Expanding the invertebrate medicine toolbox: evaluation of opisthosoma tonometry as a novel diagnostic tool for arachnids.

OBJECTIVE: The objective of this study was to analyze the use of rebound tonometry, as a noninvasive diagnostic tool, in arachnids. METHODS: 5 juvenile (yearling) female and 1 juvenile male curlyhair tarantulas (Tliltocatl albopilosus, previously Brachypelma albopilosum) were used to track estimated ventral opisthosoma pressures over a 9-month period of time. Younger, growing animals were selected as they are more likely than adults to go through multiple molts throughout the 9 months of the study length. An iCare TONOVET TV01 rebound tonometer was used to measure the estimated ventral opisthosoma pressures of the spiders. Measurements were obtained from the ventral opisthosoma, 1 of the thinnest areas of exocuticle throughout the body. Readings were obtained once per week from the ventral opisthosoma for the first 2 months, then once every 2 weeks for 1 month, then back to once per week for the remainder of the study. Additional measurements were obtained following each ecdysis, after a 2% body weight sampling of hemolymph, and at the end of the study to evaluate readings in response to induction of general anesthesia with 5% isoflurane gas and oxygen flow at 2 L/min for 10 minutes. RESULTS: The average of all estimated ventral opisthosoma pressure when spiders were not in molt was 26.19 mm Hg (SD, 3.54), with a statistically significant decrease postmolt to an average of 15.31 mm Hg (SD, 3.81), followed by a gradual increase back to premolt pressures over a 3-week period with an average of 22 days (SD, 1.93). Estimated ventral opisthosoma pressures decreased post hemolymph removal. There is not sufficient evidence that estimated ventral opisthosoma pressures changed over time following the anesthesia. CLINICAL RELEVANCE: This study demonstrates that tonometry can be used to generally assess the estimated ventral opisthosoma pressure, which could correlate with where a spider is in an ecdysis cycle.

Presumed hemocytic neoplasms in scorpions.

Although neoplasia has been documented in invertebrates, it has not been reported in scorpions. This report describes presumed hemocytic neoplasia in 2 scorpions: a >3-year-old, female emperor scorpion (Pandinus imperator) and a >4-year-old, male, Asian forest scorpion (Heterometrus sp.). The emperor scorpion had a 1-month history of body wall swelling separating the exoskeleton of the caudal opisthosoma. At necropsy, this corresponded to a white mass in the caudal coelom. The forest scorpion was found dead and processed whole for histology, at which point multiple masses were identified in the coelom and invading skeletal muscle. Histologically, both masses were composed of sheets of hemocytes with round to oval nuclei; eosinophilic, periodic acid Schiff-positive, cytoplasmic granules; mild cellular atypia; and low mitotic rates. Features of inflammation (e.g., melanization and nodulation) were not observed. These masses were diagnosed as a hemocytoma (emperor scorpion) and a hemocytic sarcoma (forest scorpion), possibly of plasmatocyte origin.

Retrospective analysis of histologic lesions in captive arachnids.

Invertebrates, including arachnids, are a common taxon in zoological collections. Invertebrate medicine and pathology are emerging subspecialties, but there is limited reference material or published resources describing histologic lesions in arachnids. Histopathology of 26 captive arachnids (20 spiders and 6 scorpions) from institutional collections was reviewed. Most animals were found dead with limited clinical signs. Tissues evaluated included body wall (cuticle and epidermis), skeletal muscle, book lungs, digestive tract (pharynx, esophagus, sucking stomach, midgut tube, midgut diverticula, and stercoral pocket), central and peripheral nervous system, heart, hemolymph vessels and sinuses, Malpighian tubules, coxal glands, and gonads. Inflammation was frequent (24/26, 92%), and seen in multiple organs (18/24, 75%) with the midgut diverticulum most commonly affected (14/24, 58%) followed by the book lungs (13/24 arachnids, 54%), and body wall (8/24 arachnids, 33%). Inflammation comprised hemocyte accumulation, hemocytic coagula, melanization, and nodulation. Infectious agents, including bacteria (11/26, 42%), fungi (10/26, 38%), and parasites (2/26, 8%), were seen within inflammatory aggregates. Coinfection with multiple infectious agents was common (6/24, 25%). No etiologic agent was identified in 7/24 (29%) cases with inflammatory lesions. Lesions suggestive of decreased nutritional status or increased metabolic rate included midgut diverticula atrophy in 11/26 (42%) animals and skeletal muscle atrophy in 6/26 (23%) animals. Atrophic lesions were seen in combination with infection (8/11, 73%), pregnancy (2/11, 18%), male sex (2/11, 18%), or without other lesions (1/11, 9%). Other suspected contributors to death included dysecdysis-associated trauma (2/26, 8%) and uterine intussusception (1/26, 4%). No animals had neoplasia.

