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Apple Snail Perivitellin Precursor Properties Help Explain Predators' Feeding Behavior
Abstract
In contrast with vitellogenin maturation, it is unknown whether gastropod perivitellin precursors are subject to large structural changes. The gastropod reproductive tract includes an accessory organ, the albumen gland (AG), that produces and secretes perivitelline fluid. In the apple snail Pomacea canaliculata, the large, reddish-pink AG provides eggs with perivitellins that are defensive against predators. Although the AG makes a considerable contribution to apple snail biomass, field observations indicate that it is rejected by avian and mammalian predators, although the underlying reason remains unknown. By analyzing the structure-function properties of P. canaliculata perivitellin precursors, we provide insight into perivitellin maturation and its relationship with apple snail predator feeding behavior. Structural analysis using small-angle X-ray scattering, absorption and fluorescence spectroscopy, circular dichroism, electrophoresis, chromatography, and partial proteolysis showed that the size, shape, and structure of perivitellin precursors resemble those of egg mature forms. Functional analysis indicates that the precursors of the defensive perivitellins ovorubin (PcOvo) and perivitellin-2 (PcPV2) are highly stable and antinutritive, withstanding proteinase digestion and displaying structural stability of their quaternary structure under a wide pH range (4.0-10.0). Furthermore, AG extracts limit a predator's ability to digest nutrients and are toxic to mice (median lethal concentration 96 h after administration: 5.9 mg/kg). Treated mice displayed neurologic signs similar to those produced by egg PcPV2. Results indicate that apple snails store active precursors of egg proteins inside the AG, providing evidence that gastropod perivitellin precursors do not experience the large structural processing of invertebrate vitellogenin maturation. These defensive proteins provide the apple snail AG with neurotoxic, antinutritive, and antidigestive activity, a likely explanation for the predators' feeding behavior.
... It is because a snail meal contains 23% protein, 3% fat, 10% fibre, 20% soluble carbohydrate, 1% calcium and 1.8% phosphorus along with other immunomodulatory components. Therefore, data on growth and reproduction of these snails are needed to be analysed carefully for their invasion management (Lo and Hsieh 2000;Huang et al. 2003;Rogevich et al. 2009;Dai et al. 2011;Giraud-Billoud et al. 2013a, b;Mu et al. 2015;Olivier et al. 2016;Xu et al. 2016;Yam et al. 2016;Cardierno et al. 2017;Gilioli et al. 2017;Arrighetti et al. 2018;Brito et al. 2019;Mansur et al. 2018;Plan et al. 2008;Salleh and Zainalabidin 2018;Yang et al. 2018;Ye et al. 2018). Undoubtedly various adaptive measures taken by the snails to enable them one of the most invasive species are reported (Albrecht and Cavicchia 2001;Rodgers et al. 2001;Dreon et al. 2004aDreon et al. , b, 2007Dreon et al. , 2008Heras et al. 2008;Dreon et al. 2010;Frassa et al. 2010;Ituarte et al. 2012;Accorsi et al. 2017;Cadierno et al. 2018;Ip et al. 2018;Ituarte et al. 2018; ...
... Similarly, Dreon et al. (2002) also confirmed that the one and only site of secretion of perivitelline 2 is albumen gland in apple snails. Therefore, invasion of apple snail can be inhibited by the inactivation of the albumen gland (Cardierno et al. 2017) which will attenuate the economic loss on the agricultural fields (Fig. 4). The reproductive activity in apple snails is influenced by environmental factors mainly by temperature as they are ectothermic in nature. ...
Apple snails are one of the most survived fresh water invasive species that causes massive economic loss to vegetations, especially to the rice fields. They survive against many environmental stressors and are considered as a major pest in water-lodged cultivated area due to their intrusiveness, adaptation mechanisms and survivability. Therefore, there is a need for strategies to control their population. Snails can also be used as pollution bioindicators. Here we review apple snails for their ecotoxic management without compromising environment and co-inhabitants, and their use as semi-sessile invasive ecotoxic marker species. Snails have been found to accumulate 0.19–0.21% biphenyl ethers, 1.65% copper sulphate with 26.7% morality, 1.53–29.7% ivermectin, 43% polybrominated biphenyl ethers, 59% triphenylphosphine oxide, 8–100% of various heavy metals and nanoparticles. Feed intake of apple snails can be enhanced up to 22% with copper supplements, while 17 and 100% mortality can be achieved with niclosamide monohydrate (0.13 mg/L) and tea seed derivatives (0.015 g/L) exposure, respectively. Snails respond to stress factors with up to 29.6% higher lipid peroxides and 1.06% protein carbonyls, along with up to 80, 240 and 127% higher activities of superoxide dismutase, catalase and glutathione peroxidase enzymes, respectively, after cypermethrin exposure. Oppositely, mercury (2–8 mg/L) and aldicarb can reduce their oxygen consumption and acetylcholinesterase activity of up to 88.1 and 96.08%, respectively.
