Female Sex-Pheromone Volatility and Male Uric Acid Excretion During Courtship in Captive African Brown House Snakes, Lamprophis Fuliginosus

Female sex-pheromone volatility and male uric acid excretion during courtship in captive African brown house snakes, Lamprophis fuliginosus

Robert D. Aldridge, Angelo P. Bufalino, Curtis Robison, Carlos Salgado, Phillip Khayyat

Social behaviour in snakes centers on reproduc- productive tract. The source of the kidney se- tive activities (Gillingham, 1987) that consist of cretions is from a specialized, androgen sen- a speciﬁc sequence of behaviours: mate search- sitive, portion of the uriniferous tubule which ing, courtship and copulation. This sequence is located at the terminal end of the nephron is triggered by sex pheromones (hereafter re- and known as the sexual segment of the kidney ferred as pheromones) released by the female (SSK; Fox, 1977). (Kubie et al., 1978; Gillingham, 1987). For the The function of these secretions is not fully majority of snake species studied to date these known, but it is believed to form a copulatory pheromones are excreted along the dorso-lateral plug in some species (Devine, 1975). The ma- trunk region and are reported to be non-volatile terials secreted by the SSK travel to the cloaca (Mason, 1992). via the ureter, which also carries uric acid to the Mate searching, the initial phase of repro- cloaca (Fox, 1977). The mechanisms by which ductive behaviour, begins when attractive fe- the ureter is able to transmit SSK secretions and males lay down pheromone trails as they move not urea to the female during copulation has not through their environment (Ford, 1986). Male been examined. snakes locate a potential mate by following The brown house snake (Lamprophis fulig- these trails using their olfactory senses (Halpern inosus: Colubridae: Lamprophiinae), is a ter- and Kubie, 1980; Ford, 1986). Once a male lo- restrial, oviparous constrictor native to Sub- cates an attractive female he begins the next Saharan Africa (Chippaux, 2001). In captivity, phase of reproductive behaviour, courtship, our snakes reproduce throughout the year (per- which may culminate in copulation. sonal observation). Knowledge of the reproduc- In many snake species, attractive females are frequently not receptive to male courtship and tive biology of this species is primarily anec- copulation is not achieved (Thamnophis sirtalis dotal. Bogert (1940) described the morphology and Storeria dekayi, Noble, 1937; Lamprophis of the hemipenis of several Lamprophis taxa fuliginosus, Walker and Ford, 1996; Nerodia from Angola. Barbour and Loveridge (1928) = sipedon, Prosser et al., 2002). If copulation is described two clutches (n 16, 7) of Lam- successful the male deposits sperm and secre- prophis from Tanzania. Nägele (1985) reported tions from the kidneys into the female’s re- that a captive female produced clutches in May (10 eggs) and July (8 eggs) and that the eggs hatched in 70-80 days. Ford (2001) described Department of Biology, Saint Louis University, St. Louis, MO 63103, USA clutch and egg characteristics of recently cap- e-mail: [email protected] tured L. fuliginosus from Tanzania.

