Methyl farnesoate couples environmental changes to testicular development in the crabCarcinus maenas

Abstract

SUMMARYCarcinus maenas males have two major color phases. Green-phase males molt frequently and tend to live in brackish estuaries during the summer. After becoming red-phase males, they molt infrequently, have higher mating success, and live in cooler, deeper water. We found profound differences between these two phases in the way salinity and temperature affect hemolymph levels of methyl farnesoate (MF), a hormone that affects crustacean reproduction. Few green-phase males (<10%) had detectable MF in 33 ppt seawater (SW) at 11 or 18°C. By contrast, about 30% of the red-phase males had detectable MF at either temperature. After transfer to 5 ppt SW, none of the green-phase males had detectable MF at 11°C whereas 100% of green-phase males did at 18°C. By contrast, 100% of the red-phase males had detectable MF in 5 ppt SW at either temperature. At 11°C, green-phase males had detectable MF after eyestalk ablation (ESA), showing that they can produce MF. There was no additional increase in MF levels when ESA animals of either color phase were transferred to 5 ppt SW, suggesting that the eyestalk is the primary regulator of the MF response to low salinity. MF levels of green-phase males were increased by injecting MF, by ESA, or by exposure to 5 ppt SW at 18°C. The testicular index of these treated animals nearly doubled after two weeks. Our results strongly suggest that environmental conditions such as temperature and salinity, affect testicular development in this crab by changing its MF levels.Key words: methyl farnesoate, testis, crustacean, salinity, reproduction, temperature. THE JOURNAL OF EXPERIMENTAL BIOLOGY 2774In the present study, we show that red-phase crabs are more likely to have detectable MF in their hemolymph than green-phase crabs. In addition, the testicular index (TI) of red-phase animals is higher than that in green-phase animals. Furthermore, our results indicate that environmental factors differentially affect MF levels in the two color phases. These results are consistent with the hypothesis that MF stimulates testicular development. To test this hypothesis, we show that environmental factors that increase MF levels also stimulate testicular development. The MF levels in these two color phases appear to be responsible, in part, for the different reproductive activities of these males. MATERIALS AND METHODS AnimalsCrabs [Carcinus maenas (Linnaeus 1758)] were generously provided by Dan Landers (Millstone Environmental Lab; Waterford, CT, USA). Most of the males used in this study were 50±5 g (carapace width, CW 5.2±0.6 cm) and were collected during the spring and early summer. Green-phase and red-phase animals were identified by the color of their ventral surface (Reid et al., 1997). None of the experimental animals was missing appendages. At the end of some experiments, the testes were removed and weighed, and the TI (% total body mass) was calculated. Crabs were kept in large tanks containing artificial SW (Instant Ocean, Spectrum Brands; Atlanta, GA, USA) at normal salinity (33 ppt) at 11 o...

Reproductive regulators in decapod crustaceans: an overview

“…did not increase its hemolymph levels of MF (Henry and Borst, 2005). Similar results were also observed in green phase crabs when they were treated with low saline water at 11°C (Nagaraju and Borst, 2008). Olmstead and LeBlanc (Olmstead and LeBlanc, 2007) found that environmental factors can cause aberrant sex determination via perturbations in MF signaling in D. magna.…”

“…Unilaterally, ESA male P. monodon showed a significantly higher sperm count, larger sperm head diameter and longer spikes in comparison to unablated ones, but no observable changes were noted in testicular index, spermatophore weight and sperm viability (Gomes and Honculada-Primavera, 1993). We also found that the ESA of C. maenas caused testicular index to increase to 2-fold higher than that of intact green crabs (Nagaraju and Borst, 2008). These and other research studies have led to the proposal that the eyestalk contains an inhibitory factor called GIH (Fingerman, 1997a; Haihui et al, 2006;Kulkarni et al, 1984), but the structural information and molecular and cellular mechanisms remain to be elucidated.…”

“…Alternative techniques have been attempted to stimulate ovarian development, such as administration of gonad inhibitory hormone (GIH) antibody, hormonal level changes by environmental factors such as temperature, salinity and photoperiod, precise functionality of neurotransmitters and double-stranded RNA interference (dsRNAi) to reduce gonad inhibitory peptide transcript. The results from these experiments have revealed the potential (Chang et al, 2001;FanjulMoles, 2006;Mazurová et al, 2008;Nagaraju, 2007;Nagaraju and Borst, 2008;Treerattrakool et al, 2008) to stimulate gonad maturity. So, in this context, I will discuss up-to-date research on gonad regulatory factors and their role in reproductive development.…”

“…Molecular analysis of VIHs isolated from females of a few crustacean species shows that they consist of signal peptides (20-31 amino acid residues) and mature peptides (77-83 amino acid residues; Fig.3). They also show a considerable degree of sequence similarity with MIH, including the preservation of six cysteine residues at the same relative locations (Demeusy, 1953;Leung-Trujillo and Lawrence, 2009;Nagaraju and Borst, 2008;Rotllant et al, 1993;Wongsawang et al, 2005;Yano et al, 1988) forming three intramolecular disulfide bonds (1-5; 2-4; 3-6 fashion); these are major chemical forces, which help to maintain the VIH tertiary structure (Fig.3) (Soyez, 2005;Udomkit, 2000;Nagaraju et al, 2009). Reverse phase high performance liquid chromatography (RP-HPLC) analysis revealed that the neuropeptide in H. americanus has two enantiomeric isoforms with the fourth residue being either ltryptophan or d-tryptophan.…”

“…High MF levels were also correlated with mating behavior; males with higher MF levels actively courted and coupled with females, while males with lower MF levels did not show this behavior (Sagi et al, 1994). In addition, in the crab O. senex senex, the green crab, C. maenus, and the prawn M. malcholumsonii, injection of MF caused an increase in the testicular index (Kalavathy et al, 1999;Nagaraju and Borst, 2008;Nagaraju et al, 2004). These results suggested that MF stimulates the testes either directly or indirectly by stimulating (GSF or AGH) or inhibiting GIH secretions.…”