Digenetic trematode community in nesting green sea turtles (Chelonia mydas) from Tortuguero National Park, Costa Rica

The digenetic community of 40 green sea turtles (Chelonia mydas) was investigated at Tortuguero National Park, Costa Rica. In total, 24,270 trematodes belonging to 29 species and 6 families including Clinostomidae 1, Microscaphidiidae 5, Paramphistomidae 2, Pronocephalidae 15, Rhytidodidae 2, and Spirorchiidae 4 were recorded from chelonians examined. Turtles harbored a mean of 7.63 +/- 3.5 SD species. Only 3 species, i.e., Learedius learedi, Microscaphidium reticulare, and Pyelosomum cochlear, infected more than 50% of the hosts sampled. Learedius learedi was the most prevalent (97.5%) and the second most abundant species with a mean of 25.6 +/- 21.6 SD. Only 1 core species, M. reticulare, was recovered from the gastrointestinal tract; it was the most abundant parasite, with a mean intensity of 477 +/- 1,180 SD, and the second most prevalent (77.5%). Diversity values ranged from 0.10 to 2.10, with a mean of 1.00 +/- 0.43 SD for the total component community and from 0.10 to 1.84, with a mean of 0.79 +/- 0.41 SD, for the gastrointestinal component community. Species richness was the highest recorded from a sea turtle species. All digenetic species were recorded from Costa Rica for the first time. This represents the first report on the helminth community of the green turtle.     

Plasmid-mediated high-level gentamicin resistance among enteric bacteria isolated from pet turtles in Louisiana

The sale of small turtles is banned by the Food and Drug Administration from the U.S. market due to concerns about their excretion of Salmonella spp. To produce a safe pet for the export market, the Louisiana pet turtle industry uses gentamicin sulfate baths (1,000 microg/ml) to eradicate Salmonella spp. from turtle eggs. In 1999, we analyzed bacterial samples recovered from turtle farms and found that strains of Salmonella enterica subsp. arizonae and other bacteria, such as Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia, were resistant to high concentrations of gentamicin (>2,000 microg/ml) and to other aminoglycosides. The goal of this study was to identify the gene(s) which contributes to the high-level gentamicin resistance phenotype observed in bacteria from environmental samples with turtle farming activity, particularly the salmonellae, and to estimate the incidence of such genes in these bacteria. R plasmids from gentamicin-resistant strains were transferred by conjugation and transformation to naive Escherichia coli cells. Cloning and sequencing of the gentamicin resistance determinants on these plasmids revealed the presence of the aminoglycoside acetyltransferase genes aac(3)-IIa and aac(3)-VIa; the latter was present as a gene cassette of a class 1 integron. Multiplex PCR assays showed that every gentamicin-resistant isolate carried one of these acetyltransferase genes. Pulsed-field gel electrophoresis and restriction enzyme digestion analysis of R plasmids carrying these genes revealed different restriction profiles and sizes, indicating a dissemination of the gentamicin resistance genes through mobile molecular elements. The data presented highlight the need to develop an alternate method for the eradication of Salmonella spp. from turtle eggs.     

The dazed and confused identity of Agassiz's land tortoise, Gopherus agassizii (Testudines, Testudinidae) with the description of a new species, and its consequences for conservation

We investigate a cornucopia of problems associated with the identity of the desert tortoise, Gopherus agassizii (Cooper). The date of publication is found to be 1861, rather than 1863. Only one of the three original cotypes exists, and it is designated as the lectotype of the species. Another cotype is found to have been destroyed in the 1906 San Francisco earthquake and subsequent fire. The third is lost. The lectotype is genetically confirmed to be from California, and not Arizona, USA as sometimes reported. Maternally, the holotype of Gopherus lepidocephalus (Ottley & Velázques Solis. 1989) from the Cape Region of Baja California Sur, Mexico is also from the Mojavian population of the desert tortoise, and not from Tiburon Island, Sonora, Mexico as previously proposed. A suite of characters serve to diagnose tortoises west and north of the Colorado River, the Mojavian population, from those east and south of the river in Arizona, USA, and Sonora and Sinaloa, Mexico, the Sonoran population. Species recognition is warranted and because Gopherus lepidocephalus is from the Mojavian population, no names are available for the Sonoran species. Thus, a new species, Gopherus morafkaisp. n., is named and this action reduces the distribution of Gopherus agassizii to only 30% of its former range. This reduction has important implications for the conservation and protection of Gopherus agassizii, which may deserve a higher level of protection.     

