Search

Small isolated populations often show lower genetic variability. Demographic bottlenecks lead to loss of genetic variation too. Arctic foxes ( Vulpes lagopus) have been isolated since the Pleistocene on Mednyi and Bering Islands ( Commander Islands). In 1970-1980, the Mednyi population passed through a severe bottleneck due to a mange outbreak. Previous studies showed lack of genetic diversity in the contemporary Mednyi population that could be due to the long history of isolation and/or the recent bottleneck. To test both factors, we analyzed the mt DNA D-loop fragment and five microsatellites in pre-bottleneck Mednyi museum samples. Also, contemporary Mednyi, Bering and mainland Alaskan Arctic foxes were analyzed. Registered genetic variability in historical Mednyi was higher than in contemporary Mednyi Arctic foxes, but lower than in contemporary the Bering population. Our data confirms that the bottleneck reduced an already depleted polymorphism in Mednyi Arctic foxes. Lack of genetic variability could be a reason why the Mednyi population did not recover following the outbreak of mange. [ABSTRACT FROM AUTHOR]

The ISAG DLA Nomenclature Committee met during the "Comparative Evolution of the Mammalian MHC" meeting in Manchester, England on 10th September 2000. The main points discussed were the naming of class I genes and alleles, and the inclusion of alleles from other canidae.

Class-II histocompatibility genes are associated with predisposition to autoimmune diseases in many mammal species. We have developed a technique using reverse transcriptase and nested-PCR for amplification from blood samples of expressed sequences encoded by canine DLA-DRB1 loci. In the first polymerase chain reaction (PCR), we utilize primers DR-SP and DR-STOP as developed by Sarmiento et al. (1990). In the nested PCR, we utilize two additional primers, namely primer 57 [5'-TCTTGGAGGCTCCTGGATGACAGC-3'] and primer 367 [5'-CACAACTACGGGGTGATTGAGAGC-3'] to produce a 334 bp amplified product. After digestion with restriction endonucleases, some of the alleles can be identified by restriction fragment length polymorphism (RFLP). The increasing information on new DLA-DRB1 alleles over the last two years renders the DLA-DRB1 too diverse for convenient use of RFLP. However, the expressed sequences amplified by our protocol can be conveniently identified by cycle sequencing. This RT n-PCR protocol will suffice for the genotyping of individual dogs at the DLA-DRB1 locus.

The D-group proteins form the major component of the proteinaceous secretion of the tubular accessory glands of the yellow mealworm beetle, Tenebrio molitor. In a previous paper, we reported the sequence of two D-protein cDNAs and their inferred translation products. Both proteins contain three highly repetitive domains (A, A' and B). In this paper, we present the cDNA-inferred sequences of 8 more D-proteins, none of which contains an A' domain. We also present the structure of a D-protein gene. Southern analysis suggests that genes coding for an A' domain are relatively rare. Genes with a total of 7 or 8 (A + B domain) repeats seem most common.

B proteins represent one of the four major protein groups secreted by the tubular accessory glands of adult, male mealworm beetles. They are acidic proteins with an apparent molecular mass of 18.8 kDa. In this paper we present the deduced amino-acid sequences of two, almost identical B proteins, termed B1 and B2. The mature proteins are 118 amino acids long. They contain 11 (B2) or 12 (B1) possible phosphorylation sites and are rich in glutamic acid (16%). Lectin binding experiments indicate the presence of asparagine linked carbohydrate. The secondary structure of the B proteins is predicted to be almost completely alpha-helical. The B proteins show significant sequence resemblance to a group of pheromone- and odorant-binding proteins in moths and Drosophila, suggesting a role as carrier proteins for lipids.

C proteins represent one of the four major protein groups secreted by the tubular accessory glands of male mealworm beetles (Tenebrio molitor). They are basic proteins with an apparent molecular mass of 21.9 kDa. In this paper we present the deduced amino-acid sequence of two, almost identical C proteins, termed C1 and C2. The C proteins contain a consensus sequence for a heparin-binding site, and they are efficiently purified from accessory gland homogenates by heparin-affinity chromatography. No sequence resemblance was found with other proteins in the databases, but their high avidity for heparin suggests a possible involvement of the C proteins in sperm capacitation.

In this paper we present the amino acid sequence of Sp23, a structural protein of the spermatophore of the mealworm beetle (Tenebrio molitor). This is the first report of the primary structure of a spermatophorin. The protein is rich in proline (24%), relatively rich in tyrosine (9%) and glutamine (10%), and does not contain sulfur-containing amino acids. In the carboxyl-terminal half of the protein a peptide motif is repeated which is similar to a repetitive motif in a group of dipteran chorion proteins.

