0.1% error or until a minimum of 20 grids had been counted. Four cellular stages were scored. (i) Pro-oestrus was characterised by low numbers of immature nucleated epithelial cells and increasing numbers of CEC. (ii) The onset of oestrus was characterised by an increasing proportion of CEC, with cell numbers per grid rising from 1 (level 1) to 2 (level 2). The oestrous period was defined as the days during which numbers of CEC were at level 2 and level 3 (3 CEC per grid). (iii) Post-oestrus was characterised by the fall in CEC from level 2 to level 1 with increasing numbers of leucocytes. (iv) Anoestrus, showing few immature nucleated epithelial cells [2]. 2.3. Preparation of faeces Faecal samples were dried at 50 °C for 24 h, sand and foreign matter removed, and macerated to a fine powder. Twenty mg

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powdered faeces were mixed with 150 lL 0.05 mol borate buffer (pH 8.0) and 1.35 mL absolute ethanol, effectively 90% ethanol [59]. Hormones were extracted by 1 min vortexing and brought to 80 °C for 20 min, and then centrifuged. The supernatant was dried under air at 37 °C and reconstituted in 500 lL 0.05 mol borate buffer. Recovery of radio-actively labelled steroid after extraction was 88 ± 3.8% for E2 and 91 ± 2.6% for P4 (n = 6). 2.4. Steroid hormone measurement 2.4.1. Cortisol Total cortisol levels were measured by radioimmunoassay in 10 lL plasma samples using a rabbit antiserum (Endocrines Sciences No. F3-314) that showed cross reactivities of 52% with prednisolone, 30% cortisone, 26% prednisone, 6.8% 21-deoxycortisol, 4.5% deoxycortisol and 2.9% with corticosterone. Unbound steroid was separated with dextran-coated charcoal and 625 lL supernatant samples were counted in 3 mL of Ultima Gold scintillant (Perkin Elmer) in a Packard Tri-Carb 2300TR scintillation counter to less than 1% error with quenching corrected by external standardisation. Free cortisol levels were estimated using a modification of the method of Boonstra and Boag [4] and the equation of Tait and Burstein [56]. The maximum corticosteroid-binding globulin (CBG) capacity of 100 lL aliquots of diluted plasma was determined by Scatchard analysis [45] and subtracted from total cortisol levels to estimate the free fraction. Non-specific binding of the plasma was determined using 10 lmol unlabelled cortisol in ethylene glycol. 2.4.2. Oestradiol-17b (E2) Antiserum (# E26-47 from Endocrine Sciences Laboratories, Calabasas, CA, USA) has a published cross-reactivity of 100% with 17boestradiol, 1.3% with oestrone, 0.6% with oestriol, 0.2% with 16keto-oestriol, 0.05% with 19-nortestosterone, and <1% with oestrone sulphate, cortisol, cortisone, corticosterone, progesterone, 17-hydroxyprogesterone, 5a-pregnanedione, 5b-pregnanedione and androstenedione. Freeze-dried antiserum was reconstituted in assay buffer (0.05 mol borate, pH 8.0) and stored frozen in 100 lL aliquots. Samples were assayed by radioimmnoassay, using the method validated in the honey possum (Tarsipes rostratus) [10] and chuditch (Dasyurus geoffroii) [53]. Briefly, the buffered faecal extracts were diluted 1:8 before assay and, together with a series of dried ethanolic oestradiol-17b standards, were incubated for 3 h at 22 °C with antiserum (1:200 dilution) in assay buffer containing 167 Bq [2,4,6,7-3H]oestradiol-17b (Amersham, UK), 0.05% bovinec-globulin, and 0.2% bovine albumin. Bound steroid was separated from unbound by incubation at 0 °C with 1 mL ice-cold dextrancoated charcoal (500 mg charcoal and 50 mg dextran per 100 mL assay buffer containing 0.1% gelatin). After centrifugation at 0 °C, an aliquot of the supernatant was counted for radioactivity (Packard Tri-Carb 2300TR, Packard Instruments, CT, USA) to <1% error and standards data were transformed using a 4-parameter logistic equation. Maximum binding averaged 61.6%. Blank values were below the level of sensitivity of the assay. All samples from an individual were measured within a single assay to avoid inter-assay variation and two internal standards were included. These were faecal extracts from one female, representing high and low values of E2. Coefficients of variation were 29.8% and 23.6% respectively (n = 3). 2.4.3. Progestagens (PM) Progesterone metabolites (PM) were measured using the commercial kit, Coat-A-Count Progesterone (Biomediq Diagnostic Products, Doncaster, Victoria, Australia) that uses a solid-phase125I radioimmunoassay with antibody-coated tubes and previously validated in the honey possum [10].

