First records of the sicklefin (Mobula tarapacana), bentfin (Mobula thurstoni), and spinetail (Mobula mobular) devil rays in the Chagos Archipelago.

Recent encounters with sicklefin (Mobula tarapacana) and bentfin (Mobula thurstoni) devil rays in the Chagos Archipelago provide the first confirmed observations of live specimens of these species in this region. Examination of illegal fishing photo archives collected during enforcement revealed these endangered species, and spinetail devil rays (Mobula mobular), are being caught within the archipelago's vast no-take marine protected area. Future cooperation between authorities and mobulid ray experts is crucial to improve the availability and accuracy of enforcement data and improve management of illegal fishing and mobulid ray conservation activities.

Comparative population genomics of manta rays has global implications for management.

Understanding population connectivity and genetic diversity is of fundamental importance to conservation. However, in globally threatened marine megafauna, challenges remain due to their elusive nature and wide-ranging distributions. As overexploitation continues to threaten biodiversity across the globe, such knowledge gaps compromise both the suitability and effectiveness of management actions. Here, we use a comparative framework to investigate genetic differentiation and diversity of manta rays, one of the most iconic yet vulnerable groups of elasmobranchs on the planet. Despite their recent divergence, we show how oceanic manta rays (Mobula birostris) display significantly higher heterozygosity than reef manta rays (Mobula alfredi) and that M. birostris populations display higher connectivity worldwide. Through inferring modes of colonization, we reveal how both contemporary and historical forces have likely influenced these patterns, with important implications for population management. Our findings highlight the potential for fisheries to disrupt population dynamics at both local and global scales and therefore have direct relevance for international conservation of marine species.

Use of underwater contactless ultrasonography to elucidate the internal anatomy and reproductive activity of manta and devil rays (family: Mobulidae).

The ability to visualise the internal anatomical structures of fish provides important information on their reproductive status and body condition and has made important contributions to many areas of fish biology. Obtaining information on the internal anatomy of fish has traditionally required euthanasia and dissection. Although ultrasonography is now increasingly used to study internal fish anatomy without the need for euthanasia, traditional techniques still require restraint and contact with the animal, both of which are known to cause stress. This has prompted the development of waterproof, contactless and portable equipment to allow ultrasonographic examinations to be carried out in free-swimming individuals, which also facilitates the application of this tool in wild populations of endangered species. This study reports the validation of this equipment using anatomical examinations of nine manta and devil ray (Mobulidae) specimens landed at fish markets in Sri Lanka. The species studied were Mobula kuhlii (n = 3), Mobula thurstoni (n = 1), Mobula mobular (n = 1), Mobula tarapacana (n = 1) and Mobula birostris (n = 3). The use of this equipment was further validated with ultrasonographic examinations in 55 free-swimming reef manta rays Mobula alfredi, which enabled maturity status to be quantified in 32 females. Structures successfully identified in free-swimming individuals were the liver, spleen, gallbladder, gastrointestinal tract, skeletal structures, developing follicles and uterus. The study demonstrated that ultrasonography provided a reliable method of determining both sexual maturity and gestational status in free-swimming M. alfredi. The methodology induced no detectable signs of disturbance to the animals involved and therefore offers a viable and practical alternative to invasive techniques currently used to study anatomical changes in both captive and wild marine organisms.

Distribution of the reef manta ray Mobula alfredi and the oceanic manta ray Mobula birostris in the Philippines: a collaborative effort for conservation.

Little is known about manta ray population size, structure and connectivity in the Philippines. In collaboration with dive operators, non-governmental organizations and authorities, sightings of manta rays were collated into a single national database. Using in-water photographs and videos gathered through citizen science and dedicated research efforts, this study compiled sightings between 2004 and 2020, showing 22 separate sites throughout the archipelago with manta rays present. A total of 392 individual reef manta rays (Mobula alfredi) and 107 oceanic manta rays (Mobula birostris) were identified from the collected footage. Four specific sites in the provinces of Masbate and Palawan together hosted 89% of all identified individuals and accounted for 95% of sightings, highlighting these areas are key aggregation sites. This study also reports the movements of M. birostris within the Philippines, based on photo-identification of three individuals moving 150 km between Cebu and Masbate. Despite the growing number of recreational divers in Daanbantayan and San Jacinto, an 80% decline in M. birostris sightings was observed at these sites. To ensure effective future conservation, it is recommended that efforts focus on the identification and protection of manta ray hotspots and migratory corridors, the creation of a sustainable tourism framework and, most important, the implementation of mitigation strategies to reduce fisheries interactions.

