Receptor-mediated yolk uptake is required for oskar mRNA localization and cortical anchorage of germ plasm components in the Drosophila oocyte.

The Drosophila germ plasm is responsible for germ cell formation. Its assembly begins with localization of oskar mRNA to the posterior pole of the oocyte. The oskar translation produces 2 isoforms with distinct functions: short Oskar recruits germ plasm components, whereas long Oskar remodels actin to anchor the components to the cortex. The mechanism by which long Oskar anchors them remains elusive. Here, we report that Yolkless, which facilitates uptake of nutrient yolk proteins into the oocyte, is a key cofactor for long Oskar. Loss of Yolkless or depletion of yolk proteins disrupts the microtubule alignment and oskar mRNA localization at the posterior pole of the oocyte, whereas microtubule-dependent localization of bicoid mRNA to the anterior and gurken mRNA to the anterior-dorsal corner remains intact. Furthermore, these mutant oocytes do not properly respond to long Oskar, causing defects in the actin remodeling and germ plasm anchoring. Thus, the yolk uptake is not merely the process for nutrient incorporation, but also crucial for oskar mRNA localization and cortical anchorage of germ plasm components in the oocyte.

Distinct vitellogenin domains differentially regulate immunological outcomes in invertebrates.

The classical role of Vitellogenin (Vg) is providing energy reserves for developing embryos, but its roles appear to extend beyond this nutritional function, and its importance in host immune defense is garnering increasing research attention. However, Vg-regulated immunological functions are dependent on three different domains within different species and remain poorly understood. In the present study, we confirmed three conserved VG domains-LPD_N, DUF1943, and VWD-in the Chinese mitten crab (Eriocheir sinensis), highlighting functional similarities of Vg in vertebrates and invertebrates. Of these three domains, DUF1943 and VWD showed definitive bacterial binding activity via interaction with the signature components on microbial surfaces, but this activity was not exhibited by the LPD_N domain. Antibacterial assays indicated that only the VWD domain inhibits bacterial proliferation, and this function may be conserved between different species due to the conserved amino acid residues. To further explore the relationship between Vg and polymeric immunoglobulin receptor (pIgR), we expressed EspIgR and the three E. sinensis Vg (EsVg) domains in HEK293T cells, and coimmunoprecipitation assay demonstrated that only the DUF1943 domain interacts with EspIgR. Subsequent experiments demonstrated that EsVg regulates hemocyte phagocytosis by binding with EspIgR through the DUF1943 domain, thus promoting bacterial clearance and protecting the host from bacterial infection. To the best of our knowledge, our work is the first to report distinct domains in Vg inducing different immunological outcomes in invertebrates, providing new evidence that pIgR acts as a phagocytic receptor for Vg.

Vitellogenin-like A-associated shifts in social cue responsiveness regulate behavioral task specialization in an ant.

Division of labor and task specialization explain the success of human and insect societies. Social insect colonies are characterized by division of labor, with workers specializing in brood care early and foraging later in life. Theory posits that this task switching requires shifts in responsiveness to task-related cues, yet experimental evidence is weak. Here, we show that a Vitellogenin (Vg) ortholog identified in an RNAseq study on the ant T. longispinosus is involved in this process: using phylogenetic analyses of Vg and Vg-like genes, we firstly show that this candidate gene does not cluster with the intensively studied honey bee Vg but falls into a separate Vg-like A cluster. Secondly, an experimental knockdown of Vg-like A in the fat body caused a reduction in brood care and an increase in nestmate care in young ant workers. Nestmate care is normally exhibited by older workers. We demonstrate experimentally that this task switch is at least partly based on Vg-like A-associated shifts in responsiveness from brood to worker cues. We thus reveal a novel mechanism leading to early behavioral maturation via changes in social cue responsiveness mediated by Vg-like A and associated pathways, which proximately play a role in regulating division of labor.

Insect tissue-specific vitellogenin facilitates transmission of plant virus.

