Unique horizontal gaze control in the box jellyfish, Tripedalia cystophora.

All known cubozoans, box jellyfish, have a similar visual system. They possess four sensory structures called rhopalia, which carry-six eyes each. Two of these six eyes are true image-forming camera type eyes in several ways similar to vertebrate eyes. The rhopalia hang by a thin flexible stalk and in the distal end, there is a high-density crystal. In an earlier study of the Caribbean species Tripedalia cystophora, we showed that the crystals act as weights ensuring that the rhopalia are always upright no matter the orientation of the medusa and the vertical part of the visual field of the eyes thus kept relatively constant. Here we have examined the horizontal part of the visual field under different experimental conditions including different visual environments. We find that the horizontal gaze direction is largely controlled by the anatomy of the rhopalium and rhopalial stalk, similar to what has previously been shown for the vertical gaze direction. In a vertically oriented medusa, the rhopalia are kept with a 90° angle between them with the lower lens eyes (LLE) pointing inwards. This 90° shift is kept in horizontally swimming medusa, resulting in the left LLE gazing right, the right gazing left, the bottom gazing orally (backwards compared to swimming direction), and the top LLE gazing aborally (forwards compared to swimming direction). The light environment was manipulated to test if the visual input influences this seemingly strict horizontal gaze direction but even in complete darkness there is tight mechanistic control.

Bay watch: Using unmanned aerial vehicles (UAV's) to survey the box jellyfish Chironex fleckeri.

Biological investigations on free ranging marine species are regarded as challenging throughout the scientific community. This is particularly true for 'logistically difficult species' where their cryptic natures, low abundance, patchy distributions and difficult and/or dangerous sampling environments, make traditional surveys near impossible. What results is a lack of ecological knowledge on such marine species. However, advances in UAV technology holds potential for overcoming these logistical difficulties and filling this knowledge gap. Our research focused on one such logistically difficult species, the Australian box Jellyfish (Chironex fleckeri), and we investigated the capacity of consumer grade UAV technology to detect this, highly venomous, target species in the inshore waters of Northern Queensland Australia. At two sites in the Weipa area, we utilized video analysis, visual count comparisons with a netted animal tally, and evaluated the role of associated environmental conditions, such as wind speed, water visibility and cloud cover on jellyfish detection rates. In total fifteen, 70 meter transects were completed between two sites, with 107 individuals captured. Drone success varied between the two sites with a significant difference between field and post-field (laboratory) counts. Animal size and cloud cover also had significant effects on detection rates with an increase in cloud cover and animal size enhancing detection probability. This study provides evidence to suggest drone surveys overcome obstacles that traditional surveys can't, with respect to species deemed logistically difficult and open scope for further ecological investigations on such species.

Tentacle Musculature in the Cubozoan Jellyfish Carybdea marsupialis.

The diploblastic cnidarian body plan comprising the epidermis and gastrodermis has remained largely unchanged since it evolved roughly 600 Ma. The origin of muscle from the mesoderm in triploblastic lineages is a central evolutionary question in higher animals. Triploblasts have three embryonic germ layers: the endoderm, mesoderm, and ectoderm, which develop into organs, muscle, and skin, respectively. Diploblasts lack the mesoderm, the layer thought to give rise to the skeletomuscular system. However, phyla such as Cnidaria and Ctenophora, which are typically classified as diploblasts, possess striated musculature. Within phylum Cnidaria, class Cubozoa includes carnivorous box jellyfish, which are capable of extending and contracting their tentacles for predation and defense mechanisms, thus suggesting a well-organized system of muscles. Here, the tentacle musculature of the cubomedusae Carybdea marsupialis is investigated using transmission electron microscopy in conjunction with light microscopy to further understand the arrangement of musculature in these primitive animals. Cross sections of tentacles confirmed that the gastrodermis is separated from the epidermis by a collagenous mesogleal layer containing numerous longitudinal muscle cells arranged in fascicles. Longitudinal muscles permit the tentacle to retract toward the bell during fast tentacle shortening and crumpling behavioral responses. Circular muscle cells were found in the gastrodermis and epidermis, encircling the layer of longitudinal muscle. These circular muscles likely enable the elongation process that allows the tentacles to return to a resting state after contraction. The presence of a definitive muscle cell layer within the mesoglea suggests that C. marsupialis has an advanced muscle morphology that is similar to triploblastic animals.

