Roles of Crustacean Female Sex Hormone 1a in a Protandric Simultaneous Hermaphrodite Shrimp

Crustacean female sex hormone (CFSH) plays a pivotal role in the development of secondary sex characteristics in dioecious crustaceans. However, until now the knowledge concerning its functions in hermaphroditic species is scanty. Herein, we explored the function of CFSH (Lvit-CFSH1a) in the peppermint shrimp Lysmata vittata, a species characterized by a rare reproductive system of protandric simultaneous hermaphroditism (PSH). Lvit-CFSH1a cDNA was 1,220-bp in length with a 720-bp ORF encoded a polypeptide of 239-aa. RT-PCR showed that Lvit-CFSH1a was exclusively expressed in the eyestalk ganglion. For female physiology, it was found that Lvit-CFSH1a was indispensable for the development of female gonopores, but it might not involve vitellogenesis of the species. For male physiology, Lvit-CFSH1a suppressed Lvit-IAG2 expression in short-term silencing experiment and recombinant protein injection experiment, but did not affect male sexual differentiation in long-term silencing experiment. In addition, silencing the Lvit-CFSH1a gene impeded individual growth in L. vittata.

X-RAY TOMOGRAPHIC MICROSCOPY AS A MEANS TO SYSTEMATICALLY TRACK EXPERIMENTAL DECAY AND FOSSILIZATION

ABSTRACT Laboratory-based decay experiments have become commonly used to supplement our understanding of how organisms enter the fossil record. Differences in how these experiments are designed and evaluated, however, including dissimilarities in qualitative decay-scoring indices superimposed on variability in model organisms, renders any semblance of comparison between studies unreliable. Here, we introduce the utility of X-ray tomographic microscopy (μCT) as a means for reliable and repeatable analysis of soft-tissue decay experiment products. As proof-of-concept, we used a relatively simple experimental design with classic studies as comparators, and present our analytical protocol using μCT for capturing the entire volume of the decay subject. Segmentation software then allows for 3D volume analysis and high-resolution internal and external character identification. We describe the workflow from sample preparation, contrast-staining, and data collection to processing and analysis of the resulting data, using peppermint shrimp (Lysmata wurdemanni) as model organisms, and compare our results to previous taphonomic studies. These methods allow for improved visualization and quantification of decay and internal volume analysis with minimal handling as compared to traditional qualitative scoring methods. Using the same scoring criteria as previous studies, this study revealed similar decay results for certain features, while we were additionally able to detect other feature loss or alteration earlier—importantly without need for potentially distortive sample handling. We conclude that μCT is a more effective, straightforward, and exact means for extracting quantitative data on the progression of decay and should be adopted in future studies, where available, to streamline and standardize comparisons.

RNAi Reveals Role of Insulin-Like Androgenic Gland Hormone 2 (IAG2) in Sexual Differentiation and Growth in Hermaphrodite Shrimp

Insulin-like androgenic gland hormone (IAG) is the most widely known hormone that regulates sexual differentiation in crustaceans. Previously, a transcriptome study described two transcripts of IAGs (Lvit-IAG1 and Lvit-IAG2) in the peppermint shrimp Lysmata vittata, a species characterized by a rare reproductive system of protandric simultaneous hermaphroditism (PSH). Herein, we explored the function of Lvit-IAG2 via RNA interference (RNAi) experiments, and then compared the functional differences between the two IAGs. We demonstrated that Lvit-IAG2 positively regulated the expression of crustacean hyperglycemic hormone (Lvit-CHH) in eyestalk ganglion but exhibited no significant effect on the expression of gonad-inhibiting hormone (Lvit-GIHs) and crustacean female sex hormone (Lvit-CFSHs). Besides, Lvit-IAG2 gene knockdown caused degeneration in appendices masculinae (AM) and suppressed germ cells at the secondary spermatocyte stage. Moreover, silencing the Lvit-IAG2 gene impeded ovarian development, including smaller previtellogenic oocytes, and lower expression of vitellogenin (Lvit-Vg) gene in hepatopancreas and vitellogenin receptor (Lvit-VgR) gene in the ovarian region. Notably, silencing the Lvit-IAG2 gene impeded individual growth of the species. Collectively, findings from this study demonstrate that Lvit-IAG2 and Lvit-IAG1 coordinatively function to modulate sexual differentiation in L. vittata; meanwhile, Lvit-IAG2 stimulates the growth of the PSH species.

Roles of gonad-inhibiting hormone in the protandric simultaneous hermaphrodite peppermint shrimp†

To date, the molecular mechanisms of the unique gonadal development mode known as protandric simultaneous hermaphroditism (PSH) are unclear in crustaceans. In this study, cDNA of a gonad-inhibiting hormone (Lv-GIH1) was isolated from the PSH peppermint shrimp Lysmata vittata, and its expression was exclusively found in the eyestalk ganglion. Real-time quantitative polymerase chain reaction (qRT-PCR) revealed that the expression of Lv-GIH1 increased during gonadal development of the functional male stages but decreased significantly at subsequent simultaneous hermaphroditism stage. Further in vitro experiment showed that recombinant GIH1 protein (rGIH1) effectively inhibited Vg expression in the cultured hepatopancreas tissues while the short-term injection of GIH1-dsRNA resulted in reduced expression of Lv-GIH1 and upregulated expression of Vg in the hepatopancreas. Moreover, long-term rGIH1 injection led to significantly reduced expression of Lv-Vg, Lv-VgR, and Lv-CFSH1, subdued growth of oocytes, and feathery setae as a secondary sexual characteristic in females. Interestingly, while germ cells in testicular part were suppressed by rGIH1 injection, the expression of Lv-IAGs showed no significant difference; and long-term GIH1-dsRNA injection results were contrary to those of rGIH1 injection. Taken together, the results of this study indicate that Lv-GIH1 is involved in gonadal development and might also participate in controlling secondary sexual characteristic development in L. vittata by inhibiting Lv-CFSH1 expression.