... Mechanisms of egg production are phylogenetically constrained and therefore intrinsic to the species and not affected by environmental factors (Eckelbarger and Watling, 1995). In this regard, recent reports indicate that apple snails and probably other gastropods have evolved an alternative vitellogenic mechanism whereby perivitellins, unlike vitellins of other invertebrates, are synthesized as already functional in the albumen gland (Cadierno et al., 2017). This provides snails with a more efficient and faster mechanism for food conversion into egg production (Cadierno et al., 2017). ...
... In this regard, recent reports indicate that apple snails and probably other gastropods have evolved an alternative vitellogenic mechanism whereby perivitellins, unlike vitellins of other invertebrates, are synthesized as already functional in the albumen gland (Cadierno et al., 2017). This provides snails with a more efficient and faster mechanism for food conversion into egg production (Cadierno et al., 2017). In this study, the decrease of AG-CG polysaccharides, soluble proteins, and the most abundant perivitellins in the ARO group reflects the massive transfer of perivitelline fluid compounds to the eggs, providing embryos with nutrients and defenses during devel-opment. ...
High fecundity often contributes to successful invasives. In molluscs, this may be facilitated by the albumen gland-capsule gland complex, which in gastropods secretes the egg perivitelline fluid that nourishes and protects embryos. The biochemistry of the albumen gland-capsule gland complex and its relationship with fecundity remain largely unknown. We addressed these issues in Pomacea canaliculata (Lamarck, 1822), a highly invasive gastropod whose fecundity and reproductive effort exceed those of ecologically similar gastropods. We evaluated the dynamics of its major secretion compounds (calcium, polysaccharides, and total proteins) as well as the gene expression and stored levels of perivitellins during key moments of the reproductive cycle, that is, before and after first copulation and at low, medium, and high reproductive output. Copulation and first oviposition do not trigger the onset of albumen gland-capsule gland complex biosynthesis. On the contrary, soon after an intermediate reproductive effort, genes encoding perivitellins overexpressed. A high reproductive effort caused a decrease in all albumen gland-capsule gland complex secretion components. Right after a high reproductive output, the albumen gland-capsule gland complex restored the main secretion components, and calcium recovered baseline reserves, but proteins and polysaccharides did not. These metabolic changes in the albumen gland-capsule gland complex after multiple ovipositions were reflected in a reduction in egg mass but did not compromise egg quality. At the end of the cycle, egg dry weight almost doubled the initial albumen glandcapsule gland complex weight. Results indicate that albumen gland-capsule gland complex biosynthesis limits a constantly high reproductive output. Therefore, lowering fecundity by targeting biosynthesis could effectively reduce the rate of this species’ spread.
... The percentage of each assessed abnormality did not exceed 10%. These findings may be attributed to the robust and adaptable structural characteristics of golden apple snails, allowing them to effectively withstand and adapt to their environment [28]. ...
This study aimed to evaluate the effects of herbicide 2, 4-D-dichlorophenoxy acetic acid on golden apple snail eggs and embryos. Additionally, the study assessed the applicability of optical coherence tomography (OCT), a non-invasive depth cross-sectional microscopic imaging technique, as a novel method, to the best of our knowledge, for studying morphological changes in golden apple snail eggs and embryos, in comparison to the conventional approach of using white light microscopy. The study revealed that the herbicide 2,4-D-dichlorophenoxy acetic acid affected the hatchery rate and morphological changes of the eggs and embryos. The lethal concentration (LC50), representing the concentration of a substance that is expected to cause death in half of the population being studied, of the golden apple eggs and embryos increased with longer exposure time and higher concentrations. The estimated median effective concentration (EC50), which denotes the concentration producing the desired effect in 50% of the exposed golden apple embryos, exhibited a similar trend of change as the LC50. When compared to the microscopic study, it was observed that OCT could be employed to investigate morphological changes of golden apple snail eggs and embryos, enabling evaluation of alterations in both 2D and 3D structures.
... S9 and data table S1, Supplementary Material online). Subsequently, we examined these genes in the context of the transition from aquatic to terrestrial egg deposition which as mentioned earlier must have required substantial changes in biochemical composition of the eggs (Cadierno et al. 2017;Pasquevich et al. 2017). Here we examined the phylogenetcis of some of these genes, and validated their functions by comparing gene expression patterns between aquatic (Lanistes and Marisa) and terrestrial egg depositors (Pomacea). ...
