Chloroplast incorporation and long-term photosynthetic performance through the life cycle in laboratory cultures of Elysia timida (Sacoglossa, Heterobranchia).

INTRODUCTION: The Mediterranean sacoglossan Elysia timida is one of the few sea slug species with the ability to sequester chloroplasts from its food algae and to subsequently store them in a functional state in the digestive gland cells for more than a month, during which time the plastids retain high photosynthetic activity (= long-term retention). Adult E. timida have been described to feed on the unicellular alga Acetabularia acetabulum in their natural environment. The suitability of E. timida as a laboratory model culture system including its food source was studied. RESULTS: In contrast to the literature reporting that juvenile E. timida feed on Cladophora dalmatica first, and later on switch to the adult diet A. acetabulum, the juveniles in this study fed directly on A. acetabulum (young, non-calcified stalks); they did not feed on the various Cladophora spp. (collected from the sea or laboratory culture) offered. This could possibly hint to cryptic speciation with no clear morphological differences, but incipient ecological differentiation. Transmission electron microscopy of chloroplasts from A. acetabulum after initial intake by juvenile E. timida showed different states of degradation - in conglomerations or singularly - and fragments of phagosome membranes, but differed from kleptoplast images of C. dalmatica in juvenile E. timida from the literature. Based on the finding that the whole life cycle of E. timida can be completed with A. acetabulum as the sole food source, a laboratory culture system was established. An experiment with PAM-fluorometry showed that cultured E. timida are also able to store chloroplasts in long-term retention from Acetabularia peniculus, which stems from the Indo-Pacific and is not abundant in the natural environment of E. timida. Variations between three experiment groups indicated potential influences of temperature on photosynthetic capacities. CONCLUSIONS: E. timida is a viable laboratory model system to study photosynthesis in incorporated chloroplasts (kleptoplasts). Capacities of chloroplast incorporation in E. timida were investigated in a closed laboratory culture system with two different chloroplast donors and over extended time periods about threefold longer than previously reported.

Transcriptomic evidence that longevity of acquired plastids in the photosynthetic slugs Elysia timida and Plakobranchus ocellatus does not entail lateral transfer of algal nuclear genes.

Sacoglossan sea slugs are unique in the animal kingdom in that they sequester and maintain active plastids that they acquire from the siphonaceous algae upon which they feed, making the animals photosynthetic. Although most sacoglossan species digest their freshly ingested plastids within hours, four species from the family Plakobranchidae retain their stolen plastids (kleptoplasts) in a photosynthetically active state on timescales of weeks to months. The molecular basis of plastid maintenance within the cytosol of digestive gland cells in these photosynthetic metazoans is yet unknown but is widely thought to involve gene transfer from the algal food source to the slugs based upon previous investigations of single genes. Indeed, normal plastid development requires hundreds of nuclear-encoded proteins, with protein turnover in photosystem II in particular known to be rapid under various conditions. Moreover, only algal plastids, not the algal nuclei, are sequestered by the animals during feeding. If algal nuclear genes are transferred to the animal either during feeding or in the germ line, and if they are expressed, then they should be readily detectable with deep-sequencing methods. We have sequenced expressed mRNAs from actively photosynthesizing, starved individuals of two photosynthetic sea slug species, Plakobranchus ocellatus Van Hasselt, 1824 and Elysia timida Risso, 1818. We find that nuclear-encoded, algal-derived genes specific to photosynthetic function are expressed neither in P. ocellatus nor in E. timida. Despite their dramatic plastid longevity, these photosynthetic sacoglossan slugs do not express genes acquired from algal nuclei in order to maintain plastid function.

Use of the intubating laryngeal mask airway in emergency pre-hospital difficult intubation.

AIM OF THE STUDY: While several techniques are used for the management of difficult intubation (DI) in planned conditions in the operating theatre, they are not always suitable or usable in pre-hospital emergencies. We decided to assess the intubating laryngeal mask airway (ILMA) after failure of tracheal intubation (TI) under direct laryngoscopy. MATERIAL AND METHODS: After emergency physicians of the mobile intensive care unit were trained (theory and training on manikin) in using the ILMA (Fastrach), prospective data were collected after each use from March 2002 to December 2005. Data included patient's age, clinical status, number of direct laryngoscopies before using ILMA, Cormack and Lehane grade, subjective and objective evaluation of ease of ILMA insertion and TI (analogue scale from 1 to 10, attempts required, failure rate). RESULTS: Over 46 months, the ILMA was used 45 times (24: cardio-respiratory arrest, 21: anaesthesia with rapid sequence induction). Median age was 59 years [range 20-86]. The number of direct laryngoscopy attempts was 3 [0-4] (76% Cormack 4). The success of ILMA insertion and TI were 96 and 91%, respectively. CONCLUSION: Emergency physicians were satisfied with using the ILMA. It allowed TI in 91% of cases of DI. The ILMA can be recommended to be included in the algorithm of DI in pre-hospital emergencies after initial training.

Mating behaviour in the sea slug Elysia timida (Opisthobranchia, Sacoglossa): hypodermic injection, sperm transfer and balanced reciprocity.

BACKGROUND: In simultaneous hermaphrodites with copulation and internal fertilization it is often unclear whether reciprocal sperm exchange results from the unconditional willingness of both partners to donate and receive sperm, or whether it follows from a more controlled process such as conditional reciprocal sperm exchange, i.e. sperm trading. While in some sea slugs mating is assumed to be based on sperm trading, it seems to be unconditional in others. Here, we describe the unusual mating behaviour of Elysia timida, a small sacoglossan, focussing on indications for conditional reciprocity. RESULTS: E. timida shows an as yet unique combination of a long series of hypodermic transfers followed by a short phase with standard insemination into a female genital aperture. Hypodermic transfer takes place in the form of repeated small injections into the dorsal surface of the partner, interrupted by synchronised circling movements. In the final mating phase sperm is transferred into the female genital aperture in a short period. In both phases the two mating individuals show a high degree of transfer symmetry and synchrony. While total duration and number of transfers were balanced within pairs, they varied significantly between pairs. Furthermore, looking at individual hypodermic transfers within pairs, reciprocal transfers lasted longer than unilateral transfers. Final sperm transfers were always reciprocal except for two cases which also diverted from the usual pattern in ways that were suggestive of a conflict over reciprocity. CONCLUSION: Our results suggest that individual mating decisions in E. timida depend on what the partner does, indicating conditional reciprocity. If hypodermic transfers also involve the transfer of sperm (which remains to be confirmed), this system represents an up to now unique transition stage between hypodermic and standard insemination, both of which are widespread in this group of sea slugs, but never have been observed to co-occur within the same species.