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The antiserum has the following cross reactivity (relative to 100% with progesterone), 43.8% with 5a-pregnan-3b-ol-20-one, 31% with 5b-pregnan-3a-ol-20-one, 23.3% with 5b-pregnan-3bol-20-one, 16% with 5a-pregnan-3a-ol-20-one, 9% with 5a-pregnan-3,20-dione, 3.5% with 5b-pregnan-3b,20a-diol, 3.4% with 17a-hydroxy-progesterone, 3.2% with 5b-pregnan-3,20-dione, 0.2% with 20a-dihydroprogesterone and <1% with 5a-pregnan3a,20a-diol, 5a-pregnan-3b,20a-diol, 17a-hydroxyprogesterone, oestradiol-17b and testosterone (kit literature and [59]. Briefly, 100 lL of faecal extract, diluted 1:5 in assay buffer, together with 100 lL each of a series of standards, were incubated with 1 kBq 125 I for 3 h at 22 °C. Anti-body-coated tubes were decanted and measured for gamma-radiation in a Prias gamma counter (PGD Auto-Gamma, Packard Instruments, Meriden, CT, USA). Radioactivity bound was analysed using a 4-parameter logistic plot. Maximum binding averaged 56.6%, non-specific binding was 3.4% ± 0.39 (n = 8) and blank values were below the level of sensitivity of the assay. All samples from an individual were measured in the same assay. Inter-assay coefficients of variation were 5.6% for a ‘high’ pool of female faeces spiked with 10,000 ng g1 of P4 and 20.1% for a ‘low’ pool of male faeces with 815 ± 111 ng g1 P4 (n = 6). 2.5. Statistical analysis Comparisons of two parameters (e.g. length of pregnancy and pseudopregnancy) used 2-tailed Student’s t-test. Significant rises in faecal steroids were identified by an iterative process [10,18]. Samples 1.75 S.D. greater than the mean were temporarily removed each time until no samples exceeded the mean + 1.75 S.D. The mean of the remaining samples represents the baseline value and samples exceeding the baseline by more than 4 S.D. were classified as statistically significantly. Differences between putative gestation lengths from mainland and island animals as assessed by the three parameters (see text) were analysed by 2-way ANOVA with locality as the source of variation. 3. Results 3.1. Males 3.1.1. Free and bound cortisol levels The affinity constant (KT) for dibbler corticosteroid-binding globulin (CBG or Transcortin) was calculated as 8.6  107 L/mol. The dissociation constant (KD) was estimated as 11.7 ± 0.9 nmol/ L, from Scatchard analysis in three male dibblers. Total cortisol levels were close to 10 lg dL1 in dibblers from Boullanger and Whitlock Islands and did not differ significantly between breeding and non-breeding seasons when data were pooled from 1998–2000 (Table 1). Free cortisol levels at 1.7 ± 0.17 lg dL1 (17.2%) were higher in the breeding season than in the non-breeding season, the latter averaging 1.4 ± 0.14 lg dL1 (14.3%), but the difference was not statistically significant due to the small sample size (n = 5). In 2000, however, when numbers of males fell after mating, with only two individuals being trapped on Boullanger Island in May, significant differences were observed with a marked increase in the free cortisol fraction during the rut in March. Free cortisol levels increased to 2.2 ± 0.27 lg dL1 (22.0%), significantly higher than recorded in the breeding (January–May) or non-breeding seasons (June–December) (Table 1). 3.2. Females 3.2.1. Pseudopregnancy No significant difference was observed between unmated and pregnant females, monitored in 1998, in the length of oestrus