Elucidation of the first definitively identified life cycle for a marine turtle blood fluke (Trematoda: Spirorchiidae) enables informed control

Blood flukes of the family Spirorchiidae are significant pathogens of both free-ranging and captive marine turtles. Despite a significant proportion of marine turtle mortality being attributable to spirorchiid infections, details of their life cycles remain almost entirely unknown. Here we report on the molecular elucidation of the complete life cycle of a marine spirorchiid, identified as Amphiorchis sp., infecting vermetid gastropods and captive hatched neonate Caretta caretta in the Oceanogràfic Aquarium, in Valencia, Spain. Specimens of a vermetid gastropod, Thylaeodus cf. rugulosus (Monterosato, 1878), collected from the aquarium filtration system housing diseased C. caretta, were infected with sporocysts and cercariae consistent with the family Spirorchiidae. We generated rDNA sequence data [internal transcribed spacer 2 (ITS2) and partial 28S rDNA] from infections from the vermetid which were identical to sequences generated from eggs from the serosa of the intestine of neonate C. caretta, and an adult spirorchiid from the liver of a C. caretta from Florida, USA. Given the reliability of these markers in the delineation of trematode species, we consider all three stages to represent the same species and tentatively identify it as a species of Amphiorchis Price, 1934. The source of infection at the Oceanogràfic Foundation Rehabilitation Centre, Valencia, Spain, is inferred to be an adult C. caretta from the western Mediterranean being rehabilitated in the same facility. Phylogenetic analysis suggests that this Amphiorchis sp. is closely related to other spirorchiids of marine turtles (species of Carettacola Manter & Larson, 1950, Hapalotrema Looss, 1899 and Learedius Price, 1934). We discuss implications of the present findings for the control of spirorchiidiasis in captivity, for the better understanding of epidemiology in wild individuals, and the elucidation of further life cycles.     

Metabolic heat estimation of the sea turtle Lepidochelys olivacea embryos

Several studies have reported the importance of metabolic heat on the increment of temperature in the sea turtle nests; however, the metabolic heat has not been calculated for sea turtle eggs. In this study, the metabolic heat generated by embryos of the sea turtle Lepidochelys olivacea was estimated from a thermal balance model by means of three measured temperatures—one in the center of the nest, and the others in the sand above and beside the nest. An experiment was conducted with a sample of 100 eggs from a Lepidochelys olivacea nest collected in the Baja Peninsula, Mexico. The results showed that during the incubation period, no metabolic heat was detected before day 19 but it increased from that day until a maximum of 0.84 W at day 34, when the incubation process was interrupted due to rain. This value corresponds to 31 emerged hatchlings. The novel model is a suitable framework to predict the temperature and metabolic heat within the nest.     

Vibrio mimicus diarrhea following ingestion of raw turtle eggs.

Clinical and epidemiological characteristics of diarrhea associated with Vibrio mimicus were identified in 33 hospitalized patients referred to the Costa Rican National Diagnostic Laboratory Network between 1991 and 1994. The relevant symptoms presented by patients included abundant watery diarrhea, vomiting, and severe dehydration that required intravenous Dhaka solution in 83% of patients but not fever. Seroconversion against V. mimicus was demonstrated in four patients, from whom acute- and convalescent-phase sera were obtained. Those sera did not show cross-reaction when tested against Vibrio cholerae O1 strain VC-12. All the V. mimicus isolates from these cases produced cholera toxin (CT) and were susceptible to commonly used antibiotics. Attempts to isolate this bacterium from stool samples of 127 healthy persons were not successful. Consumption of raw turtle eggs was recalled by 11 of the 19 (58%) individuals interviewed. All but two V. mimicus diarrheal cases were sporadic. These two had a history of a common source of turtle (Lepidochelys olivacea) eggs for consumption, and V. mimicus was isolated from eggs from the same source (a local market). Among the strains, variations in the antimicrobial susceptibility pattern were observed. None of the strains recovered from market turtle eggs nor the four isolates from river water showed CT production. Further efforts to demonstrate the presence of CT-producing V. mimicus strains in turtle eggs were made. Successful results were obtained when nest eggs were tested. In this case, it was possible to isolate CT- and non-CT-producing strains, even from the same egg. For CT detection we used PCR, enzyme-linked immunosorbent assay (ELISA), and Y-1 cell assay, obtaining a 100% correlation between ELISA and PCR results. Primers Col-1 and Col-2, originally described as specific for the V. cholerae O1 ctxA gene, also amplified a 302-bp segment with an identical restriction map from V. mimicus. These results have important implications for epidemiological surveillance in tropical countries where turtle eggs are used for human consumption, serving as potential sources of cholera-like diarrhea.     

