Asymmetric mating isolation between two caridean shrimp: Lysmata wurdemanni and Lysmata boggessi.
Shrimps (Sexual behavior)
Sexual behavior in animals (Physiological aspects)
|Publication:||Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2012 National Shellfisheries Association, Inc. ISSN: 0730-8000|
|Issue:||Date: April, 2012 Source Volume: 31 Source Issue: 1|
|Topic:||Canadian Subject Form: Animal sexual behaviour|
|Product:||Product Code: 0913080 Shrimp NAICS Code: 114112 Shellfish Fishing SIC Code: 0913 Shellfish|
|Geographic:||Geographic Scope: United States Geographic Code: 1USA United States|
ABSTRACT In many animals, sex pheromones are involved in behavioral
reproductive isolation, which is thought to be more important than other
isolation barriers in causing rapid speciation. Lysmata species living
in the western Atlantic waters have been recently redefined
taxonomically, with some of the species having overlapping distribution.
It has been found that prezygotic mating isolation is not complete
between two Lysmata species--Lysmata wurdemanni and Lysmata
boggessi--and there is asymmetry in mate recognition between the two
species. Male-role L. wurdemanni can mate with female-role L. boggessi,
but not vice versa. This study shows that the asymmetric mating
isolation is caused by the difference in sex pheromone detection (both
distance and contact sex pheromones). Male L. wurdemanni can detect and
respond to distance sex pheromones secreted by female L. boggessi, even
at low concentrations, but male L. boggessi did not respond to distance
sex pheromones of L. wurdemanni at any concentration, which suggests
that their asymmetric mate recognition is not caused by a different
response threshold to conspecific and interspecific sex pheromones.
Instead, minor differences in the molecular structure of sex pheromones
and/or a different ratio in components of sex pheromones may result in
asymmetric reproductive isolation.
KEY WORDS: mate recognition, sex pheromones, Lysmata, peppermint shrimp
Speciation in animals is often characterized by the presence of prezygotic (behavioral and/or morphological) and postzygotic reproductive isolation (infertility and/or nonviability of interspecific hybrids). In many animals, such as insects (e.g., Collins & Tuskes 1979), reptiles (e.g., Shine et al. 2002), and amphibians (e.g., Rollmann et al. 2000), sex pheromones are involved in behavioral isolation, which is thought to be more important than other isolation barriers in causing rapid speciation (Coyne & Orr 2004). Pheromonal differences may provide the basis for species recognition and avoidance of interspecific mating (Cooper & Vitt 1984, Higgie et al. 2000, Shine et al. 2002). For example, Shine et al. (2002) found that the separation between two sympatric sibling sea snake species, Laticauda colubrina and Laticauda frontalis, which overlap in both geographical distribution and breeding season, is maintained by species-specific pheromones that control male courtship. In the South American fruit fly Anastrepha fraterculus, a high level of prezygotic isolation has been maintained between the strains from Argentina and Peru, and quantitative and qualitative differences exist in the sex pheromones of the two strains (Caceres et al. 2009).
However, prezygotic isolation is not complete in many animals. Asymmetric mating isolation, which occurs when matings from one direction of an interspecific cross are more likely to occur than in the other direction, may be common, and under many conditions is perhaps expected (Ehrman & Wasserman 1987, Arnold et al. 1996). Sex pheromones are usually involved in asymmetric mate recognition. For example, in two congeneric, partially sympatric copepod species, Temora longicornis males readily detect and follow pheromone trails of Temora stylifera females, and pursue T. stylifera females, but there is scant mate-finding activity observed in the reverse cross (Goetze & Kiorboe 2008). Asymmetric reproductive isolation is also found in 4 sibling species of Drosophila (Drosophila melanogaster, Drosophila simulans, Drosophila sechellia, and Drosophila mauritiana). As in any hybridization, matings in one reciprocal cross occur far more readily than in the other (Cobb & Jallon 1990, Welbergen et al. 1992, Coyne et al. 1994). Such isolation has been attributed to the evolution of a distinct cuticular hydrocarbon profile (contact pheromones) among Drosophila species (Coyne et al. 1994).