HEMOLYMPH COLLECTION AND ISOFLURANE ANESTHESIA OF THE EMPEROR SCORPION (PANDINUS IMPERATOR).

With captive invertebrates increasing in popularity, a safe and effective anesthesia protocol is required for their veterinary care. This study investigated the safety and efficacy of 5% isoflurane anesthesia and 0.26 ml hemolymph collection in subadult to adult emperor scorpions (Pandinus imperator; n = 14). Each scorpion was placed in a 2-L anesthetic chamber and induced with 5% isoflurane at a flow rate of 2 L/min oxygen. Anesthetic depth was assessed by rolling the animal into dorsal recumbency and rating the response from one to four: 1 = successful coordinated righting movement, 2 = successful uncoordinated righting movement, 3 = unsuccessful righting movement, 4 = no movement. Ratings 3 and 4 were both adequate for the hemolymph collection. The mean induction time was 4.2 min to reach a level 3 depth and 10.5 min to reach a level 4 depth. All animals were clinically normal for at least 1 yr post experiment. A mean hemolymph volume drawn of 1.92% of body weight, and an anesthetic induction with 5% isoflurane, appear safe in subadult to adult emperor scorpions.

EFFECTS OF TRICAINE METHANESULFONATE IN A MANAGED COLLECTION OF MOON JELLYFISH (AURELIA AURITA).

The moon jellyfish (Aurelia aurita) is a scyphozoan frequently maintained in public and private aquaria. Little research has been conducted to investigate the effects of various drugs, such as anesthetics, in this species. Tricaine methanesulfonate (MS-222), a common immersion anesthetic for fish and amphibians, was evaluated in a managed population of moon jellyfish. Twenty-four clinically healthy jellyfish were assigned into three groups of eight for trials of 0.3 g/L MS-222 (low concentration [LC]), 0.6 g/L MS-222 (high concentration [HC]), and a saltwater control. The goal was to evaluate the effects of MS-222 administration on moon jellyfish movement and response to stimuli. Movement and response to stimuli were measured via rocking and probe stimulus tests and observations of bell contraction quality and body tone. These tests were performed at baseline and throughout both drug exposure and recovery periods. A threshold drug effect was defined based on systematic scoring criteria. Additionally, elastomer tags were administered to four of eight animals in each MS-222 group to evaluate response to tag placement after drug exposure. Threshold drug effect was achieved in six of eight individuals in the LC group and eight of eight individuals in the HC group. The LC group had median threshold and recovery times of 12.2 and 10.1 min, respectively, while the HC group had median threshold and recovery times of 4.0 and 19.9 min, respectively. The HC group had significantly faster time to threshold drug effect (P < 0.001) and longer recovery times (P= 0.005) than the LC group. In both the LC and HC tagged group, three of four jellyfish had no reaction to tag placement. All animals recovered uneventfully, and there were no mortalities. MS-222 at 0.3 and 0.6 g/L decreased movement and response to stimuli in moon jellyfish.

ISOFLURANE ANESTHESIA IN THE MADAGASCAR HISSING COCKROACH (GROMPHADORHINA PORTENTOSA).