The continuous reproduction we observed tal chambers. Males and females were in different rooms in in our snakes is not universal for the species. Saint Louis University’s animal care facility. The temperature of each room ranged between 22-26◦C and had a 12:12 Broadley (1983) reported that L. fuliginosus in hr light/dark cycle. Snakes were fed euthanized mice weekly southern Africa has a seasonal reproductive cy- and water was available ad libitum. cle and lays eggs (usually 8 to 10, maximum To avoid odors from non-experimental snakes, experiments were conducted in a laboratory separate from the ani- 16) in late spring to early summer. This sea- mal care facility. Experiments were conducted in a 37 l glass sonal pattern is not lost in captivity, because aquarium with the room temperature ranging between 22- ◦ Hermann (1989) reported that captive L. fulig- 26 C. In phase 2 of Experiment 1 (described below), females inosus obtained from South Africa reproduced were placed inside an opaque 4.6 l plastic storage container (in Germany) in the spring (April and June) measuring 20 × 33 × 9 cm. An opaque container was used similar to the spring breeding time (October- because Noble (1937) and Shine and Mason (2001) found that male Thamnophis sirtalis used vision to locate mov- December) in South Africa. Hermann (1989) ing/courting females. The container had four 3 mm diame- also reported that captive L. fuliginosus are able ter holes drilled near the corners of the lid and three holes to store sperm and produce viable young up to drilled along the bottom of one end of the container to fa- cilitate air circulation. The storage container containing the 2 years and 9 months after breeding. Haagner female was then placed on 55 mm high pedestals inside the (1987) reported on three clutches of L. fuligi- aquarium. The aquarium and storage container were thor- nosus eggs (from captive snakes) from Pretoria, oughly washed and allowed to air dry after each experiment. South Africa (26◦S, 28◦E). Haagner (1987) also Care was taken not to contaminate the exterior of the container with odors from the female. For each experiment two collated the data from several authors and con- randomly chosen males were tested with each female. All cluded that the main laying time for L. fuligi- courtship behaviour was recorded during each of the exper- nosus in southern Africa is September-January iments. Experiment 1 consisted of three phases: 1) males were (n = 20 clutches). acclimated to the aquarium for 10 min; 2) the female was Walker and Ford (1996) published a detailed placed in the small container (described above) on pedestals analysis of courtship behaviour in captive L. in the test aquarium for 15 min; 3) females were removed from the container and placed in the aquarium with the fuliginosus. There are no published data on males and observed for at least 30 additional min. A total the nature of pheromone communication in L. of 86 trials were conducted. Experiment 2 also consisted of fuliginosus. three phases: 1) males were acclimated to the aquarium for 10 min; 2) a double-tipped cotton swab, one end of which In our early studies of the reproductive bi- was rubbed on the dorso-lateral surface of the experimental ology of this species we frequently observed female, was placed in the aquarium with the males for 15 males opening their mouths (gaping) and also min; 3) the experimental female was placed in the aquarium with the males and observed for at least 30 additional min. excreting uric acid as they were courting an at- A total of 11 females were tested twice, several days apart, tractive female, thus we have incorporated ob- with randomly selected males. servations of these behaviors in our study. Data used to analyze male mouth gaping behaviour and uric acid/feces excretion were obtained from experiment 1. The purpose of this study is to: 1) determine Frequency of male courtship behaviour, uric acid excretion the volatility of the female pheromone, 2) deter- by male snakes, and frequency of males investigating the mine the frequency and context of male mouth swab and the pheromone donor female were analysed by the χ2 test for two independent samples. Comparisons of gaping behaviour, 3) determine the frequency uric acid versus uric acid with feces excretion was tested by and context of uric acid and feces excretion by the Binomial test. Mouth gape frequency was corrected for 2 both sexes during courtship, and 4) verify the the time observed and analyzed by χ goodness-of-fit test. The significance level was set at α = 0.05 for all analyses. reproductive anatomy and presence of the SSK Data for the histological analysis was obtained from two through histological examination of the testis, wild caught snakes from Sudan (Field Museum # 62268, kidney, vas deferens, and ureter. 62256). The right testis and anterior portion of the kidney, with vas deferens attached, were removed, dehydrated in Our captive born colony consisted of 22 adult females isopropanol, cleared in toluene, embedded in paraffin and and 17 adult males. The original parents of the colony were sectioned at 7 µm. Tissues were stained in hematoxylin, ◦ ◦ obtained from Burundi (5 S, 30 E) by N. Ford (University Biebrich scarlet, orange G, and fast green. The tubule di- of Tyler, Texas, USA) through commercial dealers. Snakes ameter and epithelial height of the seminiferous tubules and were kept in individual plastic containers in environmen- SSK were measured (x¯ ± SD are presented).