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Table 1 Total and free levels of cortisol in plasma from male dibblers (Parantechinus apicalis) from Boullanger and Whitlock Islands during the autumn breeding season (January– May) compared with the non-breeding season (June–December) and the rut of March 2000, when males were actively mating. Mean total cortisol (lg dL1) Breeding season Non-breeding season March 2000 rut

Mean free cortisol (lg dL1) (% free)

9.9 ± 0.54 (n = 32)a 9.8 ± 0.30 (n = 9)a

1.7 ± 0.17 (n = 21)a (17.2%) 1.4 ± 0.14 (n = 5)a (14.3%)

10.0 ± 0.91 (n = 10)a

2.2 ± 0.27 (n = 8)b (22.0%)

Values are mean ± SE with n = sample size. Statistical significance of differences between means within columns indicated with p < 0.05 where superscripts differ.

(Table 2). Nor was there any difference between the two groups in the number of days between the fall in body mass and birth or pouch changes that indicate the end of a non-pregnant cycle, or between the fall in CEC and birth or pouch changes. Oestrus occurs, therefore, in the absence of males and no difference was observed in any of the reproductive parameters detecting the length of a pregnancy or a pseudopregnancy.

3.2.2. Oestrus and mating A single annual oestrus was recorded during late summer to autumn in both populations, with the onset of full oestrus, as defined by peak levels of CEC, occurring by mid-February through to midMarch, with an onset earlier by 12 days in the mainland animals (Fig. 1). Oestrus, however, lasted for 16 days in the mainland animals, compared with 21 days in the island animals (p = 0.0065; Table 3). As a consequence, mating commenced earlier in the mainland animals, from 22nd February, and later, from 9th March, in the island animals. No difference was observed in the length of mating between the two groups but the interval between the last mating and birth was longer in mainland animals (52.4 ± 1.03 days) when compared with that in island animals (45.3 ± 0.85 days; p < 0.0001; Table 3).

3.2.3. Proestrus hormone levels Levels of faecal E2, before and during the appearance of CECs in the urine, were raised in all animals (Fig. 2) and ranged from 28.3 ± 2.36 in proestrus to 94.8 ± 15.59 ng g1 during oestrus in island animals, and from 20.7 ± 3.76 to 75.0 ± 2.00 ng g1 in the same phases in mainland animals (NS). Levels of PM in island animals during the same periods, rose from 153.8 ± 98.07 to significantly higher levels of 1,049.3 ± 383.17 ng g1 (p < 0.01) during oestrus, when compared to the rise in mainland animals, from 47.3 ± 8.89 to 242.1 ± 38.15 ng g1 (Fig. 2). Lower levels of PM excretion were evident in each animal at the end of oestrus, when the number of CEC fell from level 2 to level 1.

Table 2 Comparison of reproductive parameters in island female dibblers (Parantechinus apicalis), between mated females and pseudopregnant females isolated from males. Reproductive parameter

Pregnant (n = 14)

Pseudo-pregnant (n = 9)

p

Duration of CEC (d) Peak CEC to birth/pouch changes (d) Fall CEC to birth/pouch changes (d) Fall in BM after oestrus (g) Fall in BM at birth/end pseudopregnancy (g)

20.1 ± 0.88 44.7 ± 0.81

19.6 ± 1.08 45.7 ± 0.94

0.68 0.43

35.4 ± 1.03

38.3 ± 2.15

0.19

6.7 ± 0.70 5.1 ± 0.71

6.8 ± 0.70 3.8 ± 0.50

0.91 0.20

CEC, cornified epithelial cells; BM, body mass.