This study investigated the efficacy of isoflurane in Madagascar hissing cockroaches (Gromphadorhina portentosa). Cockroaches (n = 12) were placed in a 2-L anesthesia chamber with isoflurane initiated at 5% at 2 L/m. Movement of antennae, mandibles, and legs and righting reflex were assessed, with full induction achieved when all responses had ceased for 2 m. Cockroach movement became ataxic on average (±SD) 3.5 ± 0.9 m after isoflurane treatment, and induction occurred on average 18.7 ± 4.4 m after treatment. Loss of righting reflex was the most consistent indication of full induction. Cockroaches fully recovered on average 16.2 ± 5.6 m after removal of isoflurane inhalant. Induction was uneventful, and no mortality or obvious morbidity was observed in treated cockroaches up to 30 d posttreatment. The authors conclude that the use of inhalant isoflurane is both safe and effective for anesthesia of Madagascar hissing cockroaches.

ASSESSMENT OF A VISUAL SCORING SYSTEM FOR IDENTIFYING AND QUANTIFYING ANEMIA IN MALE EASTERN BOX TURTLES ( TERRAPENE CAROLINA CAROLINA).

The North Carolina State University College of Veterinary Medicine Turtle Rescue Team (TRT) regularly admits wild eastern box turtles (EBT) ( Terrapene carolina carolina) for treatment and rehabilitation. Adult males of this species generally have an impressive red coloration of the iris, a feature commonly used to differentiate them from their brown-eyed female conspecifics. Male EBTs that present often display marked pallor of the eyes, which has accompanied clinical anemia. The aim of this study was to examine the relationship between eye color and packed cell volume (PCV) in adult male EBTs, and to assess the accuracy of using eye color to predict PCV. Eye-color scoring cards were created using Adobe Creative Cloud Photoshop®. Each card was composed of blocks displaying progressively lighter tints of a red hue, with a number (score) assigned to each block. Five-block, six-block, and nine-block scoring cards were prepared, with lower numbers (scores) corresponding to lighter hues. To determine the variation in subjective color scores assigned by different human observers, 85 individuals were asked to score EBT eye color in three animals using each of the three scoring cards. Eye color of adult male EBTs that presented to the TRT ( n = 18) and those housed at the North Carolina Museum of Natural Sciences ( n = 6) were scored using each of the three cards, immediately preceding venipuncture of the occipital sinus for determination of PCV. All animals were scored by at least two individuals. Turtles with low scores (pale eyes) using all three scoring cards consistently had lower PCVs. The five- and six-block scorecards had a positive predictive value (PPV) of 100% while the nine-block score card had a PPV of 71.4%. This specific and noninvasive method for estimating PCV of male EBTs is clinically desirable in both rehabilitation and hospital settings.

A comparison of blood gases, biochemistry, and hematology to ecomorphology in a health assessment of pinfish (Lagodon rhomboides).

Despite the promise of hematological parameters and blood chemistry in monitoring the health of marine fishes, baseline data is often lacking for small fishes that comprise central roles in marine food webs. This study establishes blood chemistry and hematological baseline parameters for the pinfish Lagodon rhomboides, a small marine teleost that is among the most dominant members of near-shore estuarine communities of the Atlantic Ocean and Gulf of Mexico. Given their prominence, pinfishes are an ideal candidate species to use as a model for monitoring changes across a wide range of near-shore marine communities. However, pinfishes exhibit substantial morphological differences associated with a preference for feeding in primarily sea-grass or sand dominated habitats, suggesting that differences in the foraging ecology of individuals could confound health assessments. Here we collect baseline data on the blood physiology of pinfish while assessing the relationship between blood parameters and measured aspects of feeding morphology using data collected from 37 individual fish. Our findings provide new baseline health data for this important near shore fish species and find no evidence for a strong linkage between blood physiology and either sex or measured aspects of feeding morphology. Comparing our hematological and biochemical data to published results from other marine teleost species suggests that analyses of trends in blood value variation correlated with major evolutionary transitions in ecology will shed new light on the physiological changes that underlie the successful diversification of fishes.

TRICAINE METHANESULFONATE (MS-222) SEDATION AND ANESTHESIA IN THE PURPLE-SPINED SEA URCHIN (ARBACIA PUNCTULATA).