Insect vitellogenin (Vg) has been considered to be synthesized in the fat body. Here, we found that abundant Vg protein is synthesized in Laodelphax striatellus hemocytes as well. We also determined that only the hemocyte-produced Vg binds to Rice stripe virus (RSV) in vivo. Examination of the subunit composition of L. striatellus Vg (LsVg) revealed that LsVg was processed differently after its expression in different tissues. The LsVg subunit able to bind to RSV exist stably only in hemocytes, while fat body-produced LsVg lacks the RSV-interacting subunit. Nymph and male L. striatellus individuals also synthesize Vg but only in hemocytes, and the proteins co-localize with RSV. We observed that knockdown of LsVg transcripts by RNA interference decreased the RSV titer in the hemolymph, and thus interfered with systemic virus infection. Our results reveal the sex-independent expression and tissue-specific processing of LsVg and also unprecedentedly connect the function of this protein in mediating virus transmission to its particular molecular forms existing in tissues previously known as non-Vg producing.

Warmed Winter Water Temperatures Alter Reproduction in Two Fish Species.

We examined the spawning success of Fathead Minnows (Pimephales promelas) and Johnny Darters (Etheostoma nigrum) exposed to elevated winter water temperatures typical of streams characterized by anthropogenic thermal inputs. When Fathead Minnows were exposed to temperature treatments of 12, 16, or 20 °C during the winter, spawning occurred at 16 and 20 °C but not 12 °C. Eggs were deposited over 9 weeks before winter spawning ceased. Fathead Minnows from the three winter temperature treatments were then exposed to a simulated spring transition. Spawning occurred at all three temperature treatments during the spring, but fish from the 16° and 20 °C treatment had delayed egg production indicating a latent effect of warm winter temperatures on spring spawning. mRNA analysis of the egg yolk protein vitellogenin showed elevated expression in female Fathead Minnows at 16 and 20 °C during winter spawning that decreased after winter spawning ceased, whereas Fathead Minnows at 12 °C maintained comparatively low expression during winter. Johnny Darters were exposed to 4 °C to represent winter temperatures in the absence of thermal inputs, and 12, 16, and 20 °C to represent varying degrees of winter thermal pollution. Johnny Darters spawned during winter at 12, 16, and 20 °C but not at 4 °C. Johnny Darters at 4 °C subsequently spawned following a simulated spring period while those at 12, 16, and 20 °C did not. Our results indicate elevated winter water temperatures common in effluent-dominated streams can promote out-of-season spawning and that vitellogenin expression is a useful indicator of spawning readiness for fish exposed to elevated winter temperatures.

Tissue distribution and functional analysis of vitellogenin-6 of Toxocara canis.

Toxocara canis is an common intestinal nematode of canids and the principal causative agent of human toxocariasis. Vitellogenin (Vg), a source of amino acids and lipids in the eggs, are considered to play an important role in embryo development of a wide range of organisms. In the present study, the transcriptional levels of Tc-vit-6 gene in male and female adult T. canis were determined by quantitative real-time PCR, which indicated high transcription of Tc-vit-6 in the intestine, reproductive tract and body wall of male and female adult T. canis. The fragment of Tc-vit-6 encoding a vWD domain, was cloned and expressed to produce a rabbit anti-TcvWD polyclonal antibody. Tissue distribution of TcVg6 was detected by immunohistochemical assays, which showed predominant distribution of TcVg6 in the tissues of intestine, as well as reproductive tract (including some of the germ cells) and musculature of male and female adult worms. Collectively, these results indicated multiple biological roles of TcVg6 apart from that in the reproduction of T. canis.

Immune-Relevant and Antioxidant Activities of Vitellogenin and Yolk Proteins in Fish.

Vitellogenin (Vtg), the major egg yolk precursor protein, is traditionally thought to provide protein- and lipid-rich nutrients for developing embryos and larvae. However, the roles of Vtg as well as its derived yolk proteins lipovitellin (Lv) and phosvitin (Pv) extend beyond nutritional functions. Accumulating data have demonstrated that Vtg, Lv and Pv participate in host innate immune defense with multifaceted functions. They can all act as multivalent pattern recognition receptors capable of identifying invading microbes. Vtg and Pv can also act as immune effectors capable of killing bacteria and virus. Moreover, Vtg and Lv are shown to possess phagocytosis-promoting activity as opsonins. In addition to these immune-relevant functions, Vtg and Pv are found to have antioxidant activity, which is able to protect the host from oxidant stress. These non-nutritional functions clearly deepen our understanding of the physiological roles of the molecules, and at the same time, provide a sound basis for potential application of the molecules in human health.