Distribution and Feeding Behavior of Omorgus suberosus (Coleoptera: Trogidae) in Lepidochelys olivacea Turtle Nests

Omorgus suberosus (Fabricius, 1775) has been identified as a potential predator of the eggs of the turtle Lepidochelys olivacea (Eschscholtz, 1829) on one of the main turtle nesting beaches in the world, La Escobilla in Oaxaca, Mexico. This study presents an analysis of the spatio–temporal distribution of the beetle on this beach (in areas of high and low density of L. olivacea nests over two arrival seasons) and an evaluation, under laboratory conditions, of the probability of damage to the turtle eggs by this beetle. O. suberosus adults and larvae exhibited an aggregated pattern at both turtle nest densities; however, aggregation was greater in areas of low nest density, where we found the highest proportion of damaged eggs. Also, there were fluctuations in the temporal distribution of the adult beetles following the arrival of the turtles on the beach. Under laboratory conditions, the beetles quickly damaged both dead eggs and a mixture of live and dead eggs, but were found to consume live eggs more slowly. This suggests that O. suberosus may be recycling organic material; however, its consumption of live eggs may be sufficient in some cases to interrupt the incubation period of the turtle. We intend to apply these results when making decisions regarding the L. olivacea nests on La Escobilla Beach, one of the most important sites for the conservation of this species.     

Sperm-activating peptide type-V (SAP-V), a fifth member of the sperm-activating peptide family, purified from the egg-conditioned media of the heart urchin Brissus agassizii.

1. A novel type of sperm-activating peptide named sperm-activating peptide type-V (SAP-V) was isolated from the egg-conditioned media (egg jelly) of the heart urchin Brissus agassizii and the primary structure of the peptide was determined by fast atom bombardment mass spectrometry as follows: Gly-Cys-Glu-Gly-Leu-Phe-His-Gly-Met-Gly-Asn-Cys. 2. SAP-V and [Met(O)9]SAP-V stimulated the respiration of B. agassizii spermatozoa with half-maximal concentrations of 0.5 and 0.3 nM, respectively. However, half-maximal stimulation of the sperm respiration required 40 nM of S-carboxymethylated SAP-V. 3. SAP-V induced significant increases in the cyclic AMP and cyclic GMP levels in B. agassizii spermatozoa in a concentration-dependent manner. 4. The addition of SAP-V to B. agassizii spermatozoa resulted in a mobility shift of a major sperm protein (mol. wt from 133,000 to 129,000) on sodium dodecyl sulfate-polyacrylamide gels.     

Fluke (Spirorchiidae) infections in sea turtles stranded on Taiwan: prevalence and pathology

The prevalence of spirorchiid fluke infections of marine turtles is high and may cause the death of the hosts throughout their ranges. Virtually nothing has been reported regarding the infective status of sea turtles stranded on Taiwan. Between 2007 and 2010, 30 green turtles (Chelonia mydas) and 2 loggerhead turtles ( Caretta caretta ), stranded and dead, were examined for spirorchiid flukes and their eggs. Twenty-four of the green turtles were juveniles, and the stranded loggerhead turtles were subadults. Adult spirorchiid flukes were found in 13 green turtles but not in the loggerheads. Four species of flukes were identified, namely, Leardius learedi , Hapalotrema postorchis , H. mehrai , and Carettacola hawaiiensis . The main infection sites were the major arteries and heart. Seventy percent of the green turtles harbored spirorchiid eggs, but no eggs were found in loggerheads. The largest eggs with bipolar spines, type I eggs, were found in every case. Although more than half of the stranded turtles were infected, parasite infections were not the main cause of death in the green turtles. Fishery by-catch is probably responsible for the mortality of these stranded turtles.     