Data reported in this paper have been deposited in the GenBank database: genome assemblies and reads (Bioproject No: PRJNA523959 for P. canaliculata, PRJNA523958 for P. maculata, PRJNA445755 for M. cornuarietis, and PRJNA523095 for L. nyassanus); transcriptome sequences (Bioproject PRJNA473031 and PRJNA473253). Abstract The family Ampullariidae includes both aquatic and amphibious apple snails. They are an emerging model for evolutionary studies due to the high diversity, ancient history and wide geographical distribution. Insight into drivers of ampullariid evolution is hampered, however, by the lack of genomic resources. Here we report the genomes of four ampullariids spanning the Old World (Lanistes nyassanus) and New World (Pomacea canaliculata, Pomacea maculata and Marisa cornuarietis) clades. The ampullariid genomes have conserved ancient bilaterial karyotype features and a novel Hox gene cluster rearrangement, making them valuable in comparative genomic studies. They have expanded gene families related to environmental sensing and cellulose digestion, which may have facilitated some ampullarids to become notorious invasive pests. In the amphibious Pomacea, novel acquision of an egg neurotoxin and a protein for making the calcareous eggshell may have been key adaptations enabling their transition from underwater to terrestrial egg deposition.
... The perivitelline fluid of invasive Pomacea eggs shows neurotoxic, antidigestive and antinutritive properties to terrestrial and aquatic vertebrates [124][125][126][127][128]. Moreover, female reproductive organs involved in perivitellin synthesis have been recently shown to share these egg properties [129]. The most specialized avian predators of Pomacea along its native range, the Snail Kite Rostrhamus sociabilis and the Limpkin Aramus guarauna, are known to discard these organs despite the significant loss of captured biomass [130]. ...
Apple snails are large freshwater snails belonging to the family Ampullariidae that inhabit tropical to temperate areas. The South American apple snails Pomacea canaliculata and Pomacea maculata have been introduced to other continents where they have successfully established and spread. Our review aims to analyse the mechanisms of the impacts that these invasive Pomacea provoke or may provoke. Nine basic mechanisms were identified: grazing/herbivory/browsing, competition, predation, disease transmission, hybridisation with native species, poisoning/toxicity, interaction with other invasive species, promotion of collateral damage of control methods on non-target species and when acting as prey. The most important impacts are those related to their grazing on aquatic macrophytes, algae and rice and their competition and predation on other aquatic animals, mostly macroinvertebrates, including other apple snails. Invasive Pomacea are also responsible for outbreaks of an emergent parasitic disease (human eosinophilic meningitis). Their great abundance in invaded areas, their bioaccumulation of pollutants and their natural toxicity may impact on their predators and on trophic webs through apparent competition, trophic cascades and biomagnification. The biota from man-managed and natural wetlands may be unintentionally affected by mechanical, chemical and biological control against invasive Pomacea. Their capacity to hybridise may affect the distinctiveness and ecological traits of native Pomacea in invaded regions of America. Established populations of these invaders may either facilitate or resist the establishment of other exotic species. Field surveys and more realistic experimental approaches with multiple interacting species are needed to better understand the environmental impacts of invasive Pomacea and their underlying mechanisms.
... This might be caused by Lipoprotein such as Perivitellin 2 and Ovorubin which are important protein found in egg cell in early spawning stage (Dreon et al., 2003). Perivitellin 2 and Ovorubin are protein being high stability and resist to degradation and pH alteration (4−10) (Cadierno et al., 2017). However, the results of protein determination performed in extracts from head of snail in small, medium and large size indicated that snail size had no influence to extracted protein content. ...
The aim of this study was to evaluate pesticide exposure in egg and tissue of golden apple snail (Pomacea canaliculata), being collected from Huai−Saneng Reservoir, Surin Province by using AChE as bio−indicator. This is the pioneer work in Thailand with regards to the application of Acetylcholinesterase (AChE) as a situ biomarker in indicating pesticide contamination. The snail and its egg were sampled two times in the period of rice cultivating in June and July, 2017. There were 5 sampling stations (n = 10). The snail was classified based on its sizes: small, medium, and large. After studying the protein form by using 12.5% SDS−PAGE technique, it was that found that there were differences in protein expression from post−fertilization egg (pink color) and pre−hatching egg (white color). The results of Western blot analysis indicated that AChE of the egg in pink colored stage had 2 isoforms with different weights: 71 kDa and 66 kDa. However, there was only 71 kDa of AChE of that in white colored stage. The protein in 3 snail sizes was not different when compared to the snails from 2 sampling periods. Moreover, AChE expression in collected snail was higher than that in controlled group with having only 1 isoform (71 kDa). The AChE expression in the snail from stations 4 and 5 were lower than that from the other stations. After organochlorine and carbamate pesticide contamination in water was studied using test kit, it was found that there was contamination in every station in both periods and the highest was found in stations 4 and 5. Based on our results, we concluded that AChE expression in the egg and tissue of golden apple snail has a high potential to be used as an early warning signal indicating a pesticide contamination in the environment.
