Vitamin D3 in the Hemolymph of Goliath Birdeater Spiders (Theraphosa Blondi)
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Journal of Zoo and Wildlife Medicine 40(2): 344–346, 2009 Copyright 2009 by American Association of Zoo Veterinarians VITAMIN D3 IN THE HEMOLYMPH OF GOLIATH BIRDEATER SPIDERS (THERAPHOSA BLONDI) Trevor T. Zachariah, D.V.M., M.S., and Mark A. Mitchell, D.V.M., Ph.D. Abstract: Vitamin D3 is an important vitamin in vertebrates. This fat-soluble vitamin is associated with the regulation of many physiologic processes, most importantly calcium metabolism. The presence or importance of vitamin D3 has been determined in only a handful of invertebrate species. In this study, hemolymph was collected from six wild-caught, subadult goliath birdeater spiders (Theraphosa blondi) and analyzed for the presence of 25(OH)-vitamin D3, the precursor to the active form of vitamin D3. The metabolite 25(OH)-vitamin D3 was detected in all of the spiders (mean: 5.7 nmol/L, SD: 1.5 nmol/L, range: 3–7 nmol/L). The method by which spiders acquire vitamin D3 is unknown. It is possible, though unlikely, that they synthesize it via exposure to ultraviolet radiation. Many of the invertebrate species upon which theraphosid spiders prey are not known to have high circulating levels of vitamin D3 or its precursors. However, dietary intake is a possible means of vitamin D3 acquisition in this study. Key words: Hemolymph, spider, Theraphosidae, Theraphosa blondi,vitaminD3, 25(OH)-vitamin D3. BRIEF COMMUNICATION Calcium, Fluker Farms, Port Allen, Louisiana 70767, USA) until being offered to the spider. The physiology of spiders has been studied The cricket food also contained cholecalciferol, a extensively. Much of the research has been precursor molecule to 25(OH)-vitamin D .The focused on elucidating the metabolic processes 3 temperature and humidity in the enclosures were of these animals as a comparative method to maintained at approximately 23.9 C(75F) and further explain vertebrate physiology. However, a u u 80%, respectively. The spiders were held under literature search reveals a conspicuous absence in these conditions for 12 wk before diagnostic this body of work in the area of calcium and samples were obtained. vitamin D metabolism. This pilot study was 3 An intracardiac hemolymph sample was col- conducted to confirm the presence of vitamin D 3 lected from each of the T. blondi according to the in the goliath birdeater spider (Theraphosa following procedure: each spider was placed into blondi). a square, 3-L plastic storage container that was Six (four male and two female) wild-caught, modified into a gas anesthetic chamber. The subadult T. blondi were obtained from an container was customized by drilling a hole on invertebrate importer in Florida (LASCO, Na- one side and inserting an endotracheal tube ples, Florida 34119, USA). The spiders were adapter. Each spider was anesthetized with 5% housed in rectangular, 5.7-L plastic storage isoflurane (Isoflo, Abbott Laboratories, North containers with screen tops. A 50:50 mixture of Chicago, Illinois 60064, USA) at a flow rate of potting soil and vermiculite was used for the 1 L/min oxygen. Once the spider had lost its substrate. The spiders had ad lib access to ability to right itself, it was removed from the chlorinated tap water and were fed five adult anesthetic chamber and weighed. A 26-gauge, crickets weekly. The crickets had access to a high- 1.9-cm (3/4-inch) needle fastened to a 3-ml calcium cricket food and water source (High syringe was used to collect an intracardiac Calcium Cricket Diet and Cricket Quencher hemolymph sample. Collection of each sample was accomplished by inserting the needle at approximately a 45u angle through the exoskel- From the Department of Veterinary Clinical Scienc- eton at the midpoint of the dorsal midline of the es, School of Veterinary Medicine, Louisiana State opisthosoma. A total of 0.5 ml hemolymph was University, Baton Rouge, Louisiana 70803, USA. collected from each individual. Hemolymphstasis Present addresses (Mitchell): Department of Veterinary was accomplished by applying a small amount of Clinical Medicine, College of Veterinary Medicine, Nexaband glue (Veterinary Products Laborato- University of Illinois, Urbana, Illinois 61802, USA; ries, Phoenix, Arizona 85067, USA) to the (Zachariah): Chicago Zoological and Aquatic Animal Residency, Brookfield, Illinois 60513, USA. Corres- collection site. The spiders were recovered in pondence should be directed to Dr. Zachariah 100% oxygen, and recovery from anesthesia was ([email protected]). uneventful. 