Functional characterization of transmembrane adenylyl cyclases from the honeybee brain

The second messenger cAMP has a pivotal role in animals' physiology and behavior. Intracellular concentrations of cAMP are balanced by cAMP-synthesizing adenylyl cyclases (ACs) and cAMP-cleaving phosphodiesterases. Knowledge about ACs in the honeybee (Apis mellifera) is rather limited and only an ortholog of the vertebrate AC3 isoform has been functionally characterized, so far.Employing bioinformatics and functional expression we characterized two additional honeybee genes encoding membrane-bound (tm)ACs. The proteins were designated AmAC2t and AmAC8. Unlike the common structure of tmACs, AmAC2t lacks the first transmembrane domain. Despite this unusual topography, AmAC2t-activity could be stimulated by norepinephrine and NKH477 with EC_50s of 0.07 μM and 3 μM. Both ligands stimulated AmAC8 with EC_50s of 0.24 μM and 3.1 μM. In brain cryosections, intensive staining of mushroom bodies was observed with specific antibodies against AmAC8, an expression pattern highly reminiscent of the Drosophila rutabaga AC.In a current release of the honeybee genome database we identified three additional tmAC- and one soluble AC-encoding gene. These results suggest that (1) the AC-gene family in honeybees is comparably large as in other species, and (2) based on the restricted expression of AmAC8 in mushroom bodies, this enzyme might serve important functions in honeybee behavior.     

The chemical cue tetrabromopyrrole from a biofilm bacterium induces settlement of multiple Caribbean corals

Microbial biofilms induce larval settlement for some invertebrates, including corals; however, the chemical cues involved have rarely been identified. Here, we demonstrate the role of microbial biofilms in inducing larval settlement with the Caribbean coral Porites astreoides and report the first instance of a chemical cue isolated from a marine biofilm bacterium that induces complete settlement (attachment and metamorphosis) of Caribbean coral larvae. Larvae settled in response to natural biofilms, and the response was eliminated when biofilms were treated with antibiotics. A similar settlement response was elicited by monospecific biofilms of a single bacterial strain, Pseudoalteromonas sp. PS5, isolated from the surface biofilm of a crustose coralline alga. The activity of Pseudoalteromonas sp. PS5 was attributed to the production of a single compound, tetrabromopyrrole (TBP), which has been shown previously to induce metamorphosis without attachment in Pacific acroporid corals. In addition to inducing settlement of brooded larvae (P. astreoides), TBP also induced larval settlement for two broadcast-spawning species, Orbicella (formerly Montastraea) franksi and Acropora palmata, indicating that this compound may have widespread importance among Caribbean coral species.     

Larval Settlement: The Role of Surface Topography for Sessile Coral Reef Invertebrates

For sessile marine invertebrates with complex life cycles, habitat choice is directed by the larval phase. Defining which habitat-linked cues are implicated in sessile invertebrate larval settlement has largely concentrated on chemical cues which are thought to signal optimal habitat. There has been less effort establishing physical settlement cues, including the role of surface microtopography. This laboratory based study tested whether surface microtopography alone (without chemical cues) plays an important contributing role in the settlement of larvae of coral reef sessile invertebrates. We measured settlement to tiles, engineered with surface microtopography (holes) that closely matched the sizes (width) of larvae of a range of corals and sponges, in addition to surfaces with holes that were markedly larger than larvae. Larvae from two species of scleractinian corals (Acropora millepora and Ctenactis crassa) and three species of coral reef sponges (Luffariella variabilis, Carteriospongia foliascens and Ircinia sp.,) were used in experiments. L. variabilis, A. millepora and C. crassa showed markedly higher settlement to surface microtopography that closely matched their larval width. C. foliascens and Ircinia sp., showed no specificity to surface microtopography, settling just as often to microtopography as to flat surfaces. The findings of this study question the sole reliance on chemical based larval settlement cues, previously established for some coral and sponge species, and demonstrate that specific physical cues (surface complexity) can also play an important role in larval settlement of coral reef sessile invertebrates.     