Regeneration of the Rhopalium and the Rhopalial Nervous System in the Box Jellyfish Tripedalia cystophora.

Cubozoans have the most intricate visual apparatus within Cnidaria. It comprises four identical sensory structures, the rhopalia, each of which holds six eyes of four morphological types. Two of these eyes are camera-type eyes that are, in many ways, similar to the vertebrate eye. The visual input is used to control complex behaviors, such as navigation and obstacle avoidance, and is processed by an elaborate rhopalial nervous system. Several studies have examined the rhopalial nervous system, which, despite a radial symmetric body plan, is bilaterally symmetrical, connecting the two sides of the rhopalium through commissures in an extensive neuropil. The four rhopalia are interconnected by a nerve ring situated in the oral margin of the bell, and together these structures constitute the cubozoan central nervous system. Cnidarians have excellent regenerative capabilities, enabling most species to regenerate large body areas or body parts, and some species can regenerate completely from just a few hundred cells. Here we test whether cubozoans are capable of regenerating the rhopalia, despite the complexity of the visual system and the rhopalial nervous system. The results show that the rhopalia are readily regrown after amputation and have developed most, if not all, neural elements within two weeks. Using electrophysiology, we investigated the functionality of the regrown rhopalia and found that they generated pacemaker signals and that the lens eyes showed a normal response to light. Our findings substantiate the amazing regenerative ability in Cnidaria by showing here the complex sensory system of Cubozoa, a model system proving to be highly applicable in studies of neurogenesis.

Gonadal histology of box jellyfish (Cnidaria: Cubozoa) reveals variation between internal fertilizing species Alatina alata (Alatinidae) and Copula sivickisi (Tripedaliidae).

Cubozoans (box jellyfish) are gonochoristic cnidarians with distinct reproductive strategies. This comparative histological study examines the gonad organization of Alatina alata and Copula sivickisi, two box jellyfish species that exhibit different modes of internal fertilization. A. alata reproduces via spermcasting aggregations while C. sivickisi reproduces via copulation; in both cases, internal fertilization occurs in the gastrovascular cavity. Herein, we provide the first histological description of subgastric sacs-structures unique to C. sivickisi. Although previously thought to function as sperm storage sacs, our findings reveal that subgastric sacs are nematocyst nests lacking sperm entirely. Conversely, we discovered that velarial spots in C. sivickisi females correspond to actual sperm storage structures. Histological examination of cubozoan sperm packages revealed that while sperm packages from both species have motile flagella, A. alata males produce nonencapsulated sperm bundles (i.e., "spermatozeugmata"), and C. sivickisi males produce encapsulated packages (i.e., "spermatophores"). Our findings corroborate the presence of several types of nematocysts in C. sivickisi embryo strands and spermatophores, and indicate their provenance to be both female and male gonads respectively, as well as subgastric sacs (i.e., nematocyst nests). In contrast to our findings of velarial spots as sperm storages structures in C. sivickisi females, and of nematocysts in the gonads of both sexes, we report that A. alata medusae lack both sperm storage structures and gonadal nematocysts. Finally, we discuss our findings on reproductive morphology of C. sivickisi and A. alata in light of the respective reproductive behavior of these two cubozoan species.

Statolith Morphometrics Can Discriminate among Taxa of Cubozoan Jellyfishes.