Lysmata species is a group of caridean shrimp with protandric simultaneous hermaphroditism. A shrimp matures as a functional male (male phase (MP)) with male external characteristics, and may later change to the euhermaphrodite phase (EP), with both male and female functions (Bauer 2000). Intermolt EP shrimp function as male and mate with newly molted EP shrimp, which function as females (Lin & Zhang 2001). Lysmata species living in western Atlantic waters have recently been redefined taxonomically, with some of the species having overlapping distribution (Rhyne & Lin 2006). Several species (e.g., Lysmata boggessi (Rhyne & Lin 2006), Lysmata ankeri, and Lysmata pederseni), previously confused with Lysmata wurdemanni (Gibbes 1850) or Lysmata rathbunae, were identified as new species by Rhyne and Lin (2006). They are similar in morphology and are closely related genetically (Baeza et al. 2009).
Two types of sex pheromones are involved in mate recognition of some Lysmata species (e.g., L. wurdemanni (Zhang & Lin 2006) and L. boggessi (Zhang et al. 2007)). Distance sex pheromones are secreted in water by premolt female-role EP shrimp, whereas contact sex pheromones are coated on the body of newly molted EP shrimp and are not water soluble (Zhang & Lin 2006). Most male-role L. wurdemanni demonstrate active precopulatory behavior and follow the receptive female-role shrimp actively before the female molts (Zhang & Lin 2004), which indicates that they track and locate females by distance sex pheromones. However, some male-role shrimp do not have obvious precopulatory behaviors, and mating occurs immediately when the male-role shrimp contacts the newly molted female-role shrimp with its antennae (Zhang & Lin 2004), which indicates the presence of contact sex pheromones.
It has been found that prezygotic isolation is not complete in some species of Lysmata shrimp. Behavioral studies show that two Lysmata species, L. wurdemanni and L. boggessi, can interbreed, and there is asymmetric reproductive isolation between the two species (Zhang et al. 2009). Male-role L. boggessi, in general, do not respond to the sex pheromones secreted by female-role EP L. wurdemanni, and do not display any precopulatory behavior to the newly molted EP L. wurdemanni. However, some male-role L. wurdemanni can respond to and mate with female-role L. boggessi (Zhang et al. 2009). The asymmetric behavior of L. wurdemanni and L. boggessi indicates that there might be differences in the signal strength, sensory spectra, and/or response threshold of the sex pheromones between the two species. Zhang et al. (2009) found that distance sex pheromones are involved in asymmetric mate recognition. However, whether this results from the response threshold or the molecular structures of the distance sex pheromones is unknown. Contact sex pheromones may also be responsible for asymmetric mate recognition between the two Lysmata species. Therefore, in this study, both distance and contact sex pheromones were tested for behavior in the L. wurdemanni and L. boggessi. In addition, distance pheromones were diluted at different concentrations to test whether there is any difference in the response threshold to distance sex pheromones of these two species, because in the wild, distance pheromones are diluted at different degrees in surrounding water, depending on the water volume and current, the distance between female-role EP shrimp and male-role shrimp, and other factors.
MATERIALS AND METHODS
Distance Sex Pheromone Detection
Lysmata shrimp generally molt every 10-15 days, depending on the species, the temperature, and other environmental factors (e.g., Lin & Zhang 2001, Zhang et al. 2007). Several hours before each EP shrimp molted, it was moved to a 1-L beaker containing 200-mL filtered (0.2-gm) seawater. For each species, molting water from 10 EP shrimp (total, 2,000 mL) was collected for behavioral study. Sex pheromones are likely to degrade over time at room temperature, and because the shrimp did not molt at the same time, the collected molting water was stored in a freezer at -20[degrees]C. Before the behavioral test, the molting water was defrosted to room temperature. Of the 2,000 mL molting water for each species, 600 mL was diluted to 1,200 mL with regular seawater (2;< dilution level), and another 300 mL was diluted to 1,200 mL (4x dilution level). The remaining 1,100 mL was kept at the original concentration (1x dilution level).