Phylogenomics and species delimitation for effective conservation of manta and devil rays.

Practical biodiversity conservation relies on delineation of biologically meaningful units. Manta and devil rays (Mobulidae) are threatened worldwide, yet morphological similarities and a succession of recent taxonomic changes impede the development of an effective conservation strategy. Here, we generate genome-wide single nucleotide polymorphism (SNP) data from a geographically and taxonomically representative set of manta and devil ray samples to reconstruct phylogenetic relationships and evaluate species boundaries under the general lineage concept. We show that nominal species units supported by alternative data sources constitute independently evolving lineages, and find robust evidence for a putative new species of manta ray in the Gulf of Mexico. Additionally, we uncover substantial incomplete lineage sorting indicating that rapid speciation together with standing variation in ancestral populations has driven phylogenetic uncertainty within Mobulidae. Finally, we detect cryptic diversity in geographically distinct populations, demonstrating that management below the species level may be warranted in certain species. Overall, our study provides a framework for molecular genetic species delimitation that is relevant to wide-ranging taxa of conservation concern, and highlights the potential for genomic data to support effective management, conservation and law enforcement strategies.

Courtship and mating behaviour of manta rays Mobula alfredi and M. birostris in the Maldives.

The aim of this 14 year study was to elucidate the entire courtship and mating behaviour of manta rays Mobula alfredi and M. birostris using behavioural observations, video and photographic records. From 2003 to 2016, over 11,000 surveys were undertaken at known manta ray aggregation sites in the Maldives to record any observed manta rays reproductive activity. From 47,591 photo-ID sightings, 4,247 individual M. alfredi were identified and 226 individual M. birostris from 229 photo-ID sightings, all recorded at 22 atolls across 265 different sites. Courtship activity was observed on 206 surveys at 30 different sites. A total of 229 courtship events were recorded, with 90% (n = 205) of them occurring at cleaning sites. The observed courtship activity was categorised into seven distinct stages which are described in detail: initiation, endurance, evasion, pre-copulatory positioning, copulation, post-copulatory holding and separation. Photographs provide the first scientific record of the entirety of manta rays courtship and mating. Both M. alfredi and M. birostris appear to engage in the same elaborate courtship rituals, exhibiting the same behaviours during all stages of the courtship and mating process.

Environmental drivers of reef manta ray (Mobula alfredi) visitation patterns to key aggregation habitats in the Maldives.

A detailed understanding of the dynamics of small-scale (10s km) habitat use by the reef manta ray (Mobula alfredi) in the Maldives Archipelago is required to develop an effective national conservation management plan for this wide-ranging species. Here, a combination of photo-ID sightings data and acoustic telemetry were used to investigate both long-term M. alfredi visitation trends and small-scale movement patterns to key habitats on the eastern side of Baa Atoll (Hanifaru Bay feeding area, Dhigu Thila multifunctional site, and Nelivaru Thila cleaning station). All tagged and most of the sighted M. alfredi exhibited high affinity to the eastern side of Baa Atoll, where 99% of detections occurred, and 69% of individuals were re-sighted in multiple years. Sightings data suggests that visitation patterns may be associated with differences in habitat use by sex and maturity status. Boosted regression trees indicated that tag detection probability at Hanifaru Bay increased with increased westerly wind speed (>5ms-1) during the day, close to a new and full moon just after high tide, and when the tidal range was low. Interaction effects between predictors suggest that wind-driven oceanographic processes, such as Langmuir Circulation, maybe working to increase zooplankton concentration at this location. Tag detection probability increased at Dhigu Thila under similar conditions. At Nelivaru Thila, it increased at lower wind speeds (<5ms-1), close to a full moon, three hours after high tide. These results suggest that M. alfredi may utilise cleaning stations during the day when environmental conditions are not suitable for feeding. There was a high level of connectivity between these three locations, which suggests they form part of a network of key habitats that provide essential services to M. alfredi locally. Future conservation efforts should focus on identifying all areas of key habitat use for this species within the Maldives; applying strict protective measures to these sites and any connecting migration corridors which link them.

Fine-scale oceanographic drivers of reef manta ray (Mobula alfredi) visitation patterns at a feeding aggregation site.