Vitellogenin in the honey bee brain: Atypical localization of a reproductive protein that promotes longevity.

In comparative gerontology, highly social insects such as honey bees (Apis mellifera) receive much attention due to very different and flexible aging patterns among closely related siblings. While experimental strategies that manipulate socio-environmental factors suggest a causative link between aging and social signals and behaviors, the molecular underpinnings of this linkage are less well understood. Here we study the atypical localization of the egg-yolk protein vitellogenin (Vg) in the brain of the honey bee. Vg is known to influence honey bee social regulation and aging rate. Our findings suggest that Vg immunoreactivity in the brain is specifically localized within the class of non-neuronal glial cells. We discuss how these results can help explain the socially-dependent aging rate of honey bees.

Genetic architecture of a hormonal response to gene knockdown in honey bees.

Variation in endocrine signaling is proposed to underlie the evolution and regulation of social life histories, but the genetic architecture of endocrine signaling is still poorly understood. An excellent example of a hormonally influenced set of social traits is found in the honey bee (Apis mellifera): a dynamic and mutually suppressive relationship between juvenile hormone (JH) and the yolk precursor protein vitellogenin (Vg) regulates behavioral maturation and foraging of workers. Several other traits cosegregate with these behavioral phenotypes, comprising the pollen hoarding syndrome (PHS) one of the best-described animal behavioral syndromes. Genotype differences in responsiveness of JH to Vg are a potential mechanistic basis for the PHS. Here, we reduced Vg expression via RNA interference in progeny from a backcross between 2 selected lines of honey bees that differ in JH responsiveness to Vg reduction and measured JH response and ovary size, which represents another key aspect of the PHS. Genetic mapping based on restriction site-associated DNA tag sequencing identified suggestive quantitative trait loci (QTL) for ovary size and JH responsiveness. We confirmed genetic effects on both traits near many QTL that had been identified previously for their effect on various PHS traits. Thus, our results support a role for endocrine control of complex traits at a genetic level. Furthermore, this first example of a genetic map of a hormonal response to gene knockdown in a social insect helps to refine the genetic understanding of complex behaviors and the physiology that may underlie behavioral control in general.

Genotype effect on lifespan following vitellogenin knockdown.

Honey bee workers display remarkable flexibility in the aging process. This plasticity is closely tied to behavioral maturation. Workers who initiate foraging behavior at earlier ages have shorter lifespans, and much of the variation in total lifespan can be explained by differences in pre-foraging lifespan. Vitellogenin (Vg), a yolk precursor protein, influences worker lifespan both as a regulator of behavioral maturation and through anti-oxidant and immune functions. Experimental reduction of Vg mRNA, and thus Vg protein levels, in wild-type bees results in precocious foraging behavior, decreased lifespan, and increased susceptibility to oxidative damage. We sought to separate the effects of Vg on lifespan due to behavioral maturation from those due to immune and antioxidant function using two selected strains of honey bees that differ in their phenotypic responsiveness to Vg gene knockdown. Surprisingly, we found that lifespans lengthen in the strain described as behaviorally and hormonally insensitive to Vg reduction. We then performed targeted gene expression analyses on genes hypothesized to mediate aging and lifespan: the insulin-like peptides (Ilp1 and 2) and manganese superoxide dismutase (mnSOD). The two honey bee Ilps are the most upstream components in the insulin-signaling pathway, which influences lifespan in Drosophila melanogaster and other organisms, while manganese superoxide dismutase encodes an enzyme with antioxidant functions in animals. We found expression differences in the llps in fat body related to behavior (llp1 and 2) and genetic background (Ilp2), but did not find strain by treatment effects. Expression of mnSOD was also affected by behavior and genetic background. Additionally, we observed a differential response to Vg knockdown in fat body expression of mnSOD, suggesting that antioxidant pathways may partially explain the strain-specific lifespan responses to Vg knockdown.