To test the behavioral response, each MP or male-role EP shrimp (male shrimp) of L. boggessi and L. wurdemanni was moved to a 20-L tank (with 10 L seawater) at least 1 day before the test. A plastic tube with a constant flow of molting water or seawater (control) was used to test the response of the male shrimp (adapted from the procedure of Zhang & Lin (2006)). The speed of the flow was maintained at 2-3 drops/sec. If shrimp approached, grasped, and/or followed the tube, this was considered a positive response. Each shrimp was tested for 1-2 min with regular seawater (control), followed by 4x diluted pheromones, 2x diluted, and 1x (undiluted) molting water. If shrimp responded to lower concentrations (i.e., 4x dilution level), it was assumed they would also respond to higher concentrations (i.e., 2x or 1x dilution level) and therefore were not tested further. The concentration at which each shrimp started to respond was defined as the response threshold in this study. For each species (L. boggessi and L. wurdemanni), 10 individuals were tested with conspecific and interspecific molting water (e.g., L. wurdemanni was tested with its conspecific molting water and molting water from L. boggessi).
McNemar's test was used to compare the response of the shrimp with the distance sex pheromones and with the control. The response threshold was categorized into 4 classes. Individuals that responded to 4x, 2x, and 1x x diluted molting water were allocated to classes 1, 2, and 3, respectively. The Mann-Whitney U-test was used to compare the responses of L. wurdemanni to conspecific and interspecific distance sex pheromones, and the responses between L. wurdemanni and L. boggessi to the distance sex pheromones from L. boggessi.
Contact Sex Pheromone Detection
Contact pheromones are coated on the body of newly molted EP shrimp (Zhang & Lin 2006). For each species (L. boggessi and L. wurdemanni), the abdomen of each of the 10 newly molted EP shrimp was immersed in 1.5 mL hexane for 30 sec to extract the contact sex pheromones (Zhang et al. 2011). The extracts were stored in a -20[degrees]C freezer. Before the behavioral test, the extracts of each species were concentrated (from 15 mL to 2 mL) using a liquid nitrogen blow to evaporate the hexane.
Each MP or male-role EP shrimp (male shrimp) of L. boggessi and L. wurdemanni was moved to a 20-L tank (with 10 L seawater) 1 day before the test. The method for contact sex pheromone detection was modified from Zhang et al. (2011). Starch gel cubes were used to test the behavioral response of the shrimp. To make the starch gel cube, 10 g starch was dissolved in 50 mL distilled water and heated to 70-80[degrees]C. It was then put into a refrigerator at 4[degrees]C for at least 3-4 h until it became solid. The starch gel was cut into 3-mm cubes for use during the behavioral test. Each starch cube was immersed in the contact sex pheromone extracts or hexane (control) for 10 sec and then allowed to air-dry for 15 sec. The treated starch cube was then placed in the tank to test the behavioral response of the male shrimp. Each shrimp was tested for 2 min with control, conspecific, and interspecific contact sex pheromones in sequence. Tested cubes were removed from the tanks before introducing the new cubes. If the shrimp approached and grasped the treated cube, it was recorded as a positive response. Twelve shrimp of each species was used for the test. McNemar's test was used to compare the response of the shrimp with the conspecific and interspecific contact sex pheromones, and with the conspecific contact sex pheromones and the control.
Distance Sex Pheromone Detection
None of the shrimp tested approached and followed the tube with regular seawater (control). The individual response to the conspecific molting water was different. Some L. boggessi and L. wurdemanni showed strong responses (approached and grasped the tube), whereas others just approached and/or followed the tube. Those that showed a positive response usually responded within 10-15 sec after introduction of the molting water. McNemar's test showed that both L. wurdemanni and L. boggessi responded to their conspecific molting water (L. wurdemanni: chi-square = 5.143, P = 0.023; L. boggessi. chisquare = 5.143, P = 0.023). The response threshold to conspecific distance sex pheromones was not significantly different between L. boggessi and L. wurdemanni (Mann-Whitney U-test, U = 20.0, P = 0.62; Table 1).