... S9 and data table S1, Supplementary Material online). Subsequently, we examined these genes in the context of the transition from aquatic to terrestrial egg deposition which as mentioned earlier must have required substantial changes in biochemical composition of the eggs (Cadierno et al. 2017;Pasquevich et al. 2017). Here we examined the phylogenetcis of some of these genes, and validated their functions by comparing gene expression patterns between aquatic (Lanistes and Marisa) and terrestrial egg depositors (Pomacea). ...
The family Ampullariidae includes both aquatic and amphibious apple snails. They are an emerging model for evolutionary studies due to the high diversity, ancient history and wide geographical distribution. Insight into drivers of ampullariid evolution is hampered, however, by the lack of genomic resources. Here we report the genomes of four ampullariids spanning the Old World (Lanistes nyassanus) and New World (Pomacea canaliculata, Pomacea maculata and Marisa cornuarietis) clades. The ampullariid genomes have conserved ancient bilaterial karyotype features and a novel Hox gene cluster rearrangement, making them valuable in comparative genomic studies. They have expanded gene families related to environmental sensing and cellulose digestion, which may have facilitated some ampullarids to become notorious invasive pests. In the amphibious Pomacea, novel acquision of an egg neurotoxin and a protein for making the calcareous eggshell may have been key adaptations enabling their transition from underwater to terrestrial egg deposition.
... This might be caused by Lipoprotein such as Perivitellin 2 and Ovorubin which are important protein found in egg cell in early spawning stage (Dreon et al., 2003). Perivitellin 2 and Ovorubin are protein being high stability and resist to degradation and pH alteration (4−10) (Cadierno et al., 2017). However, the results of protein determination performed in extracts from head of snail in small, medium and large size indicated that snail size had no influence to extracted protein content. ...
The aim of this study was to evaluate pesticide exposure in egg and tissue of golden apple snail (Pomacea canaliculata), being collected from Huai−Saneng Reservoir, Surin Province by using AChE as bio−indicator. This is the pioneer work in Thailand with regards to the application of Acetylcholinesterase (AChE) as a situ biomarker in indicating pesticide contamination. The snail and its egg were sampled two times in the period of rice cultivating in June and July, 2017. There were 5 sampling stations (n = 10). The snail was classified based on its sizes: small, medium, and large. After studying the protein form by using 12.5% SDS−PAGE technique, it was that found that there were differences in protein expression from post−fertilization egg (pink color) and pre−hatching egg (white color). The results of Western blot analysis indicated that AChE of the egg in pink colored stage had 2 isoforms with different weights: 71 kDa and 66 kDa. However, there was only 71 kDa of AChE of that in white colored stage. The protein in 3 snail sizes was not different when compared to the snails from 2 sampling periods. Moreover, AChE expression in collected snail was higher than that in controlled group with having only 1 isoform (71 kDa). The AChE expression in the snail from stations 4 and 5 were lower than that from the other stations. After organochlorine and carbamate pesticide contamination in water was studied using test kit, it was found that there was contamination in every station in both periods and the highest was found in stations 4 and 5. Based on our results, we concluded that AChE expression in the egg and tissue of golden apple snail has a high potential to be used as an early warning signal indicating a pesticide contamination in the environment.
... Interestingly, several Pomacea canaliculata egg proteins are toxic, anti-nutritive and anti-digestive for different animals (Dreon et al. 2013). These defensive proteins are already active within the AG, and are the most probable reason for the rejection of the AG by predators (Cadierno et al. 2017). ...