Globally, reef manta rays (Mobula alfredi) are in decline and are particularly vulnerable to exploitation and disturbance at aggregation sites. Here, passive acoustic telemetry and a suite of advanced oceanographic technologies were used for the first time to investigate the fine-scale (5-min) influence of oceanographic drivers on the visitation patterns of 19 tagged M. alfredi to a feeding aggregation site at Egmont Atoll in the Chagos Archipelago. Boosted regression trees indicate that tag detection probability increased with the intrusion of cold-water bores propagating up the atoll slope through the narrow lagoon inlet during flood tide, potentially transporting zooplankton from the thermocline. Tag detection probability also increased with warmer near-surface temperature close to low tide, with near-surface currents flowing offshore, and with high levels of backscatter (a proxy of zooplankton biomass). These combinations of processes support the proposition that zooplankton carried from the thermocline into the lagoon during the flood may be pumped back out through the narrow inlet during an ebb tide. These conditions provide temporally limited feeding opportunities for M. alfredi, which are tied on the tides. Results also provide some evidence of the presence of Langmuir Circulation, which transports and concentrates zooplankton, and may partly explain why M. alfredi occasionally remained at the feeding location for longer than that two hours. Identification of these correlations provides unique insight into the dynamic synthesis of fine-scale oceanographic processes which are likely to influence the foraging ecology of M. alfredi at Egmont Atoll, and elsewhere throughout their range.

A hitchhiker guide to manta rays: Patterns of association between Mobula alfredi, M. birostris, their symbionts, and other fishes in the Maldives.

Despite being among the largest and most charismatic species in the marine environment, considerable gaps remain in our understanding of the behavioural ecology of manta rays (Mobula alfredi, M. birostris). Manta rays are often sighted in association with an array of smaller hitchhiker fish species, which utilise their hosts as a sanctuary for shelter, protection, and the sustenance they provide. Species interactions, rather than the species at the individual level, determine the ecological processes that drive community dynamics, support biodiversity and ecosystem health. Thus, understanding the associations within marine communities is critical to implementing effective conservation and management. However, the underlying patterns between manta rays, their symbionts, and other hitchhiker species remain elusive. Here, we explore the spatial and temporal variation in hitchhiker presence with M. alfredi and M. birostris throughout the Maldives and investigate the factors which may influence association using generalised linear mixed effects models (GLMM). For the first time, associations between M. alfredi and M. birostris with hitchhiker species other than those belonging to the family Echeneidae are described. A variation in the species of hitchhiker associated with M. alfredi and M. birostris was identified, with sharksucker remora (Echeneis naucrates) and giant remora (Remora remora) being the most common, respectively. Spatiotemporal variation in the presence of manta rays was identified as a driver for the occurrence of ephemeral hitchhiker associations. Near-term pregnant female M. alfredi, and M. alfredi at cleaning stations, had the highest likelihood of an association with adult E. naucrates. Juvenile E. naucrates were more likely to be associated with juvenile M. alfredi, and a seasonal trend in E. naucrates host association was identified. Remora were most likely to be present with female M. birostris, and a mean number of 1.5 ± 0.5 R. remora were observed per M. birostris. It is hoped these initial findings will serve as the basis for future work into the complex relationships between manta rays and their hitchhikers.

Reef manta rays forage on tidally driven, high density zooplankton patches in Hanifaru Bay, Maldives.

Manta rays forage for zooplankton in tropical and subtropical marine environments, which are generally nutrient-poor. Feeding often occurs at predictable locations where these large, mobile cartilaginous fishes congregate to exploit ephemeral productivity hotspots. Investigating the zooplankton dynamics that lead to such feeding aggregations remains a key question for understanding their movement ecology. The aim of this study is to investigate the feeding environment at the largest known aggregation for reef manta rays Mobula alfredi in the world. We sampled zooplankton throughout the tidal cycle, and recorded M. alfredi activity and behaviour, alongside environmental variables at Hanifaru Bay, Maldives. We constructed generalised linear models to investigate possible relationships between zooplankton dynamics, environmental parameters, and how they influenced M. alfredi abundance, behaviour, and foraging strategies. Zooplankton biomass changed rapidly throughout the tidal cycle, and M. alfredi feeding events were significantly related to high zooplankton biomass. Mobula alfredi switched from non-feeding to feeding behaviour at a prey density threshold of 53.7 mg dry mass m-3; more than double the calculated density estimates needed to theoretically meet their metabolic requirements. The highest numbers of M. alfredi observed in Hanifaru Bay corresponded to when they were engaged in feeding behaviour. The community composition of zooplankton was different when M. alfredi was feeding (dominated by copepods and crustaceans) compared to when present but not feeding (more gelatinous species present than in feeding samples). The dominant zooplankton species recorded was Undinula vulgaris. This is a large-bodied calanoid copepod species that blooms in oceanic waters, suggesting offshore influences at the site. Here, we have characterised aspects of the feeding environment for M. alfredi in Hanifaru Bay and identified some of the conditions that may result in large aggregations of this threatened planktivore, and this information can help inform management of this economically important marine protected area.