Half of the male L. wurdemanni (5 of 10) responded to molting water of EP L. boggessi, although McNemar's test showed that the response was not significantly different from that of the control (chi-square = 3.200, P = 0.074). Among L. wurdemanni that showed a positive response, the response threshold to its conspecific molting water was not significantly different from that of the interspecific molting water (Mann-Whitney U-test, U = 19.5, P = 0.76). When responding to the molting water of L. boggessi, L. wurdemanni also did not demonstrate a difference from L. boggessi in the response threshold (Mann-Whitney U-test, U = 23.5, P = 0.343). On the other hand, none of the male L. boggessi responded to the molting water of L. wurdemanni (Table 1).
Contact Sex Pheromone Detection
In L. wurdemanni and L. boggessi, more than half the shrimp (7 of 12) responded to conspecific sex pheromone-treated cubes. Male shrimp grasped the cubes for at least 2-3 sec after contact. None of the shrimp grasped the control cubes. McNemar's test showed that there was a significant difference between the response to sex pheromone-treated cubes and the control (L. wurdemanni: chi-square = 5.143, P = 0.023; L. boggessi: chisquare = 5.143, P = 0.023).
Among the 7 L. wurdemanni that responded to conspecific contact sex pheromones, 4 shrimp also responded to interspecific contact sex pheromones from L. boggessi. Those shrimp that did not respond to conspecific sex pheromones also showed no response to interspecific sex pheromones. Although fewer L. wurdemanni responded to interspecific sex pheromones than to conspecific sex pheromones (4 vs. 7 of 12), no significant difference was found between the 2 treatments (McNemar's test, chi-square = 1.333, P = 0.25). On the other hand, no male L. boggessi responded to interspecific contact sex pheromones by L. wurdemanni. There was a significant difference between the response to conspecific sex pheromones and interspecific sex pheromones in L. boggessi (McNemar's test, chi-square = 5.143, P = 0.023).
The shrimp, Lysmata wurdemanni and L. boggessi are similar in morphology and size, and have similar breeding seasons and mating behaviors (Rhyne & Lin 2006, Zhang & Lin 2006, Zhang et al. 2007). They are also partially overlapping in geographical distribution, especially in the Florida Keys (Rhyne & Lin 2006). Behavioral studies have shown that prezygotic reproductive isolation is not complete between the two species (Zhang et al. 2009). Some male-role L. wurdemanni attempt to mate with female-role L. boggessi, but not vice versa. Zhang et al. (2009) found that distance sex pheromones are involved in asymmetric mate recognition. Distance pheromones are thought to be responsible for precopulatory behaviors in L. wurdemanni and L. boggessi (Zhang & Lin 2006, Zhang et al. 2007). Both species can respond to conspecific distance sex pheromones at low concentrations (e.g., 4x diluted, Table 1), and there is no difference in their response threshold. The response threshold of male L. wurdemanni to conspecific and interspecific distance pheromones is similar (Table 1), which explains why male L. wurdemanni exhibit similar precopulatory mating behaviors toward conspecific and interspecific females (Zhang et al. 2009). On the other hand, none of the male L. boggessi responded to molting water secreted by female L. wurdemanni at any dilution levels, whereas some of them responded to conspecific molting water at the 4x dilution level (Table 1). This reaction indicates that asymmetric detection in distance pheromones is not the result of the different response threshold of the two species. Instead, there might be differences in molecular structure or components of distance sex pheromones of the two species that cause the differences in sex pheromone detection. Apart from distance sex pheromones, our study shows that contact sex pheromones are also responsible for asymmetric mating recognition between the two species. Male-role L. wurdemanni can detect and respond to the contact sex pheromones secreted by female L. boggessi, but male L. boggessi do not respond to contact sex pheromones of L. wurdemanni.