Proportional accumulation of yolk proteins derived from multiple vitellogenins is precisely regulated during vitellogenesis in striped bass (Morone saxatilis).

We quantified three vitellogenins (VtgAa, VtgAb, VtgC) or their derived yolk proteins (YPs) in the liver, plasma, and ovary during pre-vitellogenic (PreVG), mid-vitellogenic (MVG), and late-vitellogenic (LVG) oocyte growth and during post-vitellogenesis (PostVG) in the striped bass (Morone saxatilis) using label-free quantitative mass spectrometry (MS). Western blotting of the samples using antisera raised against gray mullet (Mugil cephalus) lipovitellins derived from VtgAa, VtgAb, and VtgC confirmed the MS results. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed liver as the primary site of expression for all three Vtgs, with extra-hepatic transcription weakly detected in ovary, foregut, adipose tissue, and brain. Quantitative real-time RT-PCR confirmed vtgAb to be primarily expressed in liver and VtgAb proteins were predominant in liver and plasma from MVG to PostVG. However, the primary period of deposition into oocytes of VtgAb occurred up until MVG, whereas VtgAa was primarily deposited from MVG to LVG. The VtgC was gradually taken up by oocytes throughout vitellogenesis and was detected at trace levels in plasma. The ratio of yolk proteins derived from VtgAa, VtgAb, VtgC (YPAa/YPAb/YPC) in PostVG ovary is 1.4:1.4:1, which differs from ratios previously reported for other fish species in that YPC comprises a greater proportion of the egg yolk. Our results indicate that proportional accumulation of multiple Vtgs in the yolk may depend both on the precise rates of their hepatic secretion and specific uptake by oocytes. Furthermore, composition of the Vtg-derived yolk may vary among Acanthomorph fishes, perhaps reflecting their different early life histories and reproductive strategies.

Interplay between insulin signaling, juvenile hormone, and vitellogenin regulates maternal effects on polyphenism in ants.

Polyphenism is the phenomenon in which alternative phenotypes are produced by a single genotype in response to environmental cues. An extreme case is found in social insects, in which reproductive queens and sterile workers that greatly differ in morphology and behavior can arise from a single genotype. Experimental evidence for maternal effects on caste determination, the differential larval development toward the queen or worker caste, was recently documented in Pogonomyrmex seed harvester ants, in which only colonies with a hibernated queen produce new queens. However, the proximate mechanisms behind these intergenerational effects have remained elusive. We used a combination of artificial hibernation, hormonal treatments, gene expression analyses, hormone measurements, and vitellogenin quantification to investigate how the combined effect of environmental cues and hormonal signaling affects the process of caste determination in Pogonomyrmex rugosus. The results show that the interplay between insulin signaling, juvenile hormone, and vitellogenin regulates maternal effects on the production of alternative phenotypes and set vitellogenin as a likely key player in the intergenerational transmission of information. This study reveals how hibernation triggers the production of new queens in Pogonomyrmex ant colonies. More generally, it provides important information on maternal effects by showing how environmental cues experienced by one generation can translate into phenotypic variation in the next generation.

Effects of partial or total fish meal replacement by agricultural by-product diets on gonad maturation, sex steroids and vitellogenin dynamics of African catfish (Clarias gariepinus).

