Unsafe management of a zoological garden as a cause of introduction of an alien species into the wild: First documented case of feral naturalized population of Lama glama in Europe

Zoological gardens host large numbers of wild species and domestic forms, mostly not indigenous to the place where the zoological garden is located. Escapes (or releases) from zoological gardens are recognized as a pathway of introduction of alien species, leading in some cases to a naturalized population. Although previously reported worldwide, scientific accounts of escapes from zoos are rare and often anecdotal. In this note, we document, for the first time in Europe and, to our knowledge, in an area outside its native range, a case of introduction and establishment of a feral population of Llama (Lama glama), originating from a group of animals living in captivity at the Zoological Park of Cavriglia (Tuscany, central Italy). We reconstruct in detail the story of the population since 1974, also reporting preliminary data on abundance, distribution and diet. Finally, we critically evaluate the causes of this introduction, highlighting how unsafe the management of a zoological garden can be, and we discuss the implications for conservation and management perspectives.     

The first report of peritoneal tetrathyridiosis in squirrel monkey (Saimiri sciureus)

This report describes a case of peritoneal larval cestodiasis caused by tetrathyridia of Mesocestoides sp. in an adult female squirrel monkey. The monkey had lived in a zoological garden in Japan and had a clinical history of wasting. At necropsy, numerous whitish oval masses were found in the liver and peritoneal cavity. These masses contained larval cestodes. Morphological observation and molecular analyses of the mitochondrial 12S ribosomal RNA gene and cytochrome c oxidase subunit 1 gene sequences allowed us to identify the larva as the tetrathyridium of Mesocestoides sp. This is the first report of Mesocestoides larvae in a squirrel monkey in Japan.     

Molecular characterization of Trichuris spp. from captive animals based on mitochondrial markers

Monoxenous parasites may easily infect animals in captivity, and nematodes belonging to the genus Trichuris are commonly reported in zoological gardens worldwide. Infections in captive animals should be accurately monitored and the characterization of pathogens is highly advisable, as a tool to infer possible routes of intra- and interspecific transmission pathways and to assess the related zoonotic potential. Whipworms are usually identified on the basis of few morphological features of adults males and eggs and by an host-affiliation criterion. Given the strong morphological convergence of adaptive traits and the possible occurrence of hybridization and/or cross-infections events, the use of molecular methods is of great utility.Here, we analysed two partial mitochondrial loci, the cytochrome c oxidase I and the cytochrome b regions, in Trichuris spp. infecting four animal species hosted in the Bioparco Zoological Garden of Rome. Results from molecular systematics, compared to previous data, suggested that the five Trichuris taxa recovered were well separated, showing a significant degree of host affiliation (herbivorous, primates/swine and rodents/canids). The screw horn antelopes and the camels were infected with two variants of Trichuris ovis; Trichuris sp. similar to those observed in rodents from South America was infecting the Patagonian maras. Moreover, Trichuris from the ring-tailed lemur showed a great similarity to Trichuris infecting the Japanese macaque previously analysed from the same zoological garden, and clustering together with Trichuris trichiura, posing a potential zoonotic threat for visitors and workers.     

Scanning electron microscopy on Parascaris equorum (Goeze, 1782), Yorke and Maplestone, 1926

Research on Ascaridinae from African zebra and zebra living in Vincennes zoological garden (Paris) and those from horses confirmed that they all belong to the same parasite, Parascaris equorum (Goeze, 1782), Yorke &; Maplestone, 1926. Study by scanning electron microscopy permitted greater precision in investigation of various formations: denticules along the peribuccal groove, sinuses separating eulabium from prelabium, intrabuccal sockets.     

Helminths: comparison of their rhodoquinone

A rhodoquinone (RQ) has been isolated from Stephanurus dentatus and has been shown to be identical with RQ-9 from Ascaris lumbricoides var. suis (A. suum). The ratio of RQ/mg N is fairly constant among larval, juvenile, and adult forms of S. dentatus with an average value of 1.63 μg/mg N. A Chromatographie survey found RQ in all eleven species of helminths tested. Ubiquinone (UQ) was found in all larval forms, a free living nematode (Rhabditis strongyloides) and helminths with prepatent periods shorter than 35 days.     

Influence of the requirement for abdominal pain in the diagnosis of irritable bowel syndrome with constipation (IBS-C) under the Rome IV criteria using data from a large Japanese population-based internet survey

Background

Rome III was revised to Rome IV in May 2016. One important change in the Rome IV criteria is that abdominal pain must be present for a diagnosis of irritable bowel syndrome (IBS). Under Rome III, in contrast, patients with abdominal discomfort only could be diagnosed with IBS, but these cases under Rome IV are now classified as unspecified functional bowel disorder (FBD). In a simple comparison of Rome III and Rome IV, it is unclear whether this difference reflects the influence of symptomatic frequency or the presence of abdominal pain. In particular, the influence of abdominal pain restriction on the diagnosis of IBS with predominant constipation (IBS-C) in the Rome IV criteria is largely unknown.