Identification of potentially harmful cubomedusae is difficult due to their gelatinous nature. The only hard structure of medusae, the statolith, has the potential to provide robust measurements for morphometric analysis. Traditional morphometric length to width ratios (L: W) and modern morphometric Elliptical Fourier Analysis (EFA) were applied to proximal, oral and lateral statolith faces of 12 cubozoan species. EFA outperformed L: W as L: W did not account for the curvature of the statolith. Best discrimination was achieved with Canonical Discriminant Analysis (CDA) when analysing proximal + oral + lateral statolith faces in combination. Normalised Elliptical Fourier (NEF) coefficients classified 98% of samples to their correct species and 94% to family group. Statolith shape agreed with currently accepted cubozoan taxonomy. This has potential to assist in identifying levels of risk and stock structure of populations in areas where box jellyfish envenomations are a concern as the severity of envenomation is family dependent. We have only studied 12 (27%) of the 45 currently accepted cubomedusae, but analyses demonstrated that statolith shape is an effective taxonomic discriminator within the Class.

Prey Capture Ecology of the Cubozoan Carukia barnesi.

Adult Carukia barnesi medusae feed predominantly on larval fish; however, their mode of prey capture seems more complex than previously described. Our findings revealed that during light conditions, this species extends its tentacles and 'twitches' them frequently. This highlights the lure-like nematocyst clusters in the water column, which actively attract larval fish that are consequently stung and consumed. This fishing behavior was not observed during dark conditions, presumably to reduce energy expenditure when they are not luring visually oriented prey. We found that larger medusae have longer tentacles; however, the spacing between the nematocyst clusters is not dependent on size, suggesting that the spacing of the nematocyst clusters is important for prey capture. Additionally, larger specimens twitch their tentacles more frequently than small specimens, which correlate with their recent ontogenetic prey shift from plankton to larval fish. These results indicate that adult medusae of C. barnesi are not opportunistically grazing in the water column, but instead utilize sophisticated prey capture techniques to specifically target larval fish.

Sexual dimorphism in Tripedaliidae (Conant 1897) (Cnidaria, Cubozoa, Carybdeida).

The family Tripedaliidae was re-defined and expanded based on a molecular phylogenetic hypothesis by Bentlage et al. (2010, Proceedings of the Royal Society Biological Science, 277: 497). Additionally, Bentlage et al. (2010) proposed that all members of the family Tripedaliidae present dimorphism in gonads and have structures that function as seminal vesicles (at least in males). Until now, no information on Tripedalia binata concerning gonad morphology, sexual dimorphism, spermatophore formation or structures that serve as seminal vesicles or spermathecae were published. We studied mature medusae of both sexes of Tripedalia cystophora, Tripedalia binata and Copula sivickisi in order to compare these structures in their stomach regions. We found sexual dimorphism and spermatophore formation in seminal vesicle-like structures in all three species. In particular, we show that along with the females of Copula sivickisi, the females of Tripedalia cystophora and Tripedalia binata also possess structures that store spermatophores and serve as spermathecae. The results are in agreement with the morphological synapomorphies for Tripedaliidae outlined in Bentlage et al. (2010), but suggest an adjustment of the diagnosis of Tripedaliidae (underlined): All carybdeids that display sexual dimorphism of the gonads, produce spermatophores and in which males and females possess subgastral sacs, pockets or purses which function as seminal vesicles or spermathecae.

Organization of the ectodermal nervous structures in medusae: cubomedusae.

At least two conducting systems are well documented in cubomedusae. A variably diffuse network of large neurons innervates the swim musculature and can be visualized immunohistochemically using antibodies against α- or ß-tubulin. Despite the non-specificity of these antibodies, multiple lines of evidence suggest that staining highlights the primary motor networks. These networks exhibit unique neurite distributions among the muscle sheets in that network density is greatest in the perradial frenula, where neurites are oriented in parallel with radial muscle fibers. This highly innervated, buttress-like muscle sheet may serve a critical role in the cubomedusan mechanism of turning. In scyphomedusae, a second subumbrellar network immunoreactive to antibodies against the neuropeptide FMRFamide innervates the swim musculature, but it is absent in cubomedusae. Immunoreactivity to FMRFamide in cubomedusae is mostly limited to a small network of neurons in the pacemaker region of the rhopalia, the pedalial apex at the nerve ring junction, and a few neuron tracts in the nerve ring. However, FMRFamide-immunoreactive networks, as well as tubulin-immunoreactive networks, are nearly ubiquitous outside of the swim muscle sheets in the perradial smooth muscle bands, manubrium, pedalia, and tentacles. Here we describe in detail the peripheral nerve nets of box jellyfish on the basis of immunoreactivity to the antibodies above. Our results offer insight into how the peripheral nerve nets are organized to produce the complex swimming, feeding, and defensive behaviors observed in cubomedusae.

