Varroa destructor mites (Acari: Varroidae) are harmful ectoparasites of Apis mellifera honey bees. Female foundresses of wax-capped pupal host cells and their daughters feed on host fluids from open wounds on the host's integument. Details of V. destructor mite nutrition are forthcoming, and little is known about the potential physical effects on hosts from mite feeding. Chemical analysis of waste excretions can infer details of animals’ nutrition. Here, chemical analysis by high-performance liquid chromatography/mass spectrometry (HPLC-MS/MS) of mite excretions showed that the purine content of V. destructor waste consists of guanine with traces of hypoxanthine. Traces of uric acid and caffeine were also detected. Concentrations of guanine attenuated over time and excretions collected from senescing mites did not contain detectable guanine. Non-reproducing individual female mites maintained in vitro, housed in gelatin capsules and provided a honey bee pupa, deposited an average of nearly 18 excretions daily, mostly on the host's integument rather than on the capsule wall. The weight and volume of excretions suggest mites can consume nearly a microlitre of host fluids each day. Compounded over 10 days, this together with open wounds, could lead to substantial water loss and stress to developing pupae.

Synchrotron powder X-ray diffraction, X-ray absorption spectroscopy (XAS), and proton nuclear magnetic resonance (1H-NMR) data have been used to examine the structure of hypoxanthine, 1,7-dihydro-6H-purin-6-one (C5H4N4O), a purine base that participates in numerous metabolic processes. XAS and 1H-NMR spectroscopy were used to determine that hypoxanthine was present in its keto form (both in solid state and dissolved in an organic solvent). Rigid body refinement was performed with the Rietveld software package GSAS yielding triclinic lattice parameters of a = 7.1179 (2) Å, b = 9.7830 (3) Å, c = 10.4009 (3) Å, α = 58.876 (1)°, β = 67.609 (1)°, and γ = 71.937 (2)° (C5H4N4O, Z = 4, space group P $\bar 1$ ).

Hypoxanthine can block preimplantation mouse embryo development in vitro at the 2- to 4-cell stages, and this has recently been shown to be reversed by cAMP-elevating agents. However, the extent of this hypoxanthine-induced arrest is determined by the culture conditions and strain of mouse. Whitten's and KSOM/AA are two embryo culture media that support preimplantation development to the blastocyst stage. This study was undertaken to examine the influence of several components in these media on hypoxanthine-arrested preimplantation mouse embryos and to test the hypothesis that reversal of the hypoxanthine block by cAMP-elevating agents requires cooperative interaction with the chelator, EDTA. Initial experiments demonstrated that embryo development was blocked in the presence of hypoxanthine in Whitten's medium but not in KSOM/AA; furthermore, removal of EDTA from KSOM/AA rendered this medium incapable of supporting high levels of development to blastocyst (9%), whereas high numbers of blastocysts (80%) formed in Whitten's medium, which does not contain the chelator. Consequently, Whitten's medium was used to test our hypothesis. It has previously been demonstrated that the phosphodiesterase inhibitor, IBMX, can reverse the developmental arrest imposed by hypoxanthine in EDTA-supplemented Earle's basic salt solution, but in the present study the addition of IBMX to Whitten's medium resulted in a block to development and failed to reverse the hypoxanthine arrest. These disparate effects can be explained by the presence or absence of EDTA. Supplementing Whitten's medium with EDTA reverses the IBMX effect, but not the hypoxanthine-induced block. While IBMX alone is unable to reverse the hypoxanthine block in Whitten's medium, development is greatly enhanced by the simultaneous addition of EDTA and IBMX. Similar results were obtained with the cAMP analogue, 8-AHA-cAMP. The data therefore support our hypothesis that the reversal of the hypoxanthine-induced arrest by cAMP-elevating agents is critically dependent on the presence of EDTA. We contrast this with the situation in mouse oocytes, where the hypoxanthine-induced meiotic arrest is not reversed by the addition of EDTA and/or cAMP-elevating agents.

Some culture systems have been shown to support oocyte growth in mice, although there has been little success in applying these systems to other species. In the present study, we compared three culture conditions for growing bovine oocytes and examined the effect of hypoxanthine on oocyte growth. In the first experiment, early antral follicles, 0.4-0.7 mm in diameter were collected, and oocyte-cumulus-granulosa cell complexes (OCGs) and oocyte-cumulus cell complexes (OCs) were dissected from the follicles. Follicles (Fs), OCGs and OCs were embedded in collagen gels and cultured in serum-supplemented medium for 16 days. In the Fs, OCGs and OCs cultured in hypoxanthine-free medium, 21%, 9% and 4% of the oocytes showed normal morphology, respectively, and hypoxanthine (4 mM) increased the percentages in all the groups (Fs, 37%; OCGs, 29%; OCs, 10%). In the second experiment, Fs were cultured in serum-free medium with or without hypoxanthine for 16 days. Histological examination demonstrated that hypoxanthine maintained the integrity of the follicular basement membrane. After a growth culture, 91% of the oocytes showed normal morphology, and 87% of the oocytes were at the germinal vesicle stage in serum-free, hypoxanthine-supplemented medium. The mean diameters of the oocytes were significantly larger (117.6 ± 5.7 mm) than they were in the other groups and than they had been before the culture (approximately 95 mm). After a subsequent maturation culture of the oocytes, 85% underwent germinal vesicle breakdown and 23% reached the second metaphase. These results demonstrate that growing bovine oocytes from early antral follicles grow efficiently in follicles cultured in serum-free, hypoxanthine-supplemented medium and acquire meiotic competence.

The microaerophilous stationary phase system (MASP) was introduced in 1980 and successfully used as a standard technique for Babesia bovis and B. bigemina in vitro culture. The percentage of serum in the medium and the dependence on specific serum donors have been recognized as important constraints both for immunochemical studies and for the logistics of culture routine. In the present study the supplementation of RPMI 1640 with hypoxanthine at a concentration between 50 and 200 μM has enabled patterns of growth of B. bovis and B. bigemina to be achieved comparable to the standard technique with a simultaneous reduction of serum concentration from 40% to 5%. With hypoxanthine-supplemented medium it was possible to either replace the bovine serum from a specific donor with horse serum or use commercial adult bovine serum or foetal calf serum at 10%. When the serum replacement media Albumax II and GF21 were used, the growth of both B. bovis and B. bigemina markedly decreased after 3×72 h cycles. However, when these species were cultivated in culture flasks previously coated with cells from a murine peritoneal lavage, continuous parasite growth was achieved.

Hypoxanthine transport in the insect trypanosome, Crithidia luciliae, was activated in purine depleted conditions. The existence of 2 hypoxanthine transport mechanisms was established. The first, a non-saturable diffusion system, present in purine replete conditions, exhibited properties that were different from the second transport system which was evident only during purine depleted conditions (purine stress). The rate of transport under purine stress was elevated approximately 8-fold over that in replete conditions. This transporter was saturable with a Km of 3·9 μM for hypoxanthine. The transporter substrate specificity included other purine bases, e.g. adenine and guanine, and the purine nucleoside, adenosine. These inhibited hypoxanthine transport competitively with Ki values of 2 μM, 3 μM and 42 μM respectively. Coincident with the increase of hypoxanthine transport under purine stress, the transport of adenosine increased 4-fold and the activity of the 3′-nucleotidase ectoenzyme also increased significantly. Under purine stress the concurrent increase of hypoxanthine and adenosine transport and the increase in 3′-nucleotidase activity could be repressed with either the supplementation of excess purines or by cycloheximide. This study of purine salvage mechanisms in Crithidia luciliae illustrates the successful adaptation of the parasite to nutritional insufficiency.