A monoclonal antibody against a structural protein in the spermatophore of Tenebrio molitor (coleoptera)

Monoclonal antibodies were produced against insoluble proteins of spermatophores of Tenebrio molitor. One hybridoma clone produced antibody which recognized two antigens (29.4 and 27.6 kd mol. wt) in the bean-shaped accessory reproductive glands (BAGs) and in the secreted precursor of the insoluble fraction of the spermatophore. At least two molecular weight variants which differed by 1.5–1.7 kd mol. wt daltons are present. Processing accompanies secretion into the BAG lumen, with a reduction of about 4000 daltons in apparent molecular weights.The amount of target antigen during reproductive maturation, its localization and its transport were studied using Western blotting and immunohistochemistry. The monoclonal antibody recognized a protein present in one of the eight secretory cell types (type 3) of the BAG, in the secretory production of this gland, and in discrete layers of the spermatophore ejected from the male. This specific probe, and others currently being produced, will facilitate detailed studies on the process of spermatophore formation.     

A Chemoreceptor That Detects Molecular Carbon Dioxide

Animals from diverse phyla possess neurons that are activated by the product of aerobic respiration, CO₂. It has long been thought that such neurons primarily detect the CO₂ metabolites protons and bicarbonate. We have determined the chemical tuning of isolated CO₂ chemosensory BAG neurons of the nematode Caenorhabditis elegans. We show that BAG neurons are principally tuned to detect molecular CO₂, although they can be activated by acid stimuli. One component of the BAG transduction pathway, the receptor-type guanylate cyclase GCY-9, suffices to confer cellular sensitivity to both molecular CO₂ and acid, indicating that it is a bifunctional chemoreceptor. We speculate that in other animals, receptors similarly capable of detecting molecular CO₂ might mediate effects of CO₂ on neural circuits and behavior.     

Aspartyl Protease-Mediated Cleavage of BAG6 Is Necessary for Autophagy and Fungal Resistance in Plants

The Bcl-2-associated athanogene (BAG) family is an evolutionarily conserved group of cochaperones that modulate numerous cellular processes. Previously we found that Arabidopsis thaliana BAG6 is required for basal immunity against the fungal phytopathogen Botrytis cinerea. However, the mechanisms by which BAG6 controls immunity are obscure. Here, we address this important question by determining the molecular mechanisms responsible for BAG6-mediated basal resistance. We show that Arabidopsis BAG6 is cleaved in vivo in a caspase-1-like-dependent manner and via a combination of pull-downs, mass spectrometry, yeast two-hybrid assays, and chemical genomics, we demonstrate that BAG6 interacts with a C2 GRAM domain protein (BAGP1) and an aspartyl protease (APCB1), both of which are required for BAG6 processing. Furthermore, fluorescence and transmission electron microscopy established that BAG6 cleavage triggers autophagy in the host that coincides with disease resistance. Targeted inactivation of BAGP1 or APCB1 results in the blocking of BAG6 processing and loss of resistance. Mutation of the cleavage site blocks cleavage and inhibits autophagy in plants; disease resistance is also compromised. Taken together, these results identify a mechanism that couples an aspartyl protease with a molecular cochaperone to trigger autophagy and plant defense, providing a key link between fungal recognition and the induction of cell death and resistance.     

Solution Structure of the SGTA Dimerisation Domain and Investigation of Its Interactions with the Ubiquitin-Like Domains of BAG6 and UBL4A

Background

The BAG6 complex resides in the cytosol and acts as a sorting point to target diverse hydrophobic protein substrates along their appropriate paths, including proteasomal degradation and ER membrane insertion. Composed of a trimeric complex of BAG6, TRC35 and UBL4A, the BAG6 complex is closely associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates.

Methodology and Principal Findings

SGTA consists of an N-terminal dimerisation domain (SGTA_NT), a central tetratricopeptide repeat (TPR) domain, and a glutamine rich region towards the C-terminus. Here we solve a solution structure of the SGTA dimerisation domain and use biophysical techniques to investigate its interaction with two different UBL domains from the BAG6 complex. The SGTA_NT structure is a dimer with a tight hydrophobic interface connecting two sets of four alpha helices. Using a combination of NMR chemical shift perturbation, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) experiments we have biochemically characterised the interactions of SGTA with components of the BAG6 complex, the ubiquitin-like domain (UBL) containing proteins UBL4A and BAG6. We demonstrate that the UBL domains from UBL4A and BAG6 directly compete for binding to SGTA at the same site. Using a combination of structural and interaction data we have implemented the HADDOCK protein-protein interaction docking tool to generate models of the SGTA-UBL complexes.

