Female preference assays showed that disturbance to the chemical environment can, indeed, affect chemical communication and mechanisms of species recognition, suggesting that disturbance may facilitate hybridization. We found that in laboratory experiments conducted in water obtained directly from the Río Calnali, female X. birchmanni, from a population allopatric to the hybrid zone, showed no preference when presented with chemical stimuli from conspecific and heterospecific males (). However, when the same females were tested in water obtained from a natural spring and in tap water, they showed a significant preference for conspecific cues over heterospecific (). The stream water is likely to contain much higher concentrations of contaminants than spring or tap water. The primary input of contaminants upstream of the sample point is human and animal waste, including sewage discharge from the small community of Ahuacatlán. Pesticides and other synthetic contaminants are rarely employed by the subsistence farmers and ranchers in the area. Our findings suggest that water within the Río Calnali is facilitating hybridization between the two species, and that the sub-lethal contamination influencing the system can have a significant effect on communication.
In laboratory tests without supplemental HA, X. birchmanni
females strongly preferred conspecific male chemical cues over heterospecific cues (). When we measured female preferences in water containing 200
HA, we found that they responded to both conspecific and heterospecific stimuli more slowly than in tests without HA (a
). All females responded to both stimuli in all clear-water trials, but significantly fewer subjects tested in HA responded to male chemical cues at all. Furthermore, the females that did respond in HA trials, did not exhibit a difference in preference between conspecific and heterospecific male odour cues (b
). Females tested at a markedly lower HA concentration, 20
, also showed a loss of preference for conspecific cues; female preference, however, was retained in water containing only 2
In order to determine whether the loss of female preference was due to specific effects on chemical communication, rather than on females' overall motivation to mate, we tested females with computer animations of courting X. birchmanni
and X. malinche
males, in water with and without 200
of supplemental HA. Females showed nearly identical, significant preferences for conspecific visual cues in both treatments (). HA therefore inhibited females' species recognition based on chemical, but not visual cues. We also found that while females showed a strong preference for food odour cues in clear-water trials, they showed no preference for food cues in water containing 20 and 200
HA. These results further support the notion that HA disrupts detection of chemical cues in aquatic animals.
Figure 4 Association time (mean±s.e.) of female X. birchmanni with computer animations of conspecific (black bars) and heterospecific X. malinche (white bars) males. ‘Humic acid’ indicates trials conducted in 200mgl−1 (more ...)
All experimental females were exposed to a single, brief (approximately 45
min) bout of elevated HA concentration during a preference trial. In order to evaluate the long-term effects of this brief exposure, we retested the females in water without supplemental HA 2 days after the 200
HA treatment. While response latency returned to prior levels (a
) and females again showed a preference for conspecific male odour (b
), the strength of the response was significantly weakened: females continued to associate significantly less with conspecific odour when compared to their responses before exposure and 10 days after exposure, and to the responses of control females never exposed to HA (b
). These results suggest that the effects of exposure to high levels of HA can persist even after a pulse of disturbance, likely due to a damaging effect of HA on the binding properties of chemoreceptor cells. In goldfish, HA reduces the response of olfactory epithelium and olfactory bulb to sexual pheromones (Hubbard et al. 2002
). Furthermore, HA is known to inhibit binding and interfere with the function of mammalian receptor cells (Yang et al. 2002
). Ten days following exposure to 200
HA, we found that females once again showed a strong preference for conspecific male odour (b
). These results suggest that if HA did inflict chemoreceptive damage, full functional recovery was obtained after 10 days. Chemoreceptor regeneration time is unknown in Xiphophorus
, but full regeneration may not even be necessary for certain olfactory discrimination tasks. In goldfish, behavioural responses and amino-acid discrimination ability persisted even after the surface of olfactory epithelium was reduced by 85% (Zippel 2000
). Control females that were tested in water without supplemental HA throughout the series of trials consistently showed a strong significant preference for conspecific male odour, suggesting that the response profile of experimental females was due to the effects of HA rather than to habituation to the stimuli.
In addition to affecting chemoreception, HA may also be interfering with chemical cues themselves. Females' latency to respond to both conspecific and heterospecific cues was significantly greater in the trials conducted with supplemental HA than in any of those without, and latency did not differ across trials without HA (a
). Humic substances are known to bind to hydrophobic chemicals and thus reduce their aqueous concentrations. Available concentrations of a steroid pheromone in goldfish were strongly reduced by the similar concentrations of HA we used in our study (Mesquita et al. 2003
). We therefore cannot exclude the possibility that HA may interfere with the concentration of available pheromone in the swordtail signalling environment.
Regardless of the mechanisms whereby HA interferes with the female reproductive behaviour in X. birchmanni
, the consequences are clear: elevated levels of HA, a natural and ubiquitous substance, compromises females' ability to discriminate between conspecific and heterospecific male odour cues without disrupting their motivation to mate (as shown in the visual preference tests). And although we cannot exclude the hypothesis that other compounds in Río Calnali may have influenced swordtail hybridization, our laboratory experiments directly test the effects of HA of species recognition of X. birchmanni
. Despite the benefits that HA may provide (Kullberg et al. 1993
), our results suggest that its impact could also be detrimental for some species. HA concentrations vary broadly in nature (Steinberg 2003
). Particular environments such as the Río Negro in the Amazon River Basin, however, contain consistently high natural HA levels (Wood et al. 2003
), and are home to diverse assemblages of animals that rely on chemical cues in social communication (Weitzman & Fink 1985
). Furthermore, other animals seem unaffected in environments contaminated with sub-lethal amounts wastewater and agricultural effluent. Such animals may have evolved mechanisms for coping with their environments.
The results presented here are particularly important and biologically relevant to the X. birchmanni
system given the recent hybridization with X. malinche
. Rosenthal et al. (2003)
found that most individuals sampled in the hybrid zone are F2
or greater, suggesting that adults of hybrid origin largely contribute to the breeding pool. Therefore, even if the disturbance to the signalling environment is not continual or wide ranging, hybrid offspring will likely mate and result in pervasive hybridization. As exploitation of natural water resources proceeds worldwide, we are gaining an increasingly clear picture of the impact of environmental disturbance on biological communities. While chemical and physical measures reflect conditions when the sample is taken, biological monitoring gives an indication of past, as well as current, conditions. Although lethal consequences are more easily recognized, subtle changes may also inflict substantial ecological disruption (Seehausen et al. 1997
, Hayes et al. 2002
). Much of the current research on the environmental causes of reproductive distress has focused on exogenous sources of contamination (Zala & Penn 2004
). Our results illustrate how even a rare disturbance event may facilitate hybridization through a breakdown in species recognition mechanisms.