Stable isotope analyses reveal unique trophic role of reef manta rays (Mobula alfredi) at a remote coral reef.

Stable isotope analyses provide the means to examine the trophic role of animals in complex food webs. Here, we used stable isotope analyses to characterize the feeding ecology of reef manta rays (Mobula alfredi) at a remote coral reef in the Western Indian Ocean. Muscle samples of M. alfredi were collected from D'Arros Island and St. Joseph Atoll, Republic of Seychelles, in November 2016 and 2017. Prior to analysis, lipid and urea extraction procedures were tested on freeze-dried muscle tissue in order to standardize sample treatment protocols for M. alfredi. The lipid extraction procedure was effective at removing both lipids and urea from samples and should be used in future studies of the trophic ecology of this species. The isotopic signatures of nitrogen (δ15N) and carbon (δ13C) for M. alfredi differed by year, but did not vary by sex or life stage, suggesting that all individuals occupy the same trophic niche at this coral reef. Furthermore, the isotopic signatures for M. alfredi differed to those for co-occurring planktivorous fish species also sampled at D'Arros Island and St. Joseph Atoll, suggesting that the ecological niche of M. alfredi is unique. Pelagic zooplankton were the main contributor (45%) to the diet of M. alfredi, combined with emergent zooplankton (38%) and mesopelagic prey items (17%). Given the extent of movement that would be required to undertake this foraging strategy, individual M. alfredi are implicated as important vectors of nutrient supply around and to the coral reefs surrounding D'Arros Island and St. Joseph Atoll, particularly where substantial site fidelity is displayed by these large elasmobranchs.

Observations on early and late post-sporozoite tissue stages in primate malaria. I. Discovery of a new latent form of Plasmodium cynomolgi (the hypnozoite), and failure to detect hepatic forms within the first 24 hours after infection.

Previous work in this laboratory has demonstrated the ability of the immunofluorescence technique to detect pre-erythrocytic stages of the primate malaria parasite, Plasmodium cynomolgi bastianellii, in hepatic tissue obtained as early as 48 hours after sporozoite inoculation. In an attempt to visualize still earlier post-sporozoite stages, hepatic tissue obtained from a rhesus monkey infected with 12,000,000 sporozoites was examined at 2, 12, 24, and 48 hours after inoculation, employing antisera reactive with both invertebrate and vertebrate stages of the parasite. Tissue was also obtained at 7, 50, 102, and 105 days after sporozoite inoculation, and was examined for adequacy of the hepatic infection and for the presence of late exoerythrocytic schizonts. Although a new, previously unrecognized, uninucleate latent stage of 5 micrometer diameter (the "hypnozoite") was detected among large maturing schizonts in the 7-day and later biopsies, no intrahepatic parasites were found in tissue taken at 24 hours or earlier, despite the presence of up to 61 7-day schizonts and eight hypnozoites per 5 X 8 mm section. Pre-erythrocytic forms again were detected at 48 hours, although in far smaller numbers than expected on the basis of the density of parasites at 7 days after infection. The significance of these observations is discussed in the context of previous negative findings.

Observations on early and late post-sporozoite tissue stages in primate malaria. II. The hypnozoite of Plasmodium cynomolgi bastianellii from 3 to 105 days after infection, and detection of 36- to 40-hour pre-erythrocytic forms.

Confirmation of the existence of a persistent, uninucleate, dormant pre-erythrocytic stage, the hypnozoite, of the relapsing simian malaria parasite, Plasmodium cynomolgi bastianellii, has been obtained by means of experiments involving the intravenous injection into susceptible monkeys of 48 to 85 x 10(6) sporozoites derived from mosquitoes of a different species and source than employed previously. The development of these hypnozoites was traced from 3 days until 105 days after sporozoite inoculation, employing a sensitive immunofluorescence technique followed by restaining with Giemsa. From an average mean diameter of 4 micrometers at 3 and 5 days, uninucleate hypnozoites grow to 5 micrometers at 7 days, then persist with little change until at least 105 days after infection. Strong evidence for the viability of these persistent forms was obtained by treatment of a host monkey with primaquine, which eliminated all trace of hypnozoites present 2 weeks before. Examination of hepatic tissue from a monkey injected with sporozoites 36 and 40 hours earlier revealed rare uninucleate pre-erythrocytic forms of 2.5-micrometers diameter. These early forms were present in hepatocytes in a density only approximately 1/30th of that expected on the basis of numbers of pre-erythrocytic stages found in the same animal's liver 7 days after infection. Nevertheless, subinoculation experiments appeared to rule out the circulation as a vehicle for dissemination of any putative early intermediate hepatotropic forms from another site.