Methods

We reclassified subjects from a Japanese internet survey experiencing abdominal pain or discomfort at least one day each week as surrogate Rome III IBS-C subjects. Among them, we then reclassified subjects experiencing abdominal pain as surrogate Rome IV IBS-C subjects and subjects not experiencing abdominal pain as surrogate Rome IV FBD subjects. Symptoms were quantified and compared between the two groups.

Results

The surrogate Rome IV IBS-C subjects felt a significantly higher degree of anxiety in their daily lives (p?<?0.001) compared with the surrogate Rome IV FBD subjects. The combined female and 20–49?years surrogate Rome IV IBS-C subjects felt a higher degree of anxiety in their daily lives (p?<?0.05) than the respective Rome IV FBD subjects.

Conclusions

These results suggest that female IBS-C patients aged 20–49?years with abdominal pain in Rome IV have more anxiety than those without abdominal pain in Rome III. Changes in the diagnostic criteria from Rome III to Rome IV will better identify candidates for the biopsychosocial approach.

Trial registration

Although this survey was an anonymous internet survey, we obtained informed consent for the study as an online response. The disclosure of this study was approved by the Ethics Committee of Tohoku University Graduate School of Medicine (approval number: 2015–1-405).

     

Plerocercoids of Nybelinia surmenicola (Cestoda: Tentacularidae) in Squids,Todarodes pacificus,from East Sea,the Republic of Korea

A visceral helminth of the squid, Todarodes pacificus, is reported from the East Sea, the Republic of Korea. Total 39 squid samples were purchased from a fish market in Jumunjin-eup, Gangneung-si (City) from August 2014 to July 2015 and were examined for helminth parasites with naked eyes and under a stereomicroscope after opening the abdominal cavity with a pair of scissors. Whitish larval worms were mainly found in the stomach and abdominal cavity of the squid. They were detected in 25 (64.1%) out of 39 squids examined, and the infection density was 7 larvae per infected squid. Spatula-shaped larvae were 8.2×2.0 mm in average size, round to slightly flattened anteriorly, with round hatching posteriorly, and had characteristic 4 tentacles with numerous hooklets in the scolex. The larvae were identified as the plerocercoid stage of Nybelinia surmenicola by their morphological features. This finding represents a new host record and the first report of N. surmenicola infection in T. pacificus squids from the east coast of Korea.     

Intraspecific Epitopic Variation in a Carbohydrate Antigen Exposed on the Surface of Trichostrongylus colubriformis Infective L3 Larvae

The carbohydrate larval antigen, CarLA, is present on the exposed surface of all strongylid nematode infective L3 larvae tested, and antibodies against CarLA can promote rapid immune rejection of incoming Trichostrongylus colubriformis larvae in sheep. A library of ovine recombinant single chain Fv (scFv) antibody fragments, displayed on phage, was prepared from B cell mRNA of field-immune sheep. Phage displaying scFvs that bind to the surface of living exsheathed T. colubriformis L3 larvae were identified, and the majority of worm-binding scFvs recognized CarLA. Characterization of greater than 500 worm surface binding phage resulted in the identification of nine different anti-CarLA scFvs that recognized three distinct T. colubriformis CarLA epitopes based on blocking and additive ELISA. All anti-CarLA scFvs were specific to the T. colubriformis species of nematode. Each of the three scFv epitope classes displayed identical Western blot recognition patterns and recognized the exposed surface of living T. colubriformis exsheathed L3 larvae. Surprisingly, each of the anti-CarLA scFvs was able to bind to only a subset of worms. Double-labelling indirect immunofluorescence revealed that the three classes of anti-CarLA scFvs recognize distinct, non-overlapping, T. colubriformis sub-populations. These results demonstrate that individual T. colubriformis L3 larvae display only one of at least three distinct antigenic forms of CarLA on their surface at any given time, and suggest that antigenic variation within CarLA is likely a mechanism of immune evasion in strongylid nematodes.     