Embryonic communication in the nest: metabolic responses of reptilian embryos to developmental rates of siblings

Incubation temperature affects developmental rates and defines many phenotypes and fitness characteristics of reptilian embryos. In turtles, eggs are deposited in layers within the nest, such that thermal gradients create independent developmental conditions for each egg. Despite differences in developmental rate, several studies have revealed unexpected synchronicity in hatching, however, the mechanisms through which synchrony are achieved may be different between species. Here, we examine the phenomenon of synchronous hatching in turtles by assessing proximate mechanisms in an Australian freshwater turtle (Emydura macquarii). We tested whether embryos hatch prematurely or developmentally compensate in response to more advanced embryos in a clutch. We established developmental asynchrony within a clutch of turtle eggs and assessed both metabolic and heart rates throughout incubation in constant and fluctuating temperatures. Turtles appeared to hatch at similar developmental stages, with less-developed embryos in experimental groups responding to the presence of more developed eggs in a clutch by increasing both metabolic and heart rates. Early hatching did not appear to reduce neuromuscular ability at hatching. These results support developmental adjustment mechanisms of the 'catch-up hypothesis' for synchronous hatching in E. macquarii and implies some level of embryo-embryo communication. The group environment of a nest strongly supports the development of adaptive communication mechanisms between siblings and the evolution of environmentally cued hatching.     

Western blot can distinguish natural and acquired antibodies to Mycoplasma agassizii in the desert tortoise (Gopherus agassizii)

Mycoplasma agassizi has been identified as a cause of upper respiratory tract disease (URTD) in the threatened Mojave population of the desert tortoise (Gopherus agassizii), and anti-M. agassizii antibodies have been found by ELISA in as many as 15% of these animals across their geographic range. Here we report that a cohort of 16 egg-reared desert tortoises never exposed to M. agassizii had ELISA antibody titers to this organism that overlapped with titers obtained from some M. agassizii-infected tortoises. These natural antibodies were predominantly of the IgM class. Western blots of plasma from these non-infected tortoises produced a characteristic banding pattern against M. agassizii antigens. A group of 38 wild-caught desert tortoises was tested by ELISA, and although some of these tortoises had antibody titers significantly higher than the non-infected tortoises, there was considerable overlap at the lower titer levels. However, Western blot analysis revealed distinct banding patterns that could readily distinguish between the non-infected tortoises and tortoises with acquired antibodies, regardless of ELISA antibody titers. We conclude that desert tortoises have natural antibodies to M. agassizii that can compromise the determination of infection status by ELISA. However, the Western blot technique can distinguish between natural and acquired antibody patterns and can be used to confirm the diagnosis of M. agassizii infections in the desert tortoise.     

A gravid fossil turtle from the Early Cretaceous reveals a different egg development strategy to that of extant marine turtles

Extant sea turtles develop and lay pliable (flexible) eggs; however, it is unknown whether they inherited this reproductive strategy from their closer fossil relatives or if it represents an evolutionary novelty. Here, we describe the first undisputable gravid marine fossil turtle ever found, from the early Cretaceous of Colombia, belonging to Desmatochelys padillai Cadena & Parham, which constitutes a representative of the Protostegidae. Using thin sectioning of one of the eggs, as well as scanning electron microscopy coupled with elemental characterization, cathodoluminescence, and computer tomography, we established that Desmatochelys padillai produced rigid eggs similar to those associated with some extant and fossil freshwater and terrestrial turtles. At least 48 spherical eggs were preserved inside this gravid turtle. We suggest that the development of rigid eggs in the extinct marine turtle Desmatochelys padillai resulted as an adaptation for egg‐embryo requirements dictated by the physical attributes of the nesting site.     