February

March

April

1999

May

June

Fig. 1. Oestrous periods in eight female mainland dibblers (Parantechinus apicalis) (black bars) and eight island females (open bars) in relation to each individual’s day of birth (circles).

Table 3 Differences between island and mainland dibblers (Parantechinus apicalis) in the presence of cornified epithelial cells (CEC), mating, onset of leucocytes, body weight (BW) changes, and faecal progestagen (PM) changes, in relation to the time of birth.

CEC Date of onset Duration at level 2 and 3 (oestrus) Interval, fall from level 2 to birth Mating Interval from first mating to birth Interval from last mating to birth Interval from first mating to leucocytes Leucocytes Interval from appearance to birth BM BM at onset of oestrus (g) Interval from low post-mating BM to birth PM Interval from low post-oestrus PM to birth Presumptive gestation length

Island

Mainland

(p)

(days) (n = 8)

(days) (n = 7)

24 February 21.0 ± 1.35 36.44 ± 0.50

12 February 16 ± 0.9 44.38 ± 0.71

46.67 ± 0.93 45.25 ± 0.74 7.67 ± 1.01

54.25 ± 1.04 52.38 ± 1.03 8.6 ± 0.87

<0.0001 <0.0001

38.33 ± 1.38

45.40 ± 1.12

<0.001

54.78 ± 1.93 37.22 ± 1.21

66.38 ± 2.59 46.71 ± 2.55

=0.002 <0.001

38.00 ± 0.61 (n = 4) 37.5 ± 0.4

44.00 (n = 4) 45.1 ± 0.6

=0.001

<0.01 <0.001

<0.0001

3.2.4. Detecting ovulation and calculating gestation length The interval between the lowest body mass post-oestrus to the day of birth was 46.7 ± 2.55 days in mainland animals, compared with 37.2 ± 1.21 days in island animals (p < 0.001; Table 3). The interval between the fall in the number of CECs in the urine and the day of birth was 44.4 ± 0.71 days in mainland animals, compared with 36.4 ± 0.50 days in island animals (p < 0.001), and polymorphonuclear leucocytes appeared in the urine 45.4 ± 1.12 days before birth in the mainland animals, compared with 38.3 ± 1.38 days in island animals (p < 0.001). Ovulation thus occurs in mainland animals approximately 45 days before birth compared with an approximate 38 days in the case of the island animals. The difference between these three parameters in island and mainland individuals is shown graphically in Fig. 3. A twoway analysis of variance gave F1,41 = 47.6 (p < 0.0001) with island and mainland as source. The interval from the first mating to the day of presumed ovulation is a measure of the time that sperm is stored in the reproductive tract and is estimated as 7–9 days, depending on the day of mating, in both island and mainland females (from Table 3).

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birth 2000

1500

faecal PM (ng.g-1)

faecal PM (ng.g- 1)

2000

1000

500

0

1500

1000

* 500

L

-500

70

60

50

40

30 0

days before birth

70

60

50

40

30

20

10

0

days before birth

faecal E2 (ng.g -1)

150 Fig. 4. Profile of mean faecal progestagens (PM) throughout proestrus, oestrus and pregnancy in 8 mainland dibblers (Parantechinus apicalis). Single dots represent levels of PM in individual island females. Baseline concentration of PM (see Section 2) represented by dotted line; oestrous period denoted by cross-hatched bar and mating by solid bar; asterisk (⁄) denotes presumed time of ovulation; L, appearance of leucocytes.