The establishment of the first sexual maturation was characterized in African catfish (Clarias gariepinus) in order to study the efficiency of replacement of fish meal (FM) by diets composed of local vegetable ingredients. Four diets were formulated containing decreasing levels of FM (50-0% for diet 1 to diet 4) and increasing proportions of vegetable ingredients (50-100%). Gonadosomatic index (GSI), diameter and percentages of developmental stages of oocytes, plasma sex steroids and vitellogenin dynamics were investigated from February to June using one-year-old fish. Fish were individually tagged, and 12 individuals from each diet were investigated monthly. Replacement of FM with plant ingredients did not affect the GSI neither in males, nor in females. All males were spermiating, and no abnormal gonads were found. In females, GSI and percentages of advanced stages of oocytes decreased during the dry season, indicating seasonal changes in gonad development. Moreover, oocytes were in late exogenous vitellogenesis, but no final maturation stages were observed, whatever the diet. Higher plasma levels of E2 in females and of androgens (T and 11-KT) in both sexes were observed in fish fed diet 4 than in those receiving diet 1 depending on the season. Levels of plasma E2 and ALP (indicator for vitellogenin) in males did not differ among treatments and seasons suggesting no phytoestrogenic activity. The results showed that total replacement of FM by vegetable diets composed of groundnut oilcakes, bean and sunflower meals has no deleterious effect on the onset of sexual maturation in African catfish but, may stimulate the sex steroid production and in turns may potentially exert some positive actions on reproductive success.

Surgically increased ovarian mass in the honey bee confirms link between reproductive physiology and worker behavior.

Honey bee (Apis mellifera L.) workers are essentially sterile females that are used to study how complex social behavior develops. Workers perform nest tasks, like nursing larvae, prior to field tasks, like foraging. Despite worker sterility, this behavioral progression correlates with ovary size: workers with larger ovaries (many ovary filaments) start foraging at younger ages on average. It is untested, however, whether the correlation confers a causal relationship between ovary size and behavioral development. Here, we successfully grafted supernumerary ovaries into worker bees to produce an artificial increase in the amount of ovary tissue. We next measured fat body mRNA levels for the yolk precursor gene vitellogenin, which influences honey bee behavioral development and can correlate with ovary size. Vitellogenin was equally expressed in surgical controls and bees with supernumerary ovaries, leading us to predict that these groups would be characterized by equal behavior. Contrary to our prediction, bees with supernumerary ovaries showed accelerated behavioral development compared to surgical controls, which behaved like reference bees that were not treated surgically. To explore this result we monitored fat body expression levels of a putative ecdysteroid-response gene, HR46, which is genetically linked to ovary size in workers. Our data establish that social insect worker behavior can be directly influenced by ovaries, and that HR46 expression changes with ovary size independent of vitellogenin.

The curious case of aging plasticity in honey bees.

As in all advanced insect societies, colony-organization in honey bees emerges through a structured division of labor between essentially sterile helpers called workers. Worker bees are sisters that conduct all social tasks except for egg-laying, for example nursing brood and foraging for food. Curiously, aging progresses slowly in workers that engage in nursing and even slower when bees postpone nursing during unfavorable periods. We, therefore, seek to understand how senescence can emerge as a function of social task performance. The alternative utilization of a common yolk precursor protein (vitellogenin) in nursing and somatic maintenance can link behavior and aging plasticity in worker bees. Beneficial effects of vitellogenin may also be mediated by inhibitory action on juvenile hormone and insulin-like signaling.

Hormonal regulation of metamorphosis and reproduction in ticks.

The presence of a "status quo" hormone like JH has not been found in ticks. The most advanced understanding of tick endocrinology is associated with female reproduction, where the sequence of the first messages for storage proteins (vitellogenin (Vg) and carrier protein), the Vg receptor, and male peptidic pheromones were recently reported. The current consensus model suggests that ecdysteroids from the epidermis regulated by a putative peptidic ecdysiotrophic hormone from the synganlion initiates the expression of the Vg messages in fat body and midgut. Vg protein, secreted into the hemolymph, requires an ovary Vg receptor to be absorbed by oocytes. Male pheromones transferred into the female genital tract during mating initiate blood feeding to repletion and vitellogenesis. The work so far on tick endocrinology is limited by the paucity of identified hormones and the small number of studies on a few tick models. The role of storage proteins in the evolution of hematophagy is discussed.

Vitellogenin functions as a multivalent pattern recognition receptor with an opsonic activity.