The evolution of the molecular structure of pheromones is relatively conservative in animals (Wyatt 2003). The distance sex pheromone in L. wurdemanni has been found to be similar to the shore crab sex pheromone uridine diphosphate (UDP) (Zhang et al. 2010). However, L. wurdemanni do not respond to UDP (Zhang et al. 2010). The contact sex pheromones in Lysmata shrimp are a multicomponent blend of cuticular hydrocarbons with major component of (Z)-9-octadecenamide and other minor components (Zhang et al. 2011). The bioactive components of contact sex pheromones also exist in intermolt EP and MP shrimp, but in different ratios (Zhang et al. 2011). The minor modifications of the major component or ratios of the multiple components in the sex pheromone bouquet may be species specific, and may cause behavioral reproductive isolation in different species. Because the difference between the sex pheromones of two species (especially between two genetically, closely related species such as L. boggessi and L. wurdemanni) may be small, it is possible that the sex pheromones of one species (e.g., L. boggessi) can still trigger the behavioral response of the other species (e.g., L. wurdemanni) as a result of the different sensory spectra of the two species. In such cases, postzygotic reproductive isolation (such as infertility and/or nonviability of hybrids) is involved. Although male L. wurdemanni can detect sex pheromones secreted by female L. boggessi, and mating was successful between the two species, their embryos do not live for more than 10 days and fail to hatch (Zhang et al. 2009).
Asymmetric mating isolation is also found in other animals. Kaneshiro (1976) studied several species of Hawaiian Drosophila and found that "ancestral" females from the species on older islands discriminated against "derived" males from species on younger islands, and "derived" females accepted the courtship and mated relatively more frequently with "ancestral" males. Therefore, he suggested that the observed pattern of mating asymmetry may provide a means by which to identify the direction of evolution in similar demographic situations (Kaneshiro 1980). Phylogenetic study shows that L. boggessi is in a derived position relative to L. wurdemanni (Baeza et al. 2009), and therefore the asymmetric reproductive isolation between L. boggessi and L. wurdemanni is in accordance with Kaneshiro's (1980) hypothesis. However, in Kaneshiro's model, asymmetric reproductive isolation is caused by loss of courtship elements in derived species during founder events resulting from genetic drift (Kaneshiro 1976, Kaneshiro 1980). In the 2 Lysmata species studied here, it is unlikely that the asymmetry in mating isolation arises from founder effects because the two species are not isolated geographically. Arnold et al. (1996) suggested that asymmetry in isolation need not to be linked with population bottlenecks or loss of courtship elements, and can arise between sister populations irrespective of the direction of evolution. In the current study, asymmetric mating isolation between L. boggessi and L. wurdemanni is more likely to be a transient phase in the divergence of sexually selected traits (e.g., sex pheromones and corresponding sensory receptors).
We thank Adeljean Ho and Dr. Mathew Wittenrich of the Florida Institute of Technology, and Dr. Andy Rhyne of Roger Williams University for help with shrimp collection. The study was supported in part by Proaquatix, Inc., and National Science Foundation of China (project no.: 30970445).
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JING ZHU, (1) * DONG ZHANG (2,3) AND JUNDA LIN (1)
(1) Department of Biological Sciences, Florida Institute of Technology, 150 West University Boulevard, Melbourne, FL 32901; (2) East China Sea Fisheries Research Institution, Chinese Academy of Fisheries Sciences, 300 Jungong Road, Shanghai, 200090, P. R. China; (3) Veto Beach Marine Laboratory, Florida Institute of Technology, 805 E. 46th Place, Veto Beach, FL 32963
* Corresponding author. E-mail: firstname.lastname@example.org
TABLE 1. Number of L. wurdemanni and L. boggessi that responded to different dilution levels of conspecific or interspecific molting water. Dilution Level Lw x Lw Lb x Lw 4x 3 0 2x 2 0 lx 2 0 Dilution Level Lb x Lb Lw x Lb 4x 4 1 2x 2 3 lx 1 1 Lw x Lw and Lb x Lb, response of L. wurdemanni and L. boggessi to conspecific molting water; Lw x Lb, response of L. wurdemanni to interspecific molting water by L. boggessi; Lb x Lw, response of L. boggessi to interspecific molting water by L. wurdemanni. If the shrimp responded to a higher dilution level (e.g., 4x), it was assumed that it would also respond to lower dilution levels (e.g., 2x or 1x) and therefore was not tested further. The number of shrimp that did not respond can be calculated by subtracting the total number of shrimp that responded to each dilution level from the sample size of 10.
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