100

50

The mean litter size in the island females (7.4 ± 0.1) was not significantly different from that of 6.3 ± 0.84 in the mainland animals. 0

4. Discussion 70

60

50

40

30

days before birth

4.1. Post-mating mortality in males

Fig. 2. Levels of faecal progestagens (PM) and faecal oestradiol-17b (E2) throughout the proestrus and oestrus phases in the dibbler, Parantechinus apicalis, in island animals (dashed line) and mainland animals (solid line). Oestrus denoted in island animals by hatched bar and by solid bar in mainland animals.

Putative Gestation Length (Days)

3.2.5. Progestagens (PM) during pregnancy A profile of mean faecal PM concentrations throughout the reproductive period of seven mainland dibblers is represented in Fig. 4. The mean baseline concentration of PM is 41 ± 11.73 (S.D.) ng g1. All values above 88 ng g1, i.e. those that exceed baseline levels by 4 S.D. are significantly raised. An obvious peak of PM (⁄) is present around the presumed time of ovulation. Systematic PM profiling was not possible with the limited data available for the island animals, but PM levels in individual females during pregnancy appeared to be higher than those in mainland animals (Fig. 4). 50

Post-mating mortality of dibblers on Boullanger Island was first reported by Dickman and Braithwaite [13] who concluded that they displayed a ‘Type I’ dasyurid population strategy in the terminology of Dickman [12] and Lee et al. [31]. This describes species where ovulation in females is highly synchronised, occurring over a short period of time, with intense competition between males for mating opportunities. Elevated levels of testosterone in male A. stuartii (now agilis) during the mating season have been shown to depress synthesis of the corticosteroid-binding globulin, CBG, by the liver with a consequent significant elevation of unbound or ‘free’ levels of cortisol [8,29,7]. These in turn impact widely on the animals’ physiology, depressing immune function, catabolising muscle protein and engendering gastric ulceration, haemorrhagic adrenals, loss of pelage and, eventually, death [30]. Similar sequelae were described in male dibblers on Boullanger Island post-mating by Dickman and Braithwaite [13], but later field

Day 1 CEC to birth Day 1 of Leuc to birth Days Lowest BM to birth

40 30 20 10 0 Mainland

Island

Source Fig. 3. Bar graph of putative gestation length in mainland and island dibblers (Parantechinus apicalis), with data of reproductive parameters indicative of time of ovulation, analysed from Table 3. Open bars depict the day of fall in urinary cornified epithelial cells (CEC) from level 2 to level 1 (see Section 2) in relation to birth. Diagonally-hatched bar denotes day of appearance of leucocytes in relation to birth. Solid bar denotes time from fall in body mass at presumed ovulation to birth.