Significance

This atomic level information contributes to our understanding of the way in which hydrophobic proteins have their fate decided by the collaboration between SGTA and the BAG6 complex.     

Repeated visitations of spermatophores and polyandry in females of eriophyoid mites

Eriophyoid females store sperm either asymmetrically in one spermatheca, or symmetrically in both spermathecae. Previous studies have suggested that species in which females store sperm asymmetrically pick up sperm from only one spermatophore, while those with symmetrical sperm storage pick up sperm from two or more spermatophores during their lifetime. The aim of this study was to examine spermatophore visitation behaviour and symmetry of sperm storage in Aculops allotrichus from the black locust tree and Cecidophyopsis hendersoni from the yucca. This would indicate monandry or polyandry in these species. In both eriophyoids, the spermatophore visitation consisted of three phases: mounting, lying on the spermatophore and dismounting. Aculops allotrichus stored sperm asymmetrically. However, nearly one-third of the observed females visited two spermatophores, rather than only one in their lives. When A. allotrichus females visited two spermatophores they spent a similar amount of time at the first and at the second visitation. Also, the times of visitation of the first of the two spermatophores and the single spermatophore in a female lifetime did not differ significantly. This would suggest that apart from monandry, double insemination also occurs in this species. By contrast, C. hendersoni females were polyandrous. They stored sperm symmetrically and visited several spermatophores, on average 1.54 (max 6) per day, and up to 33 spermatophores in their lives. The benefits of repeated spermatophore visitation and the possible mechanisms of sperm storage in both species are discussed.     

d-Isoascorbic acid fed to Spodoptera littoralis moths,induces sterility due to spermatophore malformation

d-Isoascorbic acid (d-araboascorbic acid; d-erythorbic acid) fed to Spodoptera littoralis (Boisduval) moths causes males to produce malformed spermatophores with no reduction in their mating capacity. Males fed daily on the analogue, induced sterility in females paired with them. Female fertility and fecundity were markedly reduced compared with the reproductive potential of females mated to males fed either l-ascorbic acid or sucrose only. Spermatophore pathology originated in failure to inflate the spermatophore corpus at the time of mating. This failure was produced by a single male in two females independently, when males only had access to the analogue. Thus, the effect on the spermatophore seems linked to male ingestion of the analogue. When d-isoascorbic acid-feeding of males was discontinued after 48 hr, egg fertility was restored. When l-ascorbic acid followed the analogue, the effect of spermatophore malformation was not completely erased. The mode of action of this ascorbic acid analogue is discussed.     

On the occurrence of acid mucopolysaccharides in the spermatophores of two marine prawns,Penaeus indiens (Milne-Edwards) and Metapenaeus monoceros (Fabricius) (Crustacea: Macrura)

In marine prawns, spermatophores aid in sperm transfer. The prawn spermatophores, in general, consist of two parts, sperm sac and wing. The former encloses spermatozoa and the latter serves as an adhesive pad during transfer to the female's thelycum. This paper reports on the occurrence of acid mucopolysaccharides (AMPS) in the spermatophores of two penaeid prawns, Penaeus indicus (Milne-Edwards) and Metapenaeus monoceros (Fabricius). They, being the principal component of the spermatophores, have been characterized and quantified in the wing, sperm mass, sperm sac, and crystalline structures. In both prawns, the spermatophore is a simple structure. In P. indicus, it consists of sperm mass, enclosed within sperm sac, to which is attached the foliacious wing. In M. monoceros, the freshly extruded spermatophore consists of sperm mass and milky-white crystalline structures. Isolation and purification of AMPS resulted in the resolution of two toluidine blue-positive AMPS fractions in agarose-gel electrophoresis. Densitometric study of the standard and sample AMPS fractions reveals further that the two fractions correspond to chondroitin sulfate and hyaluronic acid. Quantitative assay on total AMPS shows that the sperm sac of P. indicus contains the maximum amount of AMPS (195.50 μg/mg), whereas its wing contains the least quantity of AMPS (43.68 μg/mg). The qualitative and quantitative variations found in the AMPS content of spermatophoric components of two prawns have been discussed in relation to their adhesion to thelycum as well as sperm storage and maintenance pending fertilization.     

The Association of BAG6 with SGTA and Tail-Anchored Proteins

Background

The BAG6 protein is a subunit of a heterotrimeric complex that binds a range of membrane and secretory protein precursors localized to the cytosol, enforcing quality control and influencing their subsequent fate.

