Introduction
Many crustaceans species, such as crabs (Ryan 1966; Gleeson 1980; Seifert 1982; Hardege et al. 2002; Kamio et al. 2002), lobsters (Atema 1984 for a review), and crayfish (Ameyaw-Akumn and Hazlett 1975; Tierney et al. 1984), copulate with the females using chemical compounds as sex attractants. Pheromones as a kind of chemical signals are widespread in aquatic species. To communicate and mate, sex pheromones that emitted by male or female become the most important chemical messages and are also used in mate choice.
Sex pheromones are classified two types: distance pheromones and contact pheromones. As a kind of water-borne (aquatic species) or volatile (terrestrial species) chemical, distance pheromones can take effect within a certain range in water or in air Chang et al. 2007, Lim and Greenfield 2008). Kamio et al. (2002) found that post-molt females can release a water-borne pheromone, which is different from pre-molt females. Unlike distance pheromones, contact pheromones are insoluble and coat the female’s body (Kamiguchi 1972, Bauer 1979, Borowsky 1991, Zhang and Lin 2006).
In many animals, pheromones are detected by specialized receptor organs, such as vomeronasal systems of vertebrates (Halpern and Martinez-Marcos 2003). But crustaceans sense chemicals through an organ called cuticular sensilla. Antennules, as the first part of antennae, have been reported as pheromone receptors in many decapod crustaceans. “Male-specific sensilla” are also found in some mysidan, cumacean, and amphipodan crustaceans for detecting females (Johansson et al. 1996, Hallberg et al. 1997).
Lysmata species is a group of caridean shrimps with protandric simultaneous hermaphroditism. There are a lot of studies which can indicate that the genus shrimp matures into a male-phase (MP), and then later change sex to a euhermaphrodita -phase (EP) with both male and female functions (Bauer 2000). EP shrimps can only keep female phase during a narrow post-molt window about 3-8 hrs in each molt cycle (about 10 days), while inter-molt shrimp function as males (Lin and Zhang 2001). Male-role shrimp of L. ankeri, L. boggessi, and L. wurdemanni track and locate the receptive females by both distance and contact pheromones (Zhang and Lin 2006).
Hypotheses
I. Lysmata shrimp have obvious responses to extractive pheromones, including distance and contact pheromones. II. The more the aesthetascs are, the more obvious the responses are.
Materials and methods
Experimental animals
Four different species of shrimps were collected to determine the specificity of the shrimp pheromones: L. boggessi shrimp from Hernando Beach, Florida, U.S.A., L. ankeri shrimp from Haiti, L. debelius and L. amboinensis which are purchased from petstore because they are Indo-Pacific Ocean species.
Pheromone collection
Several hours before euhermaphrodite-phase shrimp molt (when the ovary is determined fully developed), 7-10 shrimps will be moved to 1-L beakers which each contains 200 ml filtered and one pre-copulatory shrimp. The seawater which provides environment for euhermaphrodite-phase shrimp molted (molting water) was filtered with 0.2 μm filters. After the shrimp molted, the water sample, that contains distance pheromone, will then be serially diluted (e.g. 1x, 2x and 4x) and used to test the behavioral response of MP shrimp or male-role EP shrimp. Contact pheromones are a series of hydrocarbon, which coats the surface of newly molted EP shrimp body, therefore collecting the contact pheromones can use hexane to extract. The Extracts of contact pheromones, which is obtained through the abdomen of 10 newly molted EP shrimp dipped in 1.5 mL hexane for 30 seconds, will be concentrated (e.g. from 15 mL to 2 mL) using liquid nitrogen flow to evaporate hexane in order to test the behavioral response of male-role shrimp.
Pheromone detection
Distance pheromones
All the experimental shrimps must be feed as its species in the 20-L tank (with 10 L seawater). Because distance pheromones can dissolve in water, we tried three methods to do the experiment. Dropping distance pheromone solution directly is the first way. An appropriate cylinder will be used to measure 50 mL solution and then pour the solution into the tank gently in order to keep the shrimp quiet. The second method to test distance pheromones is using a plastic tube with constant flow of solution. Behavioral responses of the male shrimp to the pheromone plume, or sea water (control) will be evaluated by both response time and behavior intensity. The third method is undertaken by sponge binding with round stone. The overall sponge is immersed for five min in either fresh seawater (control) or pheromone solution, and then put into the experimental tanks. All of the three methods will be recorded using a video camera.
Contact pheromones Because contact cues are only on the exoskeleton of newly molted EP shrimp, the main point of the test is to let the shrimp touch the pheromones. All the experimental shrimps must be feed as its species in the 20-L tank (with 10 L seawater). A piece of plastic tube (about 2-3 cm in length) will be dipped into the extract, or hexane (control) for about 10 seconds and moved to the tank. The response will be evaluated by response time. The behavior of the shrimp will be recorded using digital video camera.
Results
In Lysmata shrimp behavior to both types of pheromones was observed as three situations: fast or slow swimming, sharp swing and frequently approaching. In these Lysmata species, only L. boggessi shrimp have obvious response.
In the first experiment with L. boggessi shrimp using dropping method to test distance pheromones, 3 MP shrimps reacted as fast swimming; 5 as slow swimming rather than strong response; only 1 shrimp swung vertically. In the second treatment (tube method), only 1 of 10 MP shrimps had some responses like approaching to the plastic tube, which contains pheromone plume instead of seawater, and 2 shrimps reacted as fast swimming. However, there was no obvious response with the sponge treatment.
In the observation of contact pheromone experiment, 30% of the MP shrimps reacted to touch and approach the tube frequently. But the same response happened with the tube without contact pheromone, only containing hexane.
As one kind of sensilla, the number of aesthetascs can influence the shrimp responses to pheromones. L. boggessi have the most aesthetascs among the four Lysmata species and this species studied have stronger responses to the pheromones. The number of aesthetascs per row also has difference but not that obvious. (Figure 1)
Figure 1. Number/density of aesthetascs (mean±SD) in EP and/or MP of L. boggessi, L. amboinenesis, L. ankeri and L. debelius. A.total number of aesthetascs on a flagellum. B. number of aesthetascs per row.
Discussion
The results of this study demonstrate that pheromones do have responses to MP shrimps. But there are always differences in mating behavior. L. boggessi MP shrimp like to follow the pre-molted EP shrimp until they have successful copulatory behavior. At the same time, the EP shrimp release distance pheromones, which lead the MP shrimp to follow. But L. debelius and L. amboinensis do not have that mating behavior. The MP shrimp of L. debelius and L. amboinensis only stand by to wait the EP shrimp molted.(Zhang and Lin 2004) So this is one reason of that why only L. boggessi MP shrimp have obvious responses.
Most animals have a curious reflection of new things, including shrimps. Contact pheromones are insoluble, and the same with hexane. If we put two plastic tubes (one with contact pheromones, the other with only hexane) at one time, MP shrimps will approach both two tubes, because tubes are the new things to shrimps.
From the results we can see that the number of aesthetascs has some influences to the shrimp responses of pheromones. However, the most important problem is the total number of shrimps. To test for the pre-copulatory behavioral responses of different Lysmata species, a lot of shrimps must be used to reduce individual variation. Future studies on Lysmata species will focus on how to use biological statistic to reduce these errors.
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