Uridine-5’-tri-phosphate is a candidate component of the soluble sex pheromone bouquet in a marine shrimp, Lysmata wurdemanni

Characterisation of distance sex pheromones in decapods is challenging, although great efforts have been made in this field in the past 50 yr. In a previous study, we identified a component of the distance (soluble) sex pheromone bouquet of the peppermint shrimp Lysmata wurdemanni as being a uridine-5’-di-phosphate (UDP)-like chemical. However, UDP itself does not elicit the full pre-copulatory ‘approach and follow’ behaviour in peppermint shrimp. Here, we tested the hypothesis that the UDP-like chemical is uridine-5’-tri-phosphate (UTP), a metabolic product of chitin synthesis, and that this is the component of the distance sex pheromone of L. wurdemanni. We ran a series of bioassays to examine whether UDP, UTP or mixtures of the 2 compounds elicit male mating behaviour. Our results showed that male L. wurdemanni responded to UTP by displaying stereotyped courtship behaviour—the same behaviour elicited from males when exposed to water that previously contained moulting females. Combining UTP and UDP did not enhance the intensity of this courtship behaviour. The minimum effective concentration of UTP found to elicit courtship behaviour in the male shrimp was between 10-6 and 10-7 M. HPLC analysis showed the presence of UTP in the moulting water of female shrimp and also the partial conversion of UTP to UDP during the sample preparation procedure. Both the bioassay and chemical analysis results presented in this study suggest that UTP is a component of the distance sex pheromone in L. wurdemanni, and that the major peak in the chromatogram of a L. wurdemanni pheromone identified in previous studies might be a breakdown product of UTP.

Biological controls to manage Acropora-eating flatworms in coral aquaculture

Coral aquaculture is expanding to supply the marine ornamental trade and active coral reef restoration. A common pest of Acropora corals is the Acropora-eating flatworm Prosthiostomum acroporae, which can cause colonial mortality at high infestation densities on Acropora spp. We investigated the potential of 2 biological control organisms in marine aquaria for the control of P. acroporae infestations. A. millepora fragments infested with adult polyclad flatworms (5 flatworms fragment-1) or single egg clusters laid on Acropora skeleton were cohabited with either sixline wrasse Pseudocheilinus hexataenia or the peppermint shrimp Lysmata vittata and compared to a control (i.e. no predator) to assess their ability to consume P. acroporae at different life stages over 24 h. P. hexataenia consumed 100% of adult flatworms from A. millepora fragments (n = 9; 5 flatworms fragment-1), while L. vittata consumed 82.0 ± 26.76% of adult flatworms (mean ± SD; n = 20). Pseudocheilinus hexataenia did not consume any Prosthiostomum acroporae egg capsules, while L. vittata consumed 63.67 ± 43.48% (n = 20) of egg capsules on the Acropora skeletons. Mean handling losses in controls were 5.83% (shrimp system) and 7.50% (fish system) of flatworms and 2.39% (fish system) and 7.50% (shrimp system) of egg capsules. Encounters between L. vittata and P. hexataenia result in predation of P. acroporae on an Acropora coral host and represent viable biological controls for reducing infestations of P. acroporae in aquaculture systems.

Life history traits and reproductive performance of the caridean shrimp Lysmata boggessi, a heavily traded invertebrate in the marine aquarium industry

The most intense commercial harvest of marine aquarium species in North America occurs in the coastal waters surrounding Florida, yet very often little information exists on the life histories, population dynamics, or reproductive characteristics of these organisms. The peppermint shrimp Lysmata boggessi is one such species and is heavily targeted along the west coast of Florida. It is known primarily among aquarists for its ability to control pest anemones and in the scientific community for its unique sexual system, protandric simultaneous hermaphroditism. However, no study has addressed fishery interactions or long-term population dynamics for L. boggessi. We used monthly fisheries-dependent sampling, with a trained observer present, for a full year to assess seasonality in sex phase ratio (males to males + hermaphrodites), size at sex change, fecundity, embryo volume and reproductive output of an exploited L. boggessi population. L. boggessi exhibited distinct seasonality in size distribution, sex phase ratio, size at sex phase change and reproductive activity. The peak reproductive season was in spring, when the population was dominated by small but fecund hermaphrodites. Reproduction decreased during fall and winter and sex phase ratios favored male phase shrimp that exhibited delayed sex change. This population and individual level information is the first of its kind for L. boggessi and fills a much needed data gap for the informed management of this fishery.