Electrophoretic studies on glucosephosphate isomerase and phosphoglucomutase in two types of Anisakis larvae

Agatsuma T. 1982. Electrophoretie studies on glucosephosphate isornerase and phosphoglucomutase in two types of Anisakis larvae. International Journal for Parasitology12: 35–39. Enzyme electrophoresis was carried out between the larval forms. Type I and II, of Anisakis using starch gel. In glucose-phosphate isomerase, considerable polymorphisms were found in each type. At least 5 alleles appear to occur at this enzyme locus in natural populations of both types. Out of 5 alleles, 3 were common to both types. No significant difference was found in their frequencies. However, each larval form can be easily distinguished by the electrophoretic mobility of phosphoglucomutase. It was concluded that enzyme electrophoresis is an alternative useful tool for the identification of larval forms of Anisakis.     

Trichinella spiralis: Delayed rejection in mice concurrently infected with Nematospiroides dubius

The immune response of mice to the nematode Trichinella spiral's was markedly altered when the infection was superimposed upon an existing infection with Nematospiroides dubius. The expulsion of a primary infection of T. spiralis was delayed in such mice, and the worms persisted for at least 4 weeks longer than they did in control mice. The degree to which expulsion was suppressed was related to the number of N. dubius present. It would appear that both adult and larval stages of N. dubius can exert a suppressive effect, since the expulsion of T. spiralis was affected within days of a super-imposed (i.e., larval) N. dubius infection. When adult N. dubius were removed from mice 4 days before infection with T. spiralis, the mice expelled the latter parasite within the normal time, indicating that recovery from the suppressive effects of concurrent infection occurred rapidly. Concurrent infection with N. dubius appeared to affect both the afferent and efferent arms of the immune response to T. spiralis, since sensitization by, and memory of, prior infection were impaired and the expression of acquired immunity was inferior to that of controls.     

Trichinella spiralis and Strongyloides ratti: immune interaction in adult rats.

The consequences of prior and concurrent infection with two species of nematodes were studied in rats. Primary infection with Strongyloides ratti adversely affected the development of a secondary Trichinella spiralis infection. Both immediate and delayed challenge with T. spiralis, following the expulsion of the previous S. ratti infection, reduced the percentage of worm recovery of the former as well as their fecundities and lengths. It is suggested that nonspecific inflammation produced by one species, during the peak period of worm expulsion, was not responsible for the accelerated rate of expulsion of the other; instead a direct, specific cross-immunity was probably operative affecting the survival of the challenge species. The response elicited by previous experience of the intestinal phase was reciprocal, but there was evidence of an enhancing effect by the muscle larval stages of T. spiralis on S. ratti. Rats concurrently infected with both species expelled S. ratti more rapidly than T. spiralis. Possible mechanisms underlying the interaction between the two species are suggested and discussed.     

Trichinella spiralis: effects on the host-parasite relationship in mice of BCG (attenuated Mycobacterium bovis).

The effects of BCG (Bacillus Calmette-Guérin, i.e., attenuated Mycobacterium bovis) on the host-parasite relationship in murine trichinosis were examined. A total of 2 × 10⁷ colony forming units of BCG given iv 1 week prior to Trichinella spiralis infection delayed the expulsion of adult worms from the gut. The suppression of adult worm elimination was proportional to the dose of BCG given. This finding was associated with a reduction in the degree of partial villous atrophy induced in the small bowel by T. spiralis. Adult female worms were fecund when they were examined 1, 2, and 3 weeks after infection of mice with T. spiralis. Despite the prolongation of fecund adult worms in the gut, there were no significant differences in muscle larval counts 4 and 6 weeks after infection. When newborn larvae were cultivated in vitro and injected iv, there was a significant 25% reduction in larval numbers recovered from the muscles of BCG-treated mice 4 weeks later. The administration of BCG had no effect on the inflammatory reaction around larvae in the muscles 4 and 6 weeks after infection. It is concluded that BCG alters the host-parasite relationship producing retention of adult worms in the gut, reduction in the severity of partial villous atrophy, and increased nonspecific resistance to the systemic larval phase of this parasite.     

Development and Infectious Disease in Hosts with Complex Life Cycles

Metamorphosis is often characterized by profound changes in morphology and physiology that can affect the dynamics of species interactions. For example, the interaction between a pathogen and its host may differ depending on the life stage of the host or pathogen. One pathogen that infects hosts with complex life cycles is the emerging fungal pathogen of amphibians, Batrachochytrium dendrobatidis (Bd). We sought to determine how conditions at the larval stage can affect variation in development and patterns of Bd infection across amphibian life stages. We used outdoor experimental mesocosms to simulate natural pond habitats and manipulated the presence of Bd, the larval density, and the number of host species in larvae of two co-occurring amphibian species (Rana cascadae and Pseudacris regilla). We found that infection differed between species throughout development; P. regilla consistently had higher infection severity compared to R. cascadae. Additionally, while up to 100% of larvae were infected, only 18.2% of R. cascadae and 81.5% of P. regilla were infected after metamorphosis. This indicates that amphibians have the ability to recover from Bd infection as they undergo metamorphosis. Higher larval densities in P. regilla led to a shorter larval period, and individuals with a shorter larval period had lower infection severity. This led to a trend where P. regilla larvae reared at high densities tended to have lower infection prevalence after metamorphosis. We also found that exposure to Bd increased larval mortality and prolonged the larval period in P. regilla, indicating that P. regilla are susceptible to the negative effects of Bd as larvae. This study demonstrates that host density, species composition, and pathogen exposure may all interact to influence development and infection in hosts with complex life cycles.     