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work on the island during 1995 and 1996 recorded significant numbers of males surviving the autumn breeding season [52]. Dibbler males were also known to survive post-mating in mainland populations [64], raising the possibility that the male die-off may be facultative rather than obligatory in this species. Direct evidence of this was presented by Mills and Bencini [39] who monitored captive and island populations of dibblers over a 3year period from 1997 to 2000. There was no evidence of post-mating die-off in mainland or island animals kept in captivity, nor in the Whitlock Island population; but on Boullanger Island, male mortality was high with a complete die-off of males being recorded after the 1999 breeding season. The data on cortisol binding presented here confirm that the free levels rose significantly (22%) in males during the March 2000 rut on Boullanger Island and would be expected to have similar detrimental effects on the animals’ physiology as have been recorded for other semelparous dasyurids [5,6,12]. Interestingly, semelparity has also been recorded in the large northern quoll, Dasyurus hallucatus [13], but levels of free cortisol were not found to change as predicted [43]. The facultative nature of the male die-off of dibblers on the Jurien Bay islands suggests that resource availability may be the factor determining male survivorship. Estimates of insect abundance and dry-matter intake (DMI) on the various islands [36], using isotopic techniques developed by Moro and Bradshaw [41] suggest that Whitlock Island, which supports a large sea-bird colony, has a higher level of productivity that is reflected in the higher body condition and longevity of male dibblers (22 versus 13 months) compared with males on Boullanger Island [54]. 4.2. Oestrus and mating in females Reproduction in females was first studied by Woolley [64] who recorded matings in mainland females kept in captivity during March and April. With the discovery of the dibbler on offshore islands, March was confirmed as the time of breeding in males [11] although the reproductive period of island females was less clear. Of three captive females from the island, one entered oestrus in January, one in early February and the third in May. In the following year, one of the females entered oestrus in April and another in August [66]. In the present study, all females were in oestrus during February, March and April, although there were differences between the two populations. Island females entered oestrus 12 days later than mainland animals, and remained in oestrus for five days longer. Their extended period of oestrus was also accompanied by higher levels of progestagens (PM). Although progesterone (P4 and PM) levels are raised during proestrus in 7 other species of dasyurid, [15,22,23,24,26,35,37], its function has been largely overlooked. In the grey short-tailed opossum, Monodelphis domestica, however, raised levels of P4 (and E2) induce oestrus, as well as ovulation [14,20,25,27], but only after contact with a male. The dibbler, as with most other dasyurids, exhibits a spontaneous oestrus, as full expression of CEC in the urine of females kept in isolation from males was similar to that in mated animals. A higher level of PM in the island animals, associated with longer periods of oestrus, suggests prolonged sexual activity in island animals when compared with mainland females and may translate into heightened sexual receptivity on the part of the female. Any role P4 may have on oestrus can only be from indirect evidence in two other species of dasyurid. In the spotted-tailed quoll, Dasyurus maculatus, raised levels of proestrus P4, when higher in the presence of males, are associated with a shorter follicular phase, thereby advancing the next oestrus [23]. This is in direct contrast to the dibbler, in which the higher PM levels during proestrus are associated with a follicular phase of longer duration. Evidence in the western quoll, or chuditch, D. geoffroii, suggests that

P4 may have both roles, similar to those in the opossum, one of promoting sexual activity and the other associated with ovulation [9]. There is also some evidence in the chuditch that copulation may initiate ovulation, as, in one female that did not experience a mating, repeated periods of oestrus 12 days apart were accompanied by peaks of E2. The cycles appeared to be anovulatory as no leucocytes appeared in the urine [53]. The follicular phase in the dibbler is characterised by raised levels of PM and E2, indicating the action of these two steroids in eliciting sexually receptive behaviour and a priming of the reproductive tract. Further research is needed to define the site/s of action of P4 during proestrus in this species. 4.3. Ovulation and PM during pregnancy Both oestrus and ovulation in the dibbler are spontaneous, as the pouch of unmated females develops to the same degree and at the same time as birth in the mated females. Pouch development is taken as evidence of progesterone from a secretory corpus luteum, indicating the occurrence of an oestrous cycle, or pseudopregnancy [21,67]. In other dasyurid species, a reliable indication of ovulation has been the conjunction of three parameters, a temporary drop in body mass at the end of oestrus, the fall in CEC and the appearance of leucocytes [15,48,67]. To this now can be added a fall in proestrus levels of P4 [9]. A similar suite of changes has been observed in the dibbler in this study, with PM levels falling as for P4, thus providing an estimate of the time of ovulation in mainland animals as 45 days before birth and in the island females, 38 days before birth. An actual gestation period, defined as the time between ovulation and birth, of 45 days is the longest reported for any dasyurid and compares only with 44 days in the macropodid tree kangaroo, Dendrolagus matschiei [42]. Progestagens excreted throughout pregnancy in the mainland animals (Fig. 4) suggest a very slow development of the corpora lutea with full P4 secretion not occurring until about 8 days before birth. Such slow maturation of the corpus luteum has been linked to the slow development of the embryo in other species of dasyurid [26,35,47], and is likely to be the case in the dibbler. The higher levels of PM recorded in the island females during pregnancy may be linked to a more rapid development of embryos in the shorter pregnancy of the island population. 4.4. Sperm storage From this study, the length of time that sperm is stored in the female before ovulation (and presumed fertilisation) is calculated as 7–9 days in both mainland and island females. A related species, Pseudantechinus macdonnellensis, has a gestation period, recorded as the time between mating and birth, as 45–55 days [65]. If the number of days of sperm storage in this species is comparable with that of the dibbler, a gestation period of 36–46 days is also comparable with the length of gestation in the dibbler. 4.5. Gestation length A surprising difference to emerge between island and mainland dibblers from this study is the length of gestation, which is approximately 7 days shorter in island individuals. The length of gestation is known to vary allometrically with body size in eutherian mammals, being longer in larger species [32,33], and as island dibblers are significantly smaller than mainland animals, this could be seen as a contributing factor. Gestation lengths, however, are uniformly short in marsupials and vary much less with body mass, with log gestation time in days = 0.03 log body mass  0.22 (r2 = 0.06) [19]. Furthermore, regressing published gestation lengths against