Monoclonal antibodies were produced against the secretory product of the bean-shaped accessory gland (BAG) of male mealworm beetles (Tenebrio molitor). Antibodies from one clone (PL 6.3) recognized a 9,600 dalton protein with a pI of 6.6 which was found in homogenates of the BAG. The PL 6.3 antigen was first detected on Western blots of BAG proteins from 2-day adults, and amounts increased for the next 6 days until reproductive maturation was achieved. The antibody also recognized a polypeptide with a molecular weight (mw) of about 5,000 daltons which we believe to be derived from the larger 9,600 dalton antigen. There are eight types of secretory cells in the BAG. By using light microscopic immunohistochemistry, we localized the antigens recognized by PL 6.3 in cell type 7 (intense staining) and cell type 5 (weak staining). Results from electron microscopic immunocytochemistry showed that antigen PL 6.3 was concentrated in the secretory granules characteristic of each of these two cell types and was absent in all other cell types. PL 6.3 antigens were traced from the BAG into its secretory product and then into the prespermatophoric mass in the ejaculatory duct. The antigen was not randomly mixed with other secretory products of the accessory glands. As it flowed from the BAG and into the ejaculatory duct, it remained in a coherent, precisely localized mass. Within the definitive spermatophore, the PL 6.3 antigen was concentrated in discrete layers of material that line the lumen.

During the 9-day pupal period of Tenebrio molitor (the mealworm beetle), the cells of the male accessory glands undergo divisions for 7 days. There are two maxima in the mitotic activity in the glands in vivo, one at 1 day and the other at 4 days. The latter peak coincides with the large surge of ecdysterone occurring in the pupal stage. By the use of in vitro culture techniques, it has been demonstrated that the first bout of mitosis in both glands proceeds in basal medium, while the second bout of mitosis requires a physiological level of ecdysterone. Ecdysone was less effective than ecdysterone. Sensitivity to ecdysterone did not change significantly between Day 1 and Day 4 of the pupal stage. The results are discussed in relation to the effects of ecdysterone on cell division in mesodermal and ectodermal derivatives.

The cells of the bean-shaped accessory glands of mealworms proliferate through the first 7 days of the 9-day pupal stage. Immediately after larval-pupal ecdysis, 25-27% of the cells were in the G1 phase, 60-65% were in the G2 phase, and the balance were in S phase. Over the first 4 days of normal development, the S fraction gradually increased, to reach its highest level in the mid-pupa at the time of the major ecdysteroid peak (Delbecque et al., 1978). Thereafter, the S fraction declined until over 95% of the cells had accumulated in G2 on Day 8. When 0-day pupal glands were explanted into Landureau's S-20 medium for 6 days, the G1 fraction remained fairly constant (25-30%) while S and the G2 fractions fluctuated. On the first day in vitro, the G2 fraction declined and the S fraction rose. On the second day in basal media, the S fraction fell and G2 rose correspondingly until 70% of the cells reached G2 when cycling stopped on the third day. With addition of 20-hydroxyecdysone to 0-day cultures, the S fraction increased quite sharply. It remained large for all 6 days of the experiment in the continuing presence of hormone. A 1-day pulse of hormone produced a transient increase in S. We blocked cell cycling with hydroxyurea in a stathmokinetic experiment and showed that 20-hydroxyecdysone accelerated the flow of cells from the G2 phase to the G1 phase by 2.5-fold. An increase in the G1 fraction was detected within 10 hr of hormone administration and the effect was dose-dependent with an ED50 of 5 X 10(-7) M for 20-hydroxyecdysone. We conclude that 20-hydroxyecdysone acts at a control point in the G2 phase. Incubation of the glands with 20-hydroxyecdysone for only 30-60 min followed by washout stimulated the flow from G2 to G1 and the effect persisted after transfer of the tissues to hormone-free media. Dose-dependent stimulation also occurred with ponasterone A (ED50 3 X 10(-9] but not with cholesterol.

The tubular accessory reproductive glands of the male mealworm beetle consist of a secretory epithelium surrounded by a thin muscular sheath. Each columnar secretory cell is divisible into three zones: basal which is adjacent to the muscle layer and contains rough endoplasmic reticulum and Golgi, intermediate, which contains endoplasmic reticulum and Golgi zones in the immature gland and is filled with secretory vesicles in the mature gland, and apical. Maturation also involves proliferation and organization of the rough endoplasmic reticulum in the basal and intermediate zone. The process appears to be complete at four days after ecdysis. Parallels with other insect glands and with the mammalian prostate are striking.

