AMPHIPOD ECDYSTEROIDS IN GRAVID FEMALES AND REPRODUCTIVE MALES (Spinulogammarus subcarinatus,

Anisogammaridae) OF SOUTHEAST ALASKA

Mistee R. Vinzant Department of Natural Sciences, University of Alaska

Southeast, 11120 Glacier Hwy, Juneau Alaska, USA, 99801

INTRODUCTION

In the subphylum Crustacea, molting is regulated by the active hormone 20-hydroxyecdysone, a metabolite of the hormone ecdysone (belonging to the family of polyhydroxylated steroid hormones: the ecdysteroids), which plays a critical role in their growth and development (Chang et al.; Chang et al., 1993; Lachaise et al., 1989). Ecdysone and other ecdysteroids are synthesized by ecdysial glands called the y-organs, which is located at the base of the antennae or near the mouthparts, and is in-turn regulated by a complex neurosecretory apparatus located in or near the animal’s eyestalks (Change et al.; Lachaise et al., 1989). For both insects and crustaceans, the synthesis of ecdysteroids releases ecdysone into hemolymph where it is almost immediately converted to an active hormone, 20-hydroxyecdysone, by the peripheral tissue in very low concentrations (Lachaise et al., 1989). The circulating ecdysteroids within the hemolymph are free and not bound to carrier proteins (Chang et al.).

For crustaceans, ecdysone production by the y-organ is inhibited and regulated by the molt-inhibiting hormone (MIH), a neurosecretory peptide produced in the x-organ, which then accumulates in the sinus gland of the eyestalks (Lachaise et al., 1989). The neurosecretory function of the x-organ sinus gland complex of the eyestalk has been shown to delay or decrease the synthesis of ecdysone, and when ablated, crustaceans have shown an accelerated molting cycle and retention of juvenile characteristics (Chang et al.; Naya et al., 1989; Rotllant and Takac, 2003). A study by Chang et al. found that within 48 hours after eyestalk ablation, ecdysteroid levels elevated dramatically, which may be due to either an increase in the rate of hormone synthesis from the y-organ or a decreased rate of hormone inactivation/elimination. However, large amounts of MIH may be toxic to a molting organism, and has been recently exploited as a safer and more effective insecticide in agriculture. A handful of studies have been done to control populations of cirripeds, or barnacles, that accumulate on the bottom of ships through the use of lethal doses of juvenile molting hormones (JH) that provokes a delay in metamorphosis (Lachaise et al., 1989).

Ecdysone has also been reported to be very important in arthropod reproduction, best of which has been shown from insects, where some species can reportedly induce vitellogenic synthesis by the fat body, and oocyte maturation (Rotllant and Takac, 1999). Although far less is known about the physiological effect of ecdysone on the crustacean reproduction, ecdysteroid concentrations in some crustaceans have been shown in low concentrations in the eyestalks, ovaries, and also embryos (Chang and Bruce, 1981; Rotllant and Takac, 1999). Several studies from the early 1980’s found that within the

crabs, Gecarcinus lateralis and Carcinus maenas, hemolymphatic ecdysone increased during premolt stages and the dominant ecdysteroid before molt was Ponasterone A, a 25-deoxy counterpart of 20-hydroxyecdsyone (Lachaise et al., 1989). The Lachaise studies also noted while during intermolt stages, the concentrations of ecdysone decreased as MIH levels increased; and Ponasterone A levels increased in ovaries during ovarian maturation and in eggs during embryonic development. Another study by Chang et al. also found sufficient levels of ecdysteroids circulating throughout the intermolt cycle within the crab, Pachygrapsus crassipes.

Furthermore, the amphipod Orchestia gammarellus is reported to exhibit elevated ecdysteroid concentrations in the ovaries during previtellogenesis and during the early stages of vitellogenesis. Ecdysone is thought to play an important role in ovarian maturation, and gonadal DNA synthesis and growth of other decapods (Chang et al., 1993; Rotllant and Takac, 1999). It is hypothesized that the non-gravid Spinulogammarus subcarinatus females collected may be approaching vitellogenesis, and therefore may show an elevated ecdysteroid concentration that could be quantified by comparison to the ecdysteroid concentrations of similarly-sized males.

METHODS

In mid February, specimens were collected one hour before the morning low tide at Auke Recreational Unit, near Juneau Alaska. To minimize biological variation on data, only gravid females and reproductive males were considered for the study. Samples of amphipod hemolymph were then extracted and levels of ecdysone hormone were measured through the Enzyme-linked Immunosorbant Assay (ELISA) technique according to Tamone (2005).