Larval morphology of the scorpionfly Dicerapanorpa magna (Chou) (Mecoptera: Panorpidae) and its adaptive significance

Larval morphology can provide valuable characters for taxonomic and phylogenetic analyses of insects and reflect the adaptations to various living habits. Compared with the adult stages, larval study has lagged far behind in Mecoptera. Although several genera of Panorpidae have been studied for their larval stages, the larva of Dicerapanorpa Zhong and Hua, 2013 basically remains unclear. Here the larva of Dicerapanorpa magna (Chou) is described and illustrated in detail for the first time using light microscopy and scanning electron microscopy. The larva is eruciform, with eight pairs of abdominal prolegs in addition to three pairs of thoracic legs, as in other Panorpidae. The most remarkable characteristics of the larvae include a pair of erect subdorsal annulated processes each on abdominal segments I–IX (A1–A9) and a single middorsal annulated process on A10, as well as a pair of prominent compound eyes composed of over 40 ommatidia, which distinguish this genus from other genera of Panorpidae. The annulated processes may have adaptive significance for fossorial and soil-living habits.     

Koninklijk Zoölogisch-Botanisch Genootschap,the Zoological Garden in The Hague, 1863-1943, a Retrospect after Seventy Years from the Point of View of a Zoologist

The zoological garden in The Hague, the de facto capital of the Netherlands, founded in 1863, was forced to close during the Second World War in 1943 by order of the German occupation forces. An attempt to reconstruct its history from the point of view of a zoologist shows that in eighty years about 180 species of mammals were kept; the garden acquired a certain fame because of its primate collection, e.g., having early success with Sumatran Orang-utan (Pongo abelii), and the discovery of the Black Mangabey (Cercocebus aterrimus) in 1890. Many unusual, nowadays rare, species [e.g., Ursine Tree Kangaroo (Dendrolagus ursinus), Sumatran Bamboo Rat (Rhizomys sumatranus), Marbled Cat (Pardofelis marmorata), Malay Tapir (Tapirus indicus)] were present at times, occasionally also on deposit from animal dealers. The majority of the animals originated from the Netherlands East Indies (now Indonesia) and were donated by colonial residents, such as Sumatran Elephant (Elephas maximus sumatranus) and Orang-utan. The colony of Surinam in South America was another supplier of zoo animals and until the 1880s animals were also derived from a few Dutch trading posts in West Africa. The bird collections were always rich and varied, initially specializing in local species. Later this collection became more comprehensive; attention is drawn to early success with birds of paradise and the presence of now extinct species such as the Carolina Parakeet (Conuropsis carolinensis) and the Passenger Pigeon (Ectopistes migratorius). Series of lower vertebrates [among them the Japanese Giant Salamander (Andrias japonicus)] and invertebrates were always on display in a modest aquarium. At the turn of the century the Netherlands only harboured three zoological gardens (Amsterdam – 1838; Rotterdam – 1857; The Hague – 1863). The one in The Hague, although subject to an almost permanent financial crisis, nevertheless succeeded in playing a modest role in this field of zoological science.     

Toxocara canis: T lymphocyte function in murine visceral larva migrans and eosinophilia onset

The second larval stage of Toxocara canis, the major cause of visceral larva migrans syndrome in humans and also the common intestinal roundworm of the dog, elicits an eosinophil-rich granulomatous response in nondefinitive hosts such as man and mice. During murine infection a population of T lymphocytes was identified which responded to a preparation of T. canis larval antigens in a migration inhibition test (MIT). It was further shown that mice infected with T. canis have a substantial eosinophilia which reaches its peak approximately 2 weeks after infection. When mice are depleted of their functional T lymphocytes by use of antilymphocytic antisera, the eosinophil response of infected mice is significantly reduced. These findings are consistent with the findings of others that helminthic parasites can elicit cell-mediated immune responses and the resultant eosinophilia is but one manifestation of this response.     