During the pupal stage of Tenebrio molitor, the accessory reproductive glands of males grow by cell division. Within the secretory epithelium of the bean-shaped accessory glands (BAGs), cell numbers triple. In the tubular accessory glands (TAGs), the increase is 14-fold. There are two mitotic maxima in each gland. The first maximum occurs at 1-2 days while the second is at 4-5 days. The second maximum coincides with the major ecdysteroid peak described by Delbecque et al. [Dev. Biol. 64, 11-30 (1978)]. Nuclei were isolated from TAGs during the pupal mitotic bouts and during mitotic inactivity in the adult. After Feulgen or propidium iodide staining, the DNA content of these nuclear populations was measured by absorption cytophotometry or by fluorescence flow cytometry, respectively. The proportion of cells in each phase of the cycle was calculated using an iterative model. After mitoses have ended in the late pupa, the cells were arrested in G2. [3H]Thymidine was injected into 1- and 4-day pupae to pulse-label cells of the TAGs. After allowing various periods from 4 to 60 hr for cells to progress through G2 to reach mitosis, fractions of labelled mitoses were determined by autoradiography. From the combined cytometric and autoradiographic data, the duration of each phase of the cell cycle was calculated assuming the population was in exponential growth. Cell cycles in 4-day pupal TAGs take 48 hr. G1, S, G2, and, M lasted 13, 14, 17, and 4 hr, respectively.

To establish indices for studying the hormonal control of differentiation of the accessory reproductive glands of insects, the ultrastructural development of the spermathecal accessory gland (SAG) of female mealworm beetles has been analyzed. Over the 9 days between adult and pupal ecdysis, the SAG transforms from a stubby sac of columnar epithelium into an elongate cylindrical gland, lined with cuticle, and containing several distinct types of differentiated cells. The first phase of pupal differentiation is one of cell division and overall gland morphogenesis which lasts 3--4 days; at its close, two populations of cells can be distinguished. One of these populations will produce the cuticular ductules while the other will yield the three-cell secretory units or organules. In the second phase which lasts 2 days, the three cells of each organule become wrapped around one another and then the innermost puts out a pseudocilium and retracts within the next ensheathing cell. In the third phase which lasts 4 days, the cuticles of the axial duct, of the efferent ductule, of the vestibule upon which the ductules converge, and of the end apparatus, are deposited. The ciliary process degenerates, and after ecdysis, the secretory cells undergo peak differentiation.

In Tenebrio molitor, male adults transfer sperm to the female via a spermatophore or sperm sac. The spermatophore is formed from secretions of the bean-shaped accessory glands (BAGs) and the tubular accessory glands (TAGs) of the male beetle. Trehalase is found in the adult BAGs. During the pupal stage, the activity in the BAGs was very low. After adult ecdysis, the total activity increased 100-fold from 0 days to 6 days and reached maximum levels at 9 days. The specific activity increased 20-fold from the time of ecdysis to 6 days thereafter. In the 10 day adult, trehalase levels in testes, seminal vesicles, vas deferens, TAGs, or ejaculatory ducts, were lower by two orders of magnitude than in the BAGs. However, the specific activity in the spermatophore was similar to that in the BAGs. Trehalases in the BAGs and the spermatophores showed very similar properties (soluble, optimum pH of 5.75 and Km value of 5.4 mM for trehalose). Thus trehalase appears to be secreted from the BAGs and becomes incorporated into the spermatophores.

Spermathecal accessory glands from pupae of Tenebrio molitor were cultured in vitro in Landureau S-20 medium with or without ecdysterone at a concentration of 5 micrograms/ml medium. Morphological changes were examined by electron microscopy. Tissue taken from pupae that have not been exposed to a peak of ecdysterone in vivo is only able to differentiate in medium with hormone, and then only partially, while tissue taken from pupae that have experienced an endogenous peak of ecdysterone is able to develop maximally irrespective of the presence or absence of hormones. The specific ultrastructural changes that occur in vitro correspond to those occurring in the gland in situ during the normal course of differentiation, and are: the formation of the pseudocilium, cell retraction and formation of an end apparatus, and cuticulogenesis in the ductule and main lumen of the gland. Pseudocilium formation does not appear to be ecdysterone-dependent, while cuticulogenesis requires ecdysterone for initiation of the process. Deposition of cuticle is an expression of an earlier commitment of the cells to this process, and is initiated by elevated levels of ecdysterone. Ecdysterone is not required for completion of cuticulogenesis.

The spermatheca of the female mealworm beetle is an inflorescence of branching cuticular ducts which is connected to the bursa copulatrix via a cuticular neck surrounded by a muscular coat. The infolded bursal cuticle consists of a distinct outer epicuticle, inner epicuticle, procuticle, and a subcuticular zone; the latter is rich in mucopolysaccharides. The cuticle of the neck lacks a distinct procuticle. The cuticle of the spermatheca itself is mostly inner epicuticle with two thin underlying lamellae of procuticle. The cells of the bursa are loosely coupled to the procuticle, whereas cuticular projections bind the epithelia of the "neck" and the spermatheca proper to the underlying epithelia. The apical plasma membranes of the spermathecal epithelium are sinuous and much infolded; we believe that this epithelium controls the micro-environment within the cuticular ducts.