Excreta disposal facilities and intestinal parasitism in urban Africa: preliminary studies in Botswana, Ghana and Zambia.

The relationships between intestinal parasitism and excreta disposal technologies in Gaborone (Botswana), Ndola (Zambia) and Kumasi (Ghana) were investigated. Parasitic prevalence and intensity rates amongst groups of urban residents having similar socio-economic status and housing, but different excreta disposal technologies, were compared. In Gaborone, there was no evidence of a difference in intestinal parasitism between those using aqua privies and having access to public taps and those in identical houses enjoying flush toilets, in-house water connections and showers. In Ndola, the group with sewered aqua privies had larger houses, cleaner toilets, better water supplies, longer residence and more people in paid employment than the groups using pit latrines or communal flush toilets. Despite this, the sewered aqua privy users were not found to be different from the other groups with regard to hookworm and protozoal infection but had significantly higher Ascaris infection rates. In Kumasi, despite the differences in toilet type--from squalid communal aqua privies, through often fouled bucket latrines to well-maintained flush toilet systems--and despite also the differences in water provision, no evidence was obtained of any differences in intestinal parasitism between the groups studied. These findings suggest that the provision of superior water and sanitation facilities to a small cluster of houses, or to houses scattered through an area, may not protect those families from infection if the over-all level of faecal contamination of the environment is high. The sample sizes and response rates achieved in this study were low and follow-up studies, employing the same methodology but with larger samples, are recommended.

Observations on early and late post-sporozoite tissue stages in primate malaria. III. Further attempts to find early forms and to correlate hypnozoites with growing exo-erythrocytic schizonts and parasitaemic relapses in Plasmodium cynomolgi bastianellii infections.

Rhesus monkeys were heavily infected with sporozoites of Plasmodium cynomolgi bastianellii in an attempt to demonstrate the site of invasion of sporozoites into tissue cells and their growth there. Further attempts were made to correlate the appearance and loss of hypnozoites with parasitaemic relapses. Hypnozoites were demonstrated and once again shown to decrease in numbers over 229 days during which time the infection showed parasitaemic relapses. Liver biopsies taken at two-day intervals for 12 days showed that hypnozoites decreased in numbers over-all and growing schizonts were demonstrated in the liver. At this time a parasite the size of a hypnozoite was seen with two nuclei and another was seen with an elongate, possibly dividing nucleus in one monkey. an attempt to find the location of the early intracellular exoerythrocytic forms in the liver at various times less than 40 hours after infection using smears and immunological staining with newly prepared anti-sera failed. Large numbers of sporozoites of P. knowlesi were also injected into a rhesus monkey the liver of which on the fifth day after infection showed no hypnozoites among 157 sections of growing schizonts and no parasites at all on the 42nd day after infection. In P. cynomolgi bastianellii infections parasites, mostly hypnozoites, were found in the liver up to 229 days after infection.

A study of the pharmacokinetic profile of low-dose hepatitis B immune globulin in long-term liver transplant recipients for chronic hepatitis B infection.

Post-transplant protocols for hepatitis B (HBV) prophylaxis using high-dose intravenous hepatitis B immune globulin (10,000 IU) with or without lamivudine are commonly reported. Our centre has previously reported a low-dose intramuscular (i.m.) protocol and lamivudine with excellent results. There have been, however, no pharmacokinetic studies of i.m. hepatitis B immune globulin (HBIG) in this setting. The objective of this study was to determine the pharmacokinetic profile of i.m. HBIG in long-term post-liver-transplant recipients to determine a rational dosing protocol. Six stable liver transplant recipients receiving monthly i.m. HBIG injections for greater than one year were enrolled in this study. All patients had no detectable HBV DNA levels. HBIG titers (anti-HBs) were measured predose, then three times weekly for four weeks and then twice weekly until the serum HBIG titers were 100 IU/L or less. The pharmacokinetic parameters were calculated using noncompartment methods. The mean time to maximum concentration was 10.5 d (range 4-20 d) and the mean half-life was 20 d (range 13.5-23.5 d). Based on these pharmacokinetic parameters in stable long-term post-transplant patients, a rational dosing protocol was developed that allows for more appropriate utility of HBIG and improved patient convenience.