Narcinecotyle longifilamentus n. gen., n. sp. (Monogenea: Hexabothriidae), gill parasite of the numbfish Narcine entemedor (Torpediniformes: Narcinidae) from the Mexican Pacific coast

A new genus and species of Hexabothriidae Price, 1942 is described from specimens infecting the gill of the numbfish Narcine entemedor Jordan and Starks from the Pacific coast of Mexico. In addition to the new taxon described here, species of 8 genera also display symmetrical haptors and have eggs with two polar filaments. However, they differ in the combination of the following features: distal portion of the male copulatory organ tubular, unarmed and proximally dilated confining an internal coiled duct, as well as dorsal origin of haptoral appendix. Based on molecular data derived from 3 loci, the mitochondrial Cytochrome C oxidase subunit 1 as well as the nuclear ribosomal 18S and 28S, the new species was found nested within Hexabothriidae together with the other 4 genera with representatives in GenBank. This is the first species of Hexabothriidae reported from a species of Narcinidae.     

Vermetid gastropods as key intermediate hosts for a lineage of marine turtle blood flukes (Digenea: Spirorchiidae), with evidence of transmission at a turtle rookery

Blood flukes of the family Spirorchiidae Stunkard, 1921 are significant pathogens of marine turtles, both in the wild and in captivity. Despite causing considerable disease and mortality, little is known about the life cycles of marine species, with just four reports globally. No complete life cycle has been elucidated for any named species of marine spirorchiid, but the group is reported to use vermetid and fissurellid gastropods, and terebelliform polychaetes as intermediate hosts. Here we report molecular evidence that nine related spirorchiid species infect vermetid gastropods as first intermediate hosts from four localities along the coast of Queensland, Australia. ITS2 rDNA and cox1 mtDNA sequence data generated from vermetid infections provides the first definitive identifications for the intermediate hosts for the four species of Hapalotrema Looss, 1899 and Learedius learedi Price, 1934. Additionally, we provide a new locality report for larval stages of Amphiorchis sp., and evidence of three additional unidentified spirorchiid species in Australian waters. Based on the wealth of infections from vermetids during this study, we conclude that the previous preliminary report of a fissurellid limpet as the intermediate host for L. learedi was likely mistaken. The nine species found infecting vermetids during this study form a strongly supported clade exclusive of species of the other two marine spirorchiid genera for which sequence data are available; Carettacola Manter & Larson, 1950 which falls sister to the vermetid-infecting clade + a small clade of freshwater spirorchiids, and Neospirorchis Price, 1934 which is distantly related to the vermetid-infecting clade. We provide further evidence that spirorchiid transmission can occur in closed system aquaria and show that spirorchiid transmission occurs at both an important turtle rookery (Heron Island, southern Great Barrier Reef, Australia) and foraging ground (Moreton Bay, Australia). We discuss the implications of our findings for the epidemiology of the disease, control in captivity, and the evolution of vermetid exploitation by the Spirorchiidae.     

To Eat or not to Eat an Endangered Species: Views of Local Residents and Physicians on the Safety of Sea Turtle Consumption in Northwestern Mexico

Sea turtles have historically been an important food resource for many coastal inhabitants of Mexico. Today, the consumption of sea turtle meat and eggs continues in northwestern Mexico despite well-documented legal protection and market conditions providing easier access to other more reliable protein sources. Although there is growing evidence that consuming sea turtles may be harmful to human health due to biotoxins, environmental contaminants, viruses, parasites, and bacteria, many at-risk individuals, trusted information sources, and risk communicators may be unaware of this information. Therefore, we interviewed 134 residents and 37 physicians in a region with high rates of sea turtle consumption to: (1) examine their knowledge and perceptions concerning these risks, as a function of sex, age, occupation, education and location; (2) document the occurrence of illness resulting from consumption; and (3) identify information needs for effective risk communication. We found that 32% of physicians reported having treated patients who were sickened from sea turtle consumption. Although physicians believed sea turtles were an unhealthy food source, they were largely unaware of specific health hazards found in regional sea turtles, regardless of location. By contrast, residents believed that sea turtles were a healthy food source, regardless of sex, age, occupation, and education, and they were largely unaware of specific health hazards found in regional sea turtles, regardless of age, occupation, and education. Although most residents indicated that they would cease consumption if their physician told them it was unhealthy, women were significantly more likely to do so than men. These results suggest that residents may lack the necessary knowledge to make informed dietary decisions and physicians do not have enough accurate information to effectively communicate risks with their patients.     