Biology and ecology of Irukandji jellyfish (Cnidaria: Cubozoa).

Irukandji stings are a leading occupational health and safety issue for marine industries in tropical Australia and an emerging problem elsewhere in the Indo-Pacific and Caribbean. Their mild initial sting frequently results in debilitating illness, involving signs of sympathetic excess including excruciating pain, sweating, nausea and vomiting, hypertension and a feeling of impending doom; some cases also experience acute heart failure and pulmonary oedema. These jellyfish are typically small and nearly invisible, and their infestations are generally mysterious, making them scary to the general public, irresistible to the media, and disastrous for tourism. Research into these fascinating species has been largely driven by the medical profession and focused on treatment. Biological and ecological information is surprisingly sparse, and is scattered through grey literature or buried in dispersed publications, hampering understanding. Given that long-term climate forecasts tend toward conditions favourable to jellyfish ecology, that long-term legal forecasts tend toward increasing duty-of-care obligations, and that bioprospecting opportunities exist in the powerful Irukandji toxins, there is a clear need for information to help inform global research and robust management solutions. We synthesise and contextualise available information on Irukandji taxonomy, phylogeny, reproduction, vision, behaviour, feeding, distribution, seasonality, toxins, and safety. Despite Australia dominating the research in this area, there are probably well over 25 species worldwide that cause the syndrome and it is an understudied problem in the developing world. Major gaps in knowledge are identified for future research: our lack of clarity on the socio-economic impacts, and our need for time series and spatial surveys of the species, make this field particularly enticing.

Velarium control and visual steering in box jellyfish.

Directional swimming in the box jellyfish Tripedalia cystophora (cubozoa, cnidaria) is controlled by the shape of the velarium, which is a thin muscular sheet that forms the opening of the bell. It was unclear how different patterns of visual stimulation control directional swimming and that is the focus of this study. Jellyfish were tethered inside a small experimental tank, where the four vertical walls formed light panels. All four panels were lit at the start of an experiment. The shape of the opening in the velarium was recorded in response to switching off different combinations of panels. We found that under the experimental conditions the opening in the velarium assumed three distinct shapes during a swim contraction. The opening was (1) centred or it was off-centred and pocketed out either towards (2) a rhopalium or (3) a pedalium. The shape of the opening in the velarium followed the direction of the stimulus as long as the stimulus contained directional information. When the stimulus contained no directional information, the percentage of centred pulses increased and the shape of the off-centred pulses had a random orientation. Removing one rhopalium did not change the directional response of the animals, however, the number of centred pulses increased. When three rhopalia were removed, the percentage of centred pulses increased even further and the animals lost their ability to respond to directional information.

Propulsion in cubomedusae: mechanisms and utility.

Evolutionary constraints which limit the forces produced during bell contractions of medusae affect the overall medusan morphospace such that jet propulsion is limited to only small medusae. Cubomedusae, which often possess large prolate bells and are thought to swim via jet propulsion, appear to violate the theoretical constraints which determine the medusan morphospace. To examine propulsion by cubomedusae, we quantified size related changes in wake dynamics, bell shape, swimming and turning kinematics of two species of cubomedusae, Chironex fleckeri and Chiropsella bronzie. During growth, these cubomedusae transitioned from using jet propulsion at smaller sizes to a rowing-jetting hybrid mode of propulsion at larger sizes. Simple modifications in the flexibility and kinematics of their velarium appeared to be sufficient to alter their propulsive mode. Turning occurs during both bell contraction and expansion and is achieved by generating asymmetric vortex structures during both stages of the swimming cycle. Swimming characteristics were considered in conjunction with the unique foraging strategy used by cubomedusae.