Methodology and Principal Findings

BAG6 has an N-terminal ubiquitin-like domain, and a C-terminal Bcl-2-associated athanogene domain, separated by a large central proline-rich region. We have used in vitro binding approaches to identify regions of BAG6 important for its interactions with: i) the small-glutamine rich tetratricopeptide repeat-containing protein alpha (SGTA) and ii) two model tail-anchored membrane proteins as a paradigm for its hydrophobic substrates. We show that the BAG6-UBL is essential for binding to SGTA, and find that the UBL of a second subunit of the BAG6-complex, ubiquitin-like protein 4A (UBL4A), competes for SGTA binding. Our data show that this binding is selective, and suggest that SGTA can bind either BAG6, or UBL4A, but not both at the same time. We adapted our in vitro binding assay to study the association of BAG6 with an immobilized tail-anchored protein, Sec61β, and find both the UBL and BAG domains are dispensable for binding this substrate. This conclusion was further supported using a heterologous subcellular localization assay in yeast, where the BAG6-dependent nuclear relocalization of a second tail-anchored protein, GFP-Sed5, also required neither the UBL, nor the BAG domain of BAG6.

Significance

On the basis of these findings, we propose a working model where the large central region of the BAG6 protein provides a binding site for a diverse group of substrates, many of which expose a hydrophobic stretch of polypeptide. This arrangement would enable the BAG6 complex to bring together its substrates with potential effectors including those recruited via its N-terminal UBL. Such effectors may include SGTA, and the resulting assemblies influence the subsequent fate of the hydrophobic BAG6 substrates.     

Circadian control of spermatophore formation in the cricket Teleogryllus commodus Walker

The cricket Teleogryllus commodus has a circadian rhythm in spermatophore formation. A spermatophore is present 1–5 hr before the onset of stridulation under LL, DD and LD 12 : 12; it is retained during the whole stridulatory phase and disposed of within a variable time after termination of singing. Overwhelmingly one spermatophore is produced during a 24 hr-period. Severance of the abdominal nerve cord or removal of the accessory glands prevent spermatophore formation, but does not inhibit periodical stridulation; when a spermatophore is removed from a singing male, stridulation continues after a short time. Coagulation of the pars intercerebralis stops spermatophore production and singing; severance of the optic lobes rends both processes arhythmical. The rôle of the pars intercerebralis as an intermediary link between the timing-device and the effector organs is discussed.     

Role of arginase transferred from the vesicula seminalis during mating and changes in amino acid pools of the spermatophore after ejaculation in the silkworm,Bombyx mori

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• 1.1. Ejaculation of seminal fluid into the spermatophore formed inside the female bursa copulatrix terminates 20 min after the beginning of copulation of Bombyx mori. The amounts of amino acids transferred are small, but the amounts of amino acids in the spermatophore continue to increase after this time due to protein degradation and amino acid interconversions.
• 2.2. Arginase, which has high activity in the vesicula seminalis, is transferred to the spermatophore without loss of activity during ejaculation. During mating, ornithine and much urea are formed in the spermatophore, indicating activity of the transferred arginase.
• 3.3. In the spermatophore, marked increase of alanine with low concentrations of ornithine and glutamate suggests the presence of a pathway for the active formation of 2-oxoglutarate with pyruvate via glutamate from arginine. During mating, proline and glutamine also increase, but at low rates.
• 4.4. Of the exocrine glands in the male reproductive system, the vesicula seminalis secretes the highest concentration of glutamate (30% of the total amino acids); serine is the amino acid present at highest concentration in secretions of other glands (20–30%). No urea was found in the secretions of any of the glands.

     

Infection increases the value of nuptial gifts, and hence male reproductive success, in the Hymenolepis diminuta-Tenebrio molitor association

During copulation, male insects pass accessory gland components to the female with the spermatophore. These gifts can affect female reproductive behaviour, ovulation and oviposition. Here, we show that female mealworm beetles, Tenebrio molitor, mated with males infected with metacestodes of the rat tapeworm, Hymenolepis diminuta, produced significantly more offspring than those mated with uninfected males. There is a significant positive relationship between parasite intensity in the male and reproductive output in the female. Infection results in a significant increase in bean-shaped accessory gland (BAG) size. We suggest that infected males pass superior nuptial gifts to females and discuss the confounding effects of infection in male and female beetles upon overall fitness costs of infection for the host and the likelihood that the parasite is manipulating host investment in reproduction.     