In Vitro Cultivation of Cymatocarpus solearis (Brachycoeliidae) Metacercariae to Obtain the Adult Stage without the Marine Turtle Definitive Host

In vitro cultivation of trematodes would assist studies on the basic biology of the parasites and their hosts. This is the first study to use the yolk of unfertilized chicken eggs as a simple and successful method of ovocultivation and the first time to obtain the adult-stage of the trematode Cymatocarpus solearis Braun, 1899 (Digenea: Brachycoeliidae). Chicken eggs were inoculated with metacercariae from the muscle of the spiny lobster, Panulirus argus (Latreille, 1804). The metacercariae were excysted and incubated for 576 hr (24 days) at 38℃ to obtain the adult stage. Eggs in utero were normal in shape and light brown color. The metacercariae developed into mature parasites that have been identified as the adult-stage found in marine turtles. The adult lobsters collected in Quintana Roo State, Mexico, showed the prevalence of 49.4% and the mean intensity of 26.0 per host (n = 87). A statistical study was performed to determine that no parasitic preference was detected for male versus female parasitized lobsters. Morphometric measurements of the adult-stage of C. solearis obtained in our study have been deposited in the National Helminths Collection of the Institute of Biology of the National Autonomous University of Mexico. This study is significant because it is the first time that a digenean of the family Brachycoeliidae has been demonstrated to develop in vitro from metacercariae into adults capable of producing eggs using the yolk of unfertilized chicken eggs. Secondly, this technique allows to obtain the adult stage of C. solearis without the presence of its marine turtle host, allows us to describe the mature parasites, and thus contribute to our understanding of the biology of C. solearis.     

Cloning and expression of the turtle (<Emphasis Type="Italic">Trachemys scripta</Emphasis>) immunoglobulin joining (J)-chain cDNA

The J-chain protein is a M(r) 15000 polypeptide associated with polymeric IgA and IgM. The complete cDNA sequences of human, mouse, cow, brushtail possum, chicken and frog J chains have been previously reported, but nothing is known about the cDNA and amino acid sequences of reptilian J chain. Here, we determined a turtle J-chain cDNA sequence by RT-PCR and RACE, and examined J-chain mRNA and protein expression by Northern blotting and immunohistochemistry. This turtle J-chain cDNA was 1934 bp and had an open reading frame of 477 nucleotides, encoding 159 amino acids. The mature J-chain protein is composed of 137 amino acids, M(r) approximately 15000. The deduced amino acid sequence of the turtle J chain was highly homologous to that of human (60%), mouse (61%), cow (60%), rabbit (60%), chicken (69%), brushtail possum (65%), Rana catesbeiana (47%) and Xenopus laevis (58%). Eight cysteine residues were located at the same positions as in these other species, with the exception of X. laevis. PROSITE database analysis indicated the presence of two N-glycosylation sites in turtle, one of which was novel. Northern blot analysis revealed that turtle J-chain mRNA was expressed in lung, stomach, spleen and intestine. In addition, immunohistochemistry showed J-chain-positive plasma cells in the intestine and spleen. These results suggest the presence of a mucosal immune system mainly composed of J-chain-containing Ig in the turtle.     

Conversion of cholesterol to progesterone by turtle corpus luteum

Homogenates of corpora lutea from the snapping turtle, $\text{Chelydra}$$\text{serpentina serpentina}$ were capable of converting small (< 1%) amounts of cholesterol-³H to progesterone-³H. There was about twice as much steroidogenic activity in corpora lutea taken from animals still carrying oviducal eggs as in those from animals that had laid their eggs. However, the latter showed up to an 80% increase in conversion of cholesterol to progesterone when turtle pituitary homogenate was incubated along with the luteal homogenate. Approximately 4 and 9 mg/g of free and esterified sterol, respectively, were found in the turtle corpus luteum. These results suggest that the corpus luteum of the oviparous turtle functions as a steroidogenic organ.     

An ontogenetic shift in the response of heart rates to temperature in the developing snapping turtle (Chelydra serpentina)

The affect of acute changes in temperature on heart rates was investigated for the first time in a developing reptile. Heart rates were determined early and late in incubation in snapping turtle (Chelydra serpentina) eggs. Late in incubation heart rates at any given temperature were lower than those observed early in incubation. The results of temperature switching experiments late in incubation were consistent with thermal acclimation.