Redescription of Alatina alata (Reynaud, 1830) (Cnidaria: Cubozoa) from Bonaire, Dutch Caribbean.

Here we establish a neotype for Alatina alata (Reynaud, 1830) from the Dutch Caribbean island of Bonaire. The species was originally described one hundred and eighty three years ago as Carybdea alata in La Centurie Zoologique-a monograph published by René Primevère Lesson during the age of worldwide scientific exploration. While monitoring monthly reproductive swarms of A. alata medusae in Bonaire, we documented the ecology and sexual reproduction of this cubozoan species. Examination of forty six A. alata specimens and additional archived multimedia material in the collections of the National Museum of Natural History, Washington, DC revealed that A. alata is found at depths ranging from surface waters to 675 m. Additional studies have reported it at depths of up to 1607 m in the tropical and subtropical Atlantic Ocean. Herein, we resolve the taxonomic confusion long associated with A. alata due to a lack of detail in the original description and conflicting statements in the scientific literature. A new cubozoan character, the velarial lappet, is described for this taxon. The complete description provided here serves to stabilize the taxonomy of the second oldest box jellyfish species, and provide a thorough redescription of the species.

The pharmacology of Malo maxima jellyfish venom extract in isolated cardiovascular tissues: A probable cause of the Irukandji syndrome in Western Australia.

The in vitro cardiac and vascular pharmacology of Malo maxima, a newly described jellyfish suspected of causing Irukandji syndrome in the Broome region of Western Australia, was investigated in rat tissues. In left atria, M. maxima crude venom extract (CVE; 1-100µg/mL) caused concentration-dependent inotropic responses which were unaffected by atropine (1µM), but significantly attenuated by tetrodotoxin (TTX; 0.1µM), propranolol (1µM), Mg(2+) (6mM) or calcitonin gene-related peptide antagonist (CGRP(8-37); 1µM). CVE caused no change in right atrial rate until 100µg/mL, which elicited bradycardia. This was unaffected by atropine, TTX, propranolol or CGRP(8-37). In the presence of Mg(2+), CVE 30-100µg/mL caused tachycardia. In small mesenteric arteries CVE caused concentration-dependent contractions (pEC(50) 1.03±0.07µg/mL) that were unaffected by prazosin (0.3µM), ω-conotoxin GVIA (0.1µM) or Mg(2+) (6mM). There was a 2-fold increase in sensitivity in the presence of CGRP(8-37) (3µM). TTX (0.1µM), box jellyfish Chironex fleckeri antivenom (92.6U/mL) and benextramine (3µM) decreased sensitivity by 2.6, 1.9 and 2.1-fold, respectively. CVE-induced maximum contractions were attenuated by C. fleckeri antivenom (-22%) or benextramine (-49%). M. maxima CVE appears to activate the sympathetic, but not parasympathetic, nervous system and to stimulate sensory nerve CGRP release in left atria and resistance arteries. These effects are consistent with the catecholamine excess thought to cause Irukandji syndrome, with additional actions of CGRP release.

Temporal properties of the lens eyes of the box jellyfish Tripedalia cystophora.

Box jellyfish (Cubomedusae) are visually orientating animals which possess a total of 24 eyes of 4 morphological types; 2 pigment cup eyes (pit eye and slit eye) and 2 lens eyes [upper lens-eye (ule) and lower lens-eye (lle)]. In this study, we use electroretinograms (ERGs) to explore temporal properties of the two lens eyes. We find that the ERG of both lens eyes are complex and using sinusoidal flicker stimuli we find that both lens eyes have slow temporal resolution. The average flicker fusion frequency (FFF) was found to be approximately 10 Hz for the ule and 8 Hz for the lle. Differences in the FFF and response patterns between the two lens eyes suggest that the ule and lle filter information differently in the temporal domain and thus are tuned to perform different visual tasks. The data collected in this study support the idea that the visual system of box jellyfish is a collection of special purpose eyes.