Biochemical properties of heterologously expressed and native adenylyl cyclases from the honeybee brain (Apis mellifera L.)

Cyclic AMP is an important intracellular signaling molecule participating e.g. in sensory signal transduction, cardiac myocyte regulation, learning and memory. The formation of cAMP is catalyzed by adenylyl cyclases. A variety of factors can modulate the properties of these enzymes and lead to dynamic changes of the intracellular cAMP concentration. Here we determined the tissue distribution of a recently cloned adenylyl cyclase (AmAC3) in honeybee brain. The protein is present in all neuropils. Intensive immunoreactivity was found in parts of the proto- and deutocerebrum and in the suboesophageal ganglion. Biochemical and pharmacological properties of AmAC3 and of native adenylyl cyclases in subregions of the honeybee brain were examined. Values for half-maximal activation with NKH477 were in the low micromolar range with 10.2 μM for AmAC3 and 3.6–8.1 μM for native enzymes. Biosynthesis of cAMP was specifically blocked by P-site inhibitors. Adenylyl cyclases in antennal lobes and AmAC3 share the inhibitory profile with 2′,5′dd3′ATP > 3′AMP > 2′deoxyadenosine. In addition to P-site inhibitors AmAC3 activity was impaired by Ca²⁺/calmodulin. The results suggest that AmAC3 is a likely candidate to fulfill an integrative role in sensory, motor and higher-order information processing in the honeybee brain.     

CO2 enrichment and reduced seawater pH had no effect on the embryonic development of Acropora palmata (Anthozoa,Scleractinia).

The effects of decreased pH, caused by carbon dioxide (CO₂) dissolution in seawater (known as ocean acidification (OA)), on the development of newly fertilized eggs of the Caribbean reef-building coral, Acropora palmata, was tested in three experiments conducted during the summers of 2008 and 2009 (two repeats). Three levels of CO₂ enrichment were used: present day conditions (400?µatm, pH 8.1) and two CO₂-enriched conditions (700?µatm, pH 7.9, and 1000?µatm, pH 7.7). No effects on the progression or timing of development, or embryo and larval size, were detected in any of the three experimental runs. The results show that the embryos and larvae of A. palmata are able to develop normally under seawater pH of at least 0.4 pH units lower than the present levels. Acropora palmata larvae do not usually begin to calcify after settlement, so this study only examined the non-calcifying part of the life cycle of this species. Most of the concern about the effects of OA on marine organisms centers on its effect on calcification. Negative effects of OA on the embryonic development of this species were not found and they may not manifest until the newly settled polyps begin to calcify.     

Can benthic algae mediate larval behavior and settlement of the coral Acropora muricata?

The resilience of coral reefs relies significantly on the ability of corals to recover successfully in algal-dominated environments. Larval settlement is a critical but highly vulnerable stage in the early life history of corals. In this study, we analyzed how the presence of two upright fleshy algae, Sargassum mcclurei (SM) and Padina australis (PA), and one crustose coralline algae, Mesophyllum simulans (MS), affects the settlement of Acropora muricata larvae. Coral larvae were exposed to seawater flowing over these algae at two concentrations. Larval settlement and mortality were assessed daily through four variables related to their behavior: swimming, substratum testing, metamorphosis, and stresses. Temperature, dissolved oxygen, pH, algal growth, and photosynthetic efficiency were monitored throughout the experiment. Results showed that A. muricata larvae can settle successfully in the absence of external stimuli (63 ± 6 % of the larvae settled in control treatments). While algae such as MS may stimulate substrate testing and settlement of larvae in the first day after competency, they ultimately had a lower settlement rate than controls. Fleshy algae such as PA, and in a lesser measure SM, induced more metamorphosis than controls and seemed to eventually stimulate settlement. A diverse combination of signals and/or modifications of microenvironments by algae and their associated microbial communities may explain the pattern observed in coral settlement. Overall, this study contributes significantly to the knowledge of the interaction between coral and algae, which is critical for the resilience of the reefs.