90 ul of diluted goat-anti-rabbit antiserum was pipetted into each well of a Costar plate. In order for the antiserum to coat the sides of the wells, the antiserum was left to incubate within the wells for at least 24 hours at room temperature before the assay was set up. The solution was then gently dumped into the sink and blotted onto an absorbent pad. To prevent unnecessary binding of remaining solutions, all residual protein binding sites were blocked in each Costar well. 300 ul of blocking buffer (assay buffer with 0.5% bovine antiserum albumin-BAA) was pipetted into each well and allowed to incubate for one hour at room temperature. Since the blocking buffer contained sodium azide (NaNH3), each Costar plate was washed three times for five minutes with a plate washer.

Nine microfuge tubes were labeled appropriately. The standard curve was then prepared by using the stock solution and was inserted into each microfuge tube as a serial dilution (1,000; 500; 250; 125; 62.5; 31.25; 15.6; 7.8; and 3.9). Wells A1 and B2 were left empty to serve as blanks and wells A3 and B4 were left empty to serve as zero hormones to yield the maximal absorbance. Wells A1 and B2 received 100 ul of assay buffer (25 mM NaPO4; 0.15 M NaCl; pH 7.5) and wells A3 and B4 received 50 ul of assay buffer.

The ecdysone hormones of amphipods were extracted by homogenizing 0.5g of gravid females and reproductive males, separately, in PBS (phosphate buffered saline; 10mM)

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every seven days for a duration of 30 days. All hemolymph samples were diluted to 1:100 and 50 ul of hemolymph was pipetted into the appropriate Costar wells.

50 ul of diluted primary antiserum solution (1:1,000,000) was pipetted with a multichannel pipetteman into each well (except for wells A1 and B2). The Costar wells were then placed into a shaker for ten minutes at room temperature. Each plate was covered with plastic adhesives and was allowed to refrigerate for at least 16 hours to incubate. Each Costar plate was then washed three times with PBS/TWEEN for two minutes. The solutions in each plate were gently emptied into the sink and blotted onto an absorbent pad.

To stop the color development in the assay, 100 ul of 1 M H3PO4 was added to each well after the plates were dried. Once the blue wells turned yellow, the absorbance of each well was then measured with a spectrophotometer at a wavelength of 450 nm.

REFERENCES

Brusca R. C., and Brusca G. J. 2003. Invertebrates. Second Edition. Sinauer Associates, Inc. Sunderland, MA.

Chang E. S., and Bruce M. J. 1981. Ecdysteroid titers of larval lobsters. Comp. Biochem. Physiol. 70A: 239-241.

Chang E. S., Bruce M. J., and Tamone S. L. 1993. Regulation of crustacean molting: a multi-hormonal system. Amer. Zool. 333: 324-329.

Chang E. S., Sage B.A., and O’Conner J.D. The characterization of circulating ecdysone titers in the crab Pachygrapsus crassipes. C.N.R.S.

Chang E. S., Tamone S. L., Lin W., and Oberbauer A. M. 1995. Modifications of the paradigm of the hormonal control of crustacean molting: effects of metabolic inhibitors on the ecdysoitropic action of methyl farnesoate and evidence for an insulin-like growth factor. Proceedings of the International Symposium on Biotechnology Applications in Aquaculture. Asian Fisheries Society Special Publication No. 10: 183-195.

Lachaise F. Synthesis and effects on molting of juvenile hormone-like compounds in crustacean and chelicerata. 74-81.

Lachaise F., Carpentier Gilles, Somme G., Colardeau J., and Beydon P. 1989. Ecdysteroid synthesis by crab y-organs. The Journal of Experimental Zoology. 252: 283-292.

Naya Y., Ohnishi M., Ikeda M., Miki W., and Nakanishi K. 1989. What is the molt-inhibiting hormone? The role of an ecdysteroidgenesis inhibitor in the crustacean molting cycle. Proc. Natl. Acad. Sci. 86: 6826-6829.

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Rotllant G., and Takac P. 1999. Ecdysones in the maturational moult of juvenile females of the spider crab, Libinia emarginata Leach, 1815 (Decapoda, Majidae). Crustaceana. 72: 221-231.

Tamone, S. 2005. ELISA for arthropod molting hormone. Biology 415. University of Alaska Southeast. Juneau, Alaska.

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