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body mass for nine dasyurid species shows no significant correlation and thus the small difference of ca 20 g in body mass between island and mainland animals cannot account for the large difference in gestation lengths. The other factor that could be involved is the apparent elevation of PM levels in breeding island dibblers compared with mainland animals. Progesterone has been shown to accelerate embryonic development in marsupials [9] and if future data indicate that PM levels are indeed higher during gestation in island compared with mainland dibblers, this could account for the difference in gestation lengths. Animals living on islands display a suite of life-history traits differing from those characteristic of mainland populations, usually referred to as the ‘island syndrome’ [1,51]. These include lower rates of reproduction as a result of shorter breeding periods, delayed maturation of females and smaller litters [17,44,68]. Island/ mainland comparisons in marsupials are restricted to the quokka (Setonix brachyurus) living on Rottnest Island, 20 km off the coast of Perth in WA. Early reproductive studies established that the island population displayed marked seasonality, with all young being born in February. Breeding on the mainland, however, was continuous and island animals returned to the mainland and fed a high protein diet lost their seasonal pattern after approximately 2 years [49,50,58]. Data on variation in gestation lengths in mammals are limited, although there is evidence from a number of studies that body condition may influence the timing and length of gestation. In a study of the sika deer (Cervus nippon) in Japan, poor condition in years with heavy snowfall resulted in poor foetal growth with an extended gestation period [34]. On the other hand, induction of ketosis with an associated decrease in condition, caused a significant shortening of the gestation period in sheep from 150 to 143 days [60]. A long-term study of bison (Bison bison) found that gestation lengths were significantly shorter in females in good condition and suggested that variation in gestation length was adaptive, and facilitated reproductive synchrony [3]. The lack of post-mating mortality of dibblers on Whitlock Island has been linked to the greater availability of resources flowing from a resident colony of sea-birds that is reflected in significantly-higher condition indices of dibblers on Whitlock when compared with individuals on Boullanger Island [61]. It is thus possible that the high body condition of the island animals has resulted in a shortening of their gestation length when compared with mainland individuals. There are insufficient data on the body condition of the mainland animals at this time to test this hypothesis, but it remains a possibility. Given that the marked difference in gestation length is apparent in dibblers that have been maintained in captivity for some time, however, a genetic explanation seems more likely. Genetic differences have been measured in the dibbler in a study into the genetic variation of island and mainland populations. The mean mtDNA sequence divergence between island haplotypes was low (at 1.3%) but there was a difference of 4.1% between island and mainland haplotypes [38]. The difference in gestation length between island and mainland dibblers may therefore be further evidence that they represent taxonomically distinct populations. Acknowledgments We thank Professor Max Cake for helpful advice in the measurement of free and bound cortisol. Animals were collected under licence from the Western Australian Department of Environment and Conservation and grateful acknowledgement is made to members of the Dibbler Recovery Team. Research was supported by funding from the Marsupial Cooperative Research Centre.

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