The purple-spined sea urchin ( Arbacia punctulata ) is commonly found in shallow waters of the western Atlantic Ocean from the New England area of the United States to the Caribbean. Sea urchins play a major role in ocean ecology, echinoculture, and biomedical research. Additionally, sea urchins are commonly displayed in public aquaria. Baseline parameters were developed in unanesthetized urchins for righting reflex (time to regain oral recumbency) and spine response time to tactile stimulus. Tricaine methanesulfonate (MS-222) was used to sedate and anesthetize purple-spined sea urchins and assess sedation and anesthetic parameters, including adhesion to and release from a vertical surface, times to loss of response to tactile stimulus and recovery of righting reflex, and qualitative observations of induction of spawning and position of spines and pseudopodia. Sedation and anesthetic parameters were evaluated in 11 individuals in three circumstances: unaltered aquarium water for baseline behaviors, 0.4 g/L MS-222, and 0.8 g/L MS-222. Induction was defined as the release from a vertical surface with the loss of righting reflex, sedation as loss of righting reflex with retained tactile spine response, anesthesia as loss of righting reflex and loss of tactile spine response, and recovery as voluntary return to oral recumbency. MS-222 proved to be an effective sedative and anesthetic for the purple-spined sea urchin at 0.4 and 0.8 g/L, respectively. Sodium bicarbonate used to buffer MS-222 had no measurable sedative effects when used alone. Anesthesia was quickly reversed with transfer of each individual to anesthesia-free seawater, and no anesthetic-related mortality occurred. The parameters assessed in this study provide a baseline for sea urchin anesthesia and may provide helpful comparisons to similar species and populations that are in need of anesthesia for surgical procedures or research.

Cystic urolithiasis in captive waxy monkey frogs (Phyllomedusa sauvagii).

The waxy monkey frog (Phyllomedusa sauvagii) is an arboreal amphibian native to arid regions of South America, and it has developed behavioral and physiologic adaptations to permit survival in dry environments. These adaptations include a uricotelic nitrogen metabolism and unique cutaneous lipid excretions to prevent evaporative water loss. Uroliths are a rare finding in amphibians. Six adult, presumed wild-caught waxy monkey frogs housed in a museum animal collection were diagnosed with cystic urolithiasis over a 7-yr period, and a single animal was diagnosed with four recurrent cases. Six cases were identified incidentally at routine physical or postmortem examination and four cases were identified during veterinary evaluation for coelomic distension, lethargy, anorexia, and increased soaking behavior. Calculi were surgically removed from three frogs via cystotomy, and a single frog underwent three cystotomies and two cloacotomies for recurrent urolithiasis. Two frogs died within the 24-hr postoperative period. Two representative calculi from a single frog were submitted for component analysis and found to consist of 100% ammonium urate. In the present report, cystic calculi are proposed to be the result of a high-protein diet based on a single invertebrate source, coupled with uricotelism, dehydration, increased cutaneous water loss, body temperature fluctuations facilitating supersaturation of urine, and subsequent accumulation and precipitation of urogenous wastes within the urinary bladder. Surgical cystotomy represents a short-term treatment strategy for this condition. Preventative measures, such as supplying a diversified and balanced diet in addition to environmental manipulation aimed at promoting adequate hydration, are anticipated to be more-rewarding management tools for cystic urolithiasis in the waxy monkey frog.

Comparison of isoflurane and carbon dioxide anesthesia in Chilean rose tarantulas (Grammostola rosea).

This study investigated the use of two anesthetic agents, isoflurane and carbon dioxide, in Chilean rose tarantulas (Grammostola rosea). We compared the onset, duration of anesthesia, and recovery time with both gases, and made observations regarding the effects of the anesthetic protocols. Subjectively, episodes for the isoflurane animals were uneventful. The spiders were calm throughout and did not respond adversely to gas exposure. Conversely, animals anesthetized with carbon dioxide experienced violent inductions and recoveries; the tarantulas appeared agitated when the carbon dioxide flow began. Seizure-like activity and defecation would frequently be noted prior to induction with carbon dioxide. Neither isoflurane nor carbon dioxide seemed to have any clinically apparent short- or long-term impact. The animals were all normal for at least 1-year postexperiment. Future studies should focus on defining the impact, if any, that these anesthetic agents have on the health of invertebrate species.