The effects of Farinocystis tribolii on the growth and development of the flour beetle Tribolium castaneum

Farinocystis tribolii multiplied only in the fatbody of Tribolium castaneum larvae. In the advanced stages of infection, the fatbody was destroyed and the albuminoid granules were completely depleted. Larvae in late stages of infection were “C” shaped. Diseased pupae were flat and straight with the head and mouth parts not completely flexed with the pupal body. Infected pupae failed to develop into adults. Heavily infected adults were distended. Larvae of third, fourth, and fifth instars, although succumbing to the infection ultimately, continued to live even after the healthy larvae of the same age had entered pupation, indicating a prolongation of larval period. Molting was either affected or delayed in infected insects. F. tribolii infection induced a juvenile hormonal effect producing larval-pupal and pupal-adult intermediate forms. Ether extract of spores of F. tribolii when applied on pupae of T. castaneum and final instar nymphs of Dysdercus cingulatus produced adultoids.     

Interaction of Xenorhabdus nematophilus subsp. nematophilus with the haemolymph of Galleria mellonella

The lethal dose (LD)₅₀ values and probit-mortality regression slopes of the primary and secondary forms of Xenorhabdus nematophilus subsp. nematophilus for Galleria mellonella were equal. The two bacterial forms grew at equal rates in larval serum-supplemented media. The secondary form grew less well in larval serum-supplemented media than in synthetic larval serum.The secondary bacteria adhered to the haemocytes to a greater extent than did the primary bacteria. Both types of bacteria did not produce metabolites suppressing the ability of the haemocytes to respond to Bacillus cerues.Differences were observed in the rate of clearance of the primary and secondary bacteria from and their subsequent re-entrance into the haemolymph in vivo. This appeared to be independent of bacterial metabolism. Evidence is presented showing multiplication of the primary bacteria during their association with the haemocytes.The total haemocyte counts increased during bacterial infection. All the haemocytes were killed. The rate and pattern of change of the total haemocyte counts was influenced by the form of bacteria and independent of bacterial metabolism.     

Homeotic gene function in the muscles of Drosophila larvae

The segmental musculature of Drosophila melanogaster larvae consists of 24-30 muscles per segment. Unique patterns of muscles are found in the three thoracic segments and the first and last abdominal segments; the remaining abdominal segments share the same pattern. Mutations in Ultrabithorax (Ubx) cause partial transformation of the muscle pattern of larval abdominal segments towards metathorax. The muscles of the thorax are not affected. In the first two abdominal segments the changes include the loss of at least 11 `abdominal' muscles and the gain of 11 `thoracic' muscles. Less extensive transformations are seen in more posterior abdominal segments. Anterobithorax, bithorax, postbithorax and bithoraxoid mutations also induce transformations of the larval musculature. Each allelic group affects a domain that is a subset of the entire Ubx domain but these domains are not restricted to compartments or segments and may extend through as many as five segments. In the muscles the segmental distribution of Ubx antigen correlates with the segments affected by Ubx mutations. The different domains of Ubx in mesoderm and ectoderm argue that the segmental diversity of the muscle pattern is not simply induced by the overlying epidermis and that Ubx function in the mesoderm is required for the correct development of abdominal segments.     

Variable correspondence of female host preference and larval performance in a phytophagous ladybird beetle Epilachna pustulosa (Coleoptera: Coccinellidae)

Genetic and environmental factors causing intraspecific variation of the thistle Cirsium kamtschaticum Ledeb. as a host plant of the phytophagous ladybird Epilachna pustulosa Kôno were investigated through simple food-choice tests and rearing of larvae. Two thistle clones (T1U₁ and T4H₂) were used, originally growing approximately 12 km apart. A previous study showed that adult female ladybirds preferred T1U₁ to T4H₂, and that larval performance was better on T1U₁, when leaves from the clones in situ were examined. The two clones retained their characteristics with respect to beetle preference after transplantation into a common garden. However, the difference between T1U₁ and T4H₂ with respect to larval performance was reduced after the transplantation. When leaves from shoots of T1U₁ exposed to different sunlight intensities were offered, adult female ladybirds did not show obvious preferences. Larval eclosion rates increased significantly with the increase in leaf sunlight intensity exposure. These results suggest strongly that both genetic and environmental factors are involved in interclonal variation of thistle quality in beetle preference and/or performance. It is suggested that the quality of thistle leaves for larval performance is largely affected by environmental factors, while leaf quality for beetle preference may be determined strictly by genetic factors. Under certain conditions, E. pustulosa females may behave maladaptively, preferring plants not appropriate for larval growth, or not choosing plants appropriate for the larval growth.