Ordered flow of secretion from accessory glands to specific layers of the spermatophore of mealworm beetles: demonstration with a monoclonal antibody

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.     

When do the Costs of Spermatogenesis Constrain Sperm Expenditure? Remarks on the Pattern of the Spermatogenic Cycle

The costs of spermatogenesis constrain sperm expenditure when sperm production per day is limited. Thus, males are challenged to allocate available resources to sperm production and other life history functions. However, this prevailing assumption is not applicable to species in which spermatogenesis becomes quiescent during the breeding season. Males of these species prepare large quantities of sperm before the breeding season. Among these species, constraints on ejaculates have been intensively investigated in salamanders that deposit spermatophores. Although it is predicted that sperm expenditure should not be limited because of abundantly prepared sperm, spermatophore deposition is often limited during the breeding season when vas deferens are full of sperm. We tested a hypothesis regarding limited spermatophore deposition by measuring sperm quantity and volume of spermatophores sequentially deposited by male eastern newts Notophthalmus viridescens. A male newt rarely deposits more than three spermatophores per mating. If depletion of non‐sperm components of spermatophores limits spermatophore deposition, we predicted that spermatophore volume decreases while sperm quantity remains constant as a male deposits more spermatophores. Alternatively, some regulative mechanisms allow a limited portion of available sperm to be expended per mating, in which sperm quantity is predicted to decrease while the spermatophore volume remains constant. Finally, depletion of non‐sperm components may regulate sperm expenditure, which predicted that both spermatophore volume and sperm quantity decrease. We found that both sperm quantity and the spermatophore volume decreased as a male deposited more spermatophores during a single mating. Sperm expenditure was constrained without the costs involved in active spermatogenesis, and depletion of non‐sperm components likely regulate sperm quantity loaded in spermatophores. In dissociated spermatogenesis, constrained sperm expenditure do not mean that costly spermatogenesis is directly limiting male mating capacity but rather suggest that the evolution of physiological mechanisms regulating sperm expenditure per mating maximizes male reproductive success.     

The effect of the presence of quiescent female nymphs, males and their spermatophores on spermatophore placement in two species of eriophyoid mites

Under sex dissociated sperm transfer, females seek spermatophores and pick up sperm without male assistance. In several species males adjust spermatophore deposition rate to the presence of conspecifics. It is not known, however, which factors could favor such elasticity in non-pairing males. In this paper, we compare male response towards conspecifics between the sex dissociated eriophyoid mites Aculus fockeui (Nalepa and Trouessart) and Aculops allotrichus (Nalepa). The species differ significantly in male reproductive strategies and, consequently, the intensity of male–male-competition. Aculus fockeui males deposit spematophores all over the leaves and occasionally leave single spermatophores beside quiescent female nymphs (QFNs). In contrast, A. allotrichus males guard QFNs and encircle them with spermatophores. In this study, males of both species deposited spermatophores close to and apart from the rival spermatophores. Aculops allotrichus males had similar spermatophore output whether they were kept alone or in a group of seven males. They did not change spermatophore output in the presence of five rival spermatophores, a QFN or a QFN and varying number of rivals, either. In contrast, A. fockeui males increased spermatophore output in the presence of rival spermatophores or when on the arena with a QFN the male number increased to eight males. They did not respond, however, to the presence of a QFN and one rival or a QFN alone. The possible effect of the species-specific intensity of male–male competition, population density, the availability of receptive females and the rate of spermatophore output on the flexibility of eriophyoid spermatophore deposition is discussed.     

Peculiar features of the sperm of a calanoid copepod Arctodiaptomus salinus

The spermatophore and spermatozoon of the calanoid copepod A. salinus, were investigated by light and electron microscopies. Mature spermatozoa were found within the spermatophore in the seminal vesicle at the end of the deferent duct. The cells measured 2–3 μm in diameter and were almost spherical; the nucleus was mixed with the cytoplasm as no envelope was present to separate them. They lack flagellum and any acrosome-like structure. The dispersed chromatin forms small granules surrounding the mitochondria, characterised by a small number of highly modified cristae. Under the plasmatic membrane, a group of electron-dense lamina enclose the whole cytoplasm. In the final phase of spermiogenesis, the dismantling of the nuclear envelope, the formation of chromatin granules and the modification of mitochondria occur. This process takes place simultaneously with the synthesis of the components of the spermatophore by the epithelial cells of the deferent duct. All these characteristics of the sperm of A. salinus identify it as being a peculiar case among copepoda.     