Body temperatures of selected amphibian and reptile species.

Ectothermic vertebrates are a diverse group of animals that rely on external sources to maintain a preferred body temperature. Amphibians and reptiles have a preferred optimal temperature zone that allows for optimal biological function. Physiologic processes in ectotherms are influenced by temperature; these animals have capabilities in which they make use of behavioral and physiologic mechanisms to thermoregulate. Core body, ambient air, body surface, and surface/water temperatures were obtained from six ectothermic species including one anuran, two snakes, two turtles, and one alligator. Clinically significant differences between core body temperature and ambient temperature were noted in the black rat snake, corn snake, and eastern box turtle. No significant differences were found between core body and ambient temperature for the American alligator, bullfrog, mata mata turtle, dead spotted turtle, or dead mole king snake. This study indicates some ectotherms are able to regulate their body temperatures independent of their environment. Body temperature of ectotherms is an important component that clinicians should consider when selecting and providing therapeutic care. Investigation of basic physiologic parameters (heart rate, respiratory rate, and body temperature) from a diverse population of healthy ectothermic vertebrates may provide baseline data for a systematic health care approach.

Dorsal laminectomy in a two-toed amphiuma (Amphiuma means).

An adult male two-toed amphiuma (Amphiuma means) was evaluated for kyphosis and a self-inflicted ulcerative dermal lesion at the site of a midbody spinal deformity. Radiology revealed a proliferative bony lesion at the 39th vertebra. A dorsal laminectomy was performed to alleviate pressure on the spinal cord believed to be secondary to bone remodeling. Resolution of the scoliosis occurred at the surgical site, and the ulcerative lesion of the body wall healed. Three weeks postoperatively, the amphiuma resumed self-mutilation caudal to the surgery site. Euthanasia was elected. Necropsy revealed focal osteonecrosis and vertebral fractures at the laminectomy site with regionally extensive fibrosis, fibrin deposition, and focal spinal cord compression. Resolution of clinical signs suggests that surgical decompression of the spine was clinically successful short term, but the patient deteriorated for reasons possibly related, although not directly attributable, to the surgery.

Experimental infection of native north Carolina salamanders with Batrachochytrium dendrobatidis.

Chytridiomycosis is an often fatal fungal disease of amphibians caused by Batrachochytrium dendrobatidis. This disease has been implicated in the worldwide decline of many anuran species, but studies of chytridiomycosis in wild salamanders are limited. Between August 2006 and December 2006, we tested wild amphibians in North Carolina, USA (n=212) by polymerase chain reaction (PCR). We identified three PCR-positive animals: one Rana clamitans and two Plethodontid salamanders. We experimentally infected two species of native North Carolina Plethodontid salamanders, the slimy salamander (Plethodon glutinosus) and the Blue Ridge Mountain dusky salamander (Desmognathus orestes) with 1,000,000 zoospores of B. dendrobatidis per animal. Susceptibility was species dependent; all slimy salamanders developed clinical signs of chytridiomycosis, and one died, whereas dusky salamanders remained unaffected. In a second experiment, we challenged naïve slimy salamanders with either 10,000 or 100,000 motile zoospores per animal. Clinical signs consistent with chytridiomycosis were not observed at either dose or in uninfected controls during the 45 days of this experiment. All animals inoculated with B. dendrobatidis in both experiments, regardless of dose, tested positive by PCR. Our study indicates that slimy salamanders are more susceptible to clinical chytridiomycosis than dusky salamanders, and in a laboratory setting, a dose greater than 100,000 zoospores per animal is required to induce clinical disease. This study also indicates that PCR is a very sensitive tool for detecting B. dendrobatidis infection, even in animals that are clinically unaffected, thus positive results should be interpreted with caution.