BACKGROUND: Vitellogenin (Vg), a major reproductive protein, has been associated with infection-resistant response in fish. However, the underlying mechanisms by which Vg is involved in anti-infectious response are not understood. METHODOLOGY/RESULTS: By both protein-microbe interaction analysis and enzyme-linked immunosorbent assay as well as phagocytosis test, we demonstrate for the first time that fish Vg acts as a pattern recognition molecule with multiple specificities that can recognize bacteria as well as fungus rather than self components from fish, and functions as an opsonin that can enhance macrophage phagocytosis. CONCLUSIONS: This study shows that fish Vg plays an integrative function in regulating immunity via its pleiotropic effects on both recognizing pathogen-associated molecular patterns and promoting macrophage phagocytosis. It also supports the notion that factors normally involved in control of female reproduction are associated with immunity in organisms that rely on Vg for oocyte development.

Vertebrate yolk complexes and the functional implications of phosvitins and other subdomains in vitellogenins.

In nonplacental or nontrophotenic vertebrates, early development depends on the maternal provision of egg yolk, which is mainly derived from large multidomain vitellogenin (Vtg) precursors. To reveal the molecular nature of the protein pools in vertebrate oocytes, published data on the N-termini of yolk proteins has been mapped to the deduced primary structures of their parent Vtgs. The available evidence shows that the primary cleavage sites of Vtgs are conserved, whereas the cleavage products exist as multidomain variants in the yolk protein pool. The serine-rich phosvitin (Pv) domains are linearly related to the molecular masses of the lipovitellin heavy chain. The 3-D localization of Pv maps to the outer edges of the Vtg monomer, where it is proposed to form amphipathic structures that loop up over the lipid pocket. At this locus, it is proposed that Pv stabilizes the nascent Vtg while it receives its lipid cargo, thereby facilitating the hepatic loading and locking of lipid within the Vtg (C-sheet)-(A-sheet)-(LvL) cavity, and enhances its solubility following secretion to the circulating plasma. The C-terminal regions of Vtgs are homologous to human von Willebrand factor type D domains (Vwfd), which are conserved cysteine-rich molecules with homologous regions that are prevalent in Vtgs, lipophorins, mucins, integrins, and zonadhesins. Unlike human VWFD, lower vertebrate Vwfds do not contain RGD motifs, which are associated with extracellular matrix binding. Although its function in Vtg is unknown, the lubricant properties associated with mucins and the cell adhesion properties associated with integrins and zonadhesins implicate Vwfd in the genesis of hemostatic platelet aggregation. Similarly, the proteolytic inhibitory properties associated with the binding of factor VIII in humans suggest that Vwfd stabilizes Vtg during passage in the systemic circulation.

Molecular diversity and evolution of the large lipid transfer protein superfamily.

Circulatory lipid transport in animals is mediated to a substantial extent by members of the large lipid transfer (LLT) protein (LLTP) superfamily. These proteins, including apolipoprotein B (apoB), bind lipids and constitute the structural basis for the assembly of lipoproteins. The current analyses of sequence data indicate that LLTPs are unique to animals and that these lipid binding proteins evolved in the earliest multicellular animals. In addition, two novel LLTPs were recognized in insects. Structural and phylogenetic analyses reveal three major families of LLTPs: the apoB-like LLTPs, the vitellogenin-like LLTPs, and the microsomal triglyceride transfer protein (MTP)-like LLTPs, or MTPs. The latter are ubiquitous, whereas the two other families are distributed differentially between animal groups. Besides similarities, remarkable variations are also found among LLTPs in their major lipid-binding sites (i.e., the LLT module as well as the predicted clusters of amphipathic secondary structure): variations such as protein modification and number, size, or occurrence of the clusters. Strikingly, comparative research has also highlighted a multitude of functions for LLTPs in addition to circulatory lipid transport. The integration of LLTP structure, function, and evolution reveals multiple adaptations, which have come about in part upon neofunctionalization of duplicated genes. Moreover, the change, exchange, and expansion of functions illustrate the opportune application of lipid-binding proteins in nature. Accordingly, comparative research exposes the structural and functional adaptations in animal lipid carriers and brings up novel possibilities for the manipulation of lipid transport.