Evolution of a novel carotenoid-binding protein responsible for crustacean shell color.

Carotenoids are commonly used by disparate metazoans to produce external coloration, often in direct association with specific proteins. In one such example, crustacyanin (CRCN) and the carotenoid astaxanthin combine to form a multimeric protein complex that is critical for the array of external shell colors in clawed lobsters. Through a combined biochemical, molecular genetic, and bioinformatic survey of the distribution of CRCN across the animal kingdom, we have found that CRCNs are restricted to, but widespread among, malacostracan crustaceans. These crustacean-specific genes separate into two distinct clades within the lipocalin protein superfamily. We show that CRCN differentially localizes to colored shell territories and the underlying epithelium in panulirid lobsters. Given the paramount importance of CRCN in crustacean shell colors and patterns and the critical role these play in survival, reproduction, and communication, we submit that the origin of the CRCN gene family early in the evolution of malacostracan crustaceans significantly contributed to the success of this group of arthropods.

Microbial diversity within early-stage cultured Panulirus ornatus phyllosomas.

A thorough understanding of the microorganisms and pathogens associated with the larval stage of the tropical ornate rock lobster, Panulirus ornatus, is required to overcome disease outbreaks that currently block aquaculture attempts. This study used microscopy in addition to culture and molecularly based microbiological techniques to characterize the bacterial community associated with cultured, developmental stage PI to PII P. ornatus phyllosomas. Scanning electron microscopy demonstrated colonization of phyllosomas by filamentous, rod-shaped, and coccus-shaped bacteria. A clone library constructed from dead phyllosomas sampled from the larval rearing tank on day 10 was dominated by Thiothrix-affiliated sequences (56% of clones). A comparable library from live phyllosomas also contained Thiothrix-affiliated sequences, though these only represented 19% of clones within the library. Fluorescent in situ hybridization (FISH) confirmed identification of the filamentous bacteria as Thiothrix sp., being present on dead phyllosomas. FISH also identified Leucothrix sp. and Vibrio sp., as well as a range of other rod- and coccus-shaped bacteria, colonizing both live and dead phyllosomas. The development of the microbial community associated with phyllosomas was monitored through a standard larval rearing run using denaturing gradient gel electrophoresis (DGGE). Vibrio sp.-affiliated bands dominated the profiles of live animals through the rearing period and dead phyllosomas sampled on selected days. The population of Vibrio sp. associated with phyllosomas was monitored with culture-based analysis on selective media and demonstrated to increase significantly on day 7, coinciding with the beginning of the larval molt. An isolated Vibrio harveyi strain demonstrated an identical 16S rRNA sequence with retrieved DGGE and clone library sequences. Colonization of phyllosomas with filamentous bacterial species potentially hinders the ability of the animals to molt and, combined with the added stress of the molt process, likely results in reduced immune function, allowing opportunistic pathogenic Vibrio sp. to cause larval mortalities.

Esterified astaxanthin levels in lobster epithelia correlate with shell colour intensity: potential role in crustacean shell colour formation.

Carotenoids, particularly astaxanthin, are the primary pigment in crustacean shell colour. Sub-adults of the western rock lobster, Panulirus cygnus, moult from a deep red colour (termed the red phase) to a much paler colour (the white phase) at sexual maturation. We observe a 2.4-fold difference in the amount of total carotenoid present in the shell extracts of reds compared to whites, as might be expected. However, analysis of the underlying epithelium shows that there is no correlation with shell colour and the amount of free (unesterified) astaxanthin-the level of free astaxanthin in reds and whites is not significantly different. Instead, we observe a correlated two-fold difference in the amount of esterified astaxanthin present in the epithelium of red versus white individuals. These data suggest a role for esterified astaxanthin in regulating shell colour formation and suggest that esterification may promote secretion and eventual incorporation of unesterified astaxanthin into the exoskeleton.