BAG3 directly associates with guanine nucleotide exchange factor of Rap1, PDZGEF2, and regulates cell adhesion

BAG3, a member of the Hsc70 binding co-chaperone BAG-family proteins, has critical roles in regulating actin organization, cell adhesion, cell motility and tumor metastasis. The PDZ domain containing guanine nucleotide exchange factor 2 (PDZGEF2) was cloned as a BAG3-interacting protein. PDZGEF2 induces activation of Rap1 and increases integrin-mediated cell adhesion. The PPDY motif at the C-terminus of PDZGEF2 binds to the WW domain of BAG3 in vitro and in vivo. BAG3 deletion mutant lacking the WW domain lose its cell adhesion and motility activity. Gene knockdown of PDZGEF2 leads to the loss of cell adhesion on fibronectin-coated plates while BAG3 overexpression increases cell adhesion in Cos7 cells, but not in PDZGEF2 gene knockdown cells indicating that PDZGEF2 is a critical partner for BAG3 in regulating cell adhesion.     

Gene and protein expression of Drosophila Starvin during cold stress and recovery from chill coma

In Drosophila melanogaster, the sole member of the Bcl-2-associated anthanogene (BAG)-family proteins, called Starvin (Stv), has only been recently described. BAG proteins regulate a large range of physiological processes including life/death cell balance and stress response. The role of Stv has been poorly studied in the context of abiotic stress and particularly during and after cold stress. In this study we investigated the temporal expression of Stv gene and protein in adult flies during both the cold stress (up to 9 h at 0 °C) and the subsequent recovery phase (up to 8 h at 25 °C). Because BAG proteins can regulate positively and negatively the function of Hsp70/Hsc70, we also checked whether Stv expression was related to Hsp70 and Hsc70. Stv mRNA and Stv protein both showed a similar expression pattern: no modulation during the cold period followed by a significant up-regulation during the recovery period. A coordinated response of Stv and Hsp70 mRNA was observed, but not for Hsc70. Our findings indicate that Stv expression is part of a stress-induced program in D. melanogaster. It probably acts as a co-chaperone modulating the activity of Hsp70 chaperone machinery during recovery from cold stress. Finally our results support the suggestion that Stv and human BAG3 may be functional homologs.     

Arachidonic and other long-chain polyunsaturated fatty acids in spermatophores and spermathecae of Teleogryllus commodus: Significance in prostaglandin-mediated reproductive behaviour

Estimates of weights of fatty acids, especially arachidonic acid, in spermathecae from virgin and mated T. commodus indicate substantial elevation in all fatty acids and particularly arachidonic acid following mating. Analysis of spermatophore lipids suggests that this can be in part accounted for by the contents of one or several spermatophores. Fractionation of total lipids from spermatophores showed that arachidonic acid comprised 24% of phosphotidylcholine and 4% of phosphotidylethanolamine, but was not detected in neutral lipids whereas it was approximately equally distributed over phosphotidylcholine and phosphotidylethanolamine in lipids from spermathecae. These data indicate that in addition to prostaglandin synthetase, the spermatophore contains a physiologically significant quantity of prostaglandin precursor, arachidonic acid, esterified to phospholipid and presumably unavailable for enzymatic action during mating transfer. We also note that proportions of arachidonic acid in the phosphotidylcholine of spermatophores are the highest recorded for this fatty acid in the insect literature, which in conjunction with recent work emphasizes the likely physiological significance of long-chain polyunsaturated fatty acids in insects generally.     

Morphological and morphometric appraisal of the spermatophore of the southern hermit crab Isocheles sawayai (Anomura: Diogenidae), with comments on gonopores in both sexes

The spermatophore morphology of the hermit crab Isocheles sawayai from southwestern Atlantic (Brazil) is described. The spermatophores show similarities with those described for other members of the family Diogenidae, especially with the recently described Loxopagurus loxochelis. The spermatophore is composed of three major regions: a sperm filled head or ampulla, a columnar stalk and a foot or pedestal. The spermatophores show specific morphology in having a circular ampulla, and a constriction or neck between the ampulla (100 μm) and the thin (27 μm), long stalk (500 μm). The stalk penetrates less than half way into the spermatophore head. Most spermatophores show one of the small posterior projections on the underside of the ampulla as being bigger than the other, making it asymmetrical. The size of the spermatophore is related to hermit crab size with direct relationships found between spermatophore ampulla width, total length, and peduncle length with shield length of the hermit crab. The morphological characteristics of the spermatophore of I. sawayai are species-specific distinguishing it from other members of the family, and are useful to infer further phylogenetic relationships.