Structure and optics of the eyes of the box jellyfish Chiropsella bronzie.

Cubomedusae have a total of 24 eyes of four morphologically different types. Two of these eye types are camera-type eyes (upper and lower lens-eye), while the other two eye types are simpler pigment pit eyes (pit and slit eye). Here, we give a description of the visual system of the box jellyfish species Chiropsella bronzie and the optics of the lens eyes in this species. One aim of this study is to distinguish between general cubozoan features and species-specific features in the layout and optics of the eyes. We find that both types of lens eyes are more severely under-focused in C. bronzie than those in the previously investigated species Tripedalia cystophora. In the lower lens-eye of C. bronzie, blur circles subtend 20 and 52 degrees for closed and open pupil, respectively, effectively removing all but the coarsest structures of the image. Histology reveals that the retina of the lower lens-eye, in addition to pigmented photoreceptors, also contains long pigment-cells, with both dark and white pigment, where the dark pigment migrates on light/dark adaptation. Unlike the upper lens-eye lens of T.cystophora, the same eye in C.bronzie did not display any significant optical power.

Unique structure and optics of the lesser eyes of the box jellyfish Tripedalia cystophora.

The visual system of box jellyfish comprises a total of 24 eyes. These are of four types and each probably has a special function. To investigate this hypothesis the morphology and optics of the lesser eyes, the pit and slit eyes, were examined. The pit eyes hold one cell type only and are probably mere light meters. The slit eyes, comprising four cell types, are complex and highly asymmetric. They also hold a lens-like structure, but its optical power is minute. Optical modeling suggests spatial resolution, but only in one plane. These unique and intriguing traits support strong peripheral filtering.

The lens eyes of the box jellyfish Tripedalia cystophora and Chiropsalmus sp. are slow and color-blind.

Box jellyfish, or cubomedusae, possess an impressive total of 24 eyes of four morphologically different types. Compared to other cnidarians they also have an elaborate behavioral repertoire, which for a large part seems to be visually guided. Two of the four types of cubomedusean eyes, called the upper and the lower lens eye, are camera type eyes with spherical fish-like lenses. Here we explore the electroretinograms of the lens eyes of the Caribbean species, Tripedalia cystophora, and the Australian species, Chiropsalmus sp. using suction electrodes. We show that the photoreceptors of the lens eyes of both species have dynamic ranges of about 3 log units and slow responses. The spectral sensitivity curves for all eyes peak in the blue-green region, but the lower lens eye of T. cystophora has a small additional peak in the near UV range. All spectral sensitivity curves agree well with the theoretical absorbance curve of a single opsin, strongly suggesting color-blind vision in box jellyfish with a single receptor type. A single opsin is supported by selective adaptation experiments.

Venom ontogeny, diet and morphology in Carukia barnesi, a species of Australian box jellyfish that causes Irukandji syndrome.

Venom profiles of two age groups of the medically important Australian box jellyfish Carukia barnesi [Southcott, R.V., 1967. Revision of some Carybdeidae (Scyphozoa, Cubomedusae), including description of jellyfish responsible for the 'Irukandji' syndrome. Aust. J. Zool. 15, 651-657] were compared. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed differences in protein banding of tentacular venom between immature and mature animals. This correlates to a change in diet from invertebrate prey in immature C. barnesi medusae to vertebrate prey in mature medusae. Unlike other cubozoan studies, a change in venom did not equate to a change in nematocyst types or their relative frequencies. Additionally, comparison of tentacle structure and bell wart number showed developmental differences between the two age classes. Observations of prey capture in mature individuals and differences in bell warts between immature and mature medusae suggest different methods of prey capture are employed at different life stages of C. barnesi.