344 ZACHARIAH AND MITCHELL—VITAMIN D3 IN A THERAPHOSID 345 Each sample was placed in a lithium heparin metabolism in these species are not well studied. Microtainer tube (Becton Dickinson, Franklin Spiders probably require calcium to ensure Lakes, New Jersey 07417, USA) and centrifuged striated muscle function and various other for 10 min at 1,411 g. Once the sample was physiological cell processes. In a study involving centrifuged, the supernatant was removed and T. blondi and Chilean rose spiders (Grammostola frozen in a Cryovial (Nalge Nunc International, rosea), it was found that hemolymph calcium Rochester, New York 14625, USA) at 262.2uC levels were greater (11.9 6 1.7 mg/dL and 16.9 6 (280uF). The samples were then transported on 1.8 mg/dL, respectively)13 than those of typical frozen gel packs to the Endocrine Service of the mammals (e.g., dogs and cats, 9.0–11.5 mg/dL).8 Diagnostic Center for Population and Animal For the theraphosid spider Eurypelma californi- Health at Michigan State University for detection cum (nomen dubium, unknown species probably 9 of 25(OH)-vitamin D3 by quantitative radioim- belonging to the genus Aphonopelma ), hemo- munoassay analysis. lymph calcium levels have also been found to be The metabolite 25(OH)-vitamin D3 was detect- greater than those in mammals (15.76 6 0.52 mg/ ed in all six spiders. The Shapiro-Wilk test was dL).10 These findings suggest that theraphosid used to assess the normality of the data, and the spiders require relatively high levels of calcium, data were found to follow a Gaussian distribu- although the method by which they achieve them tion (P 5 0.212). Measures of central tendency is unknown. and dispersion were then calculated: mean, The 25(OH)-vitamin D3 levels measured in 5.7 nmol/L; SD, 1.5 nmol/L; and range, 3– these theraphosid spiders was low in comparison 7 nmol/L. The significance level was set at a 5 to mammalian (dog: 60–215 nmol/L, cat: 65– 0.05. The statistical analysis was performed using 170 nmol/L)6 and reptilian (red-eared slider a commercial software package (SPSS 15.0, SPSS turtle: 31.4 6 13.2 nmol/L)1 vertebrates. Verte- Inc., Chicago, Illinois 60606, USA). brates have the opportunity to store calcium in To the authors’ knowledge, this is the first time their skeletons. The absence of such a storage that vitamin D3 has been measured in any species depot in spiders may be associated with the of spider. Limited studies in other taxa reveal that higher circulating levels of calcium in the body. there may be some variability in vitamin D levels This physiologic finding may also play a role in in invertebrates. Vitamin D3 and a vitamin D- the intertaxa vitamin D3 level differences detect- dependent calcium binding protein have been ed. These facts also suggest that vitamin D3 may found in two species of terrestrial snails.12 Seven play a minimal or no role in calcium metabolism species of insect, both adults and larvae, were in spiders. 4 found to have no detectable levels of vitamin D3. Most spiders prey on arthropods, although It has been stated that there is no evidence that theraphosid spiders will occasionally capture and 7 insects require vitamin D3. Also, it has been eat vertebrates. Due to the previously stated lack shown that vitamin D2 may have a role in the of vitamin D3 in insects, it is unlikely that wild nutrition of copepods.5 spiders ingest the compound from their prey. In Vitamin D3 plays an important role in calcium captivity, however, the insect prey may serve as a metabolism of vertebrate animals. Whether it is source of vitamin D3 if they are offered commer- absorbed from the diet or synthesized in the skin cial diets with cholecalciferol. From this study, it due to ultraviolet-B (UVB) radiation exposure, is not possible to determine whether the crickets the liver hydroxylates the compound to 25(OH)- or the spiders possess 25-hydroxylase, but one of vitamin D3 via the enzyme 25-hydroxylase. This the species may convert cholecalciferol to compound is either stored in adipose tissue when 25(OH)-vitamin D3. Where the hydroxylation of calcium levels are adequate, or hydroxylated by cholecalciferol occurs, and whether it is further the kidneys to form 1,25(OH)2-vitamin D3 when processed to 1,25(OH)2-vitamin D3, remains to be increased calcium absorption is required. The investigated. 1,25(OH)2-vitamin D3, also known as calcitriol, is Spiders may synthesize vitamin D3; however, the active form of vitamin D3 and is responsible the process may not be similar to that previously for promoting calcium uptake and transport in described for vertebrate species. Anecdotally, the body. In addition to this function, vitamin D3 theraphosid spiders in captivity are usually kept promotes various other metabolic functions in without the presence of a source of UVB, and tissues throughout the body.3 these animals appear to be clinically normal. The Many invertebrates possess tissues containing spiders in this study were kept without UVB calcium, though the mechanisms of calcium radiation and still maintained the reported 346 JOURNAL OF ZOO AND WILDLIFE MEDICINE calcium levels. Many species of theraphosid 4.