Snail Kites (Rostrhamus sociabilis) feed almost exclusively on Apple Snails (Pomacea spp.). While field observations indicate they discard the noxious albumen gland (AG) when feeding on female snails, there is no information on the energy lost by this behavior, the gland composition, or if there are snail sex preferences associated. We addressed for the first time these aspects for Snail Kites foraging on Pomacea canaliculata in southern South America. Whole snail’s biochemical composition exhibited significant differences between sexes. Proteins and carbohydrates were the major energy-providing components. Soft parts provide ~2.91±0.16 and 2.50±0.14 Kcal g-1 dw for male and female without AG, respectively while AG accounts for 15 % of available energy. Size and sex of prey consumed, determined at foraging perches sampled monthly, showed that Kites preferentially feed on the largest snails available throughout the sampling period. Even though the remains of female body without the AG have less energy than males, sex preferential predation toward females was observed. Our study demonstrates for the first time the optimal foraging trade-off between prey size/sex and nutrition/energy in Snail Kites. After discarding the albumen gland, male and female snails of equivalent size provide different nutrients and energy, though Snail Kite foraging is generally biased toward females mostly due to their bigger size suggesting Snail Kites are unable to distinguish between sex.
Some, probably most and perhaps all, members of the phylum Nemertea are poisonous, documented so far from marine and benthic specimens. Although the toxicity of these animals has been long known, systematic studies on the characterization of toxins, mechanisms of toxicity, and toxin evolution for this group are scarce. Here, we present the first investigation of the molecular evolution of toxins in Nemertea. Using a proteo-transcriptomic approach, we described toxins in the body and poisonous mucus of the pilidiophoran Lineus sanguineus and the hoplonemertean Nemertopsis pamelaroeae. Using these new and publicly available transcriptomes, we investigated the molecular evolution of six selected toxin gene families. In addition, we also characterized in silico the toxin genes found in the interstitial hoplonemertean, Ototyphlonemertes erneba, a meiofaunal taxa. We successfully identified over 200 toxin transcripts in each of these species. Evidence of positive selection and gene duplication was observed in all investigated toxin genes. We hypothesized that the increased rates of gene duplications observed for Pilidiophora could be involved with the expansion of toxin genes. Studies concerning the natural history of Nemertea are still needed to understand the evolution of their toxins. Nevertheless, our results show evolutionary mechanisms similar to other venomous groups.
Apple snails (ASs), the freshwater snails of the family Ampullariidae, are widely spread in tropical and subtropical regions. Owing to their strong invasiveness and harmful effects on agricultural production and ecosystems, they have attracted considerable attention. However, less research has been conducted on the whole picture of the current research status and development trends in the ASs field. For this purpose, we conducted a bibliometric analysis based on a total of 1028 publications regarding ASs published between 1900 and 2021 using VOSviewer 1.6.18 and CiteSpace 6.1.R3 software. The results suggest that ASs research has received increasing attention in recent years. The most productive country has been the USA, while the institution with the most publications was Consejo Nacional de Investigaciones Científicas y Técnicas Conicet. “Zoology” was the category of the most popular subject, Journal of Molluscan Studies was the primary journal, and P.R. Martin was the most outstanding author in ASs field. Invasive alien species, alarm response, copper (Cu) exposure, taxonomy and phylogenetic analysis, Angiostrongylus cantonensis (parasite), control (including molluscicidal activity), growth and reproduction, and feeding preferences were the main research hotspots. Further, “Pomacea maculata”, “Evolution”, “Biology”, “Digestive gland”, “Phylogeny”, “Identification”, “Pomacea canaliculata caenogastropoda”, “Diversity”, and “Nonnative apple snail” have been emerging active topics in recent years. These findings can help researchers in this area to better understand the direction and advances of ASs research and are valuable for future research and agricultural practice of apple snail control.
The only wetland in Europe colonized successfully by the apple snail Pomacea maculata Perry, 1810, is the Ebro Delta (Spain). After ten years it has become widespread there, despite significant eradication attempts. In spite of its great negative ecological and economic impacts, its incorporation into food webs can result in an abundant potential resource for native and exotic predators. We identified the potential predators, including fishes, amphibians, crustaceans, birds, turtles and mammals in natural conditions using stable isotopic analyses (δ13C and δ15N). Six predator species, three native (two birds and one frog) and three invasive (one crustacean, one turtle and one mammal), were confirmed to consume apple snails. None of the 10 fish species analyzed consumed apple snails, although some have been previously observed consuming this snail under laboratory conditions. This study emphasizes the need to assess the consumption of apple snail, as well as other invasive organisms, by potential predators in natural conditions to confirm the establishment of new trophic relationships and to understand whether these potential predators actually act as biological control agents in nature.
Covering: 1951 to 2022Packed with nutrients and unable to escape, eggs are the most vulnerable stage of an animal's life cycle. Consequently, many species have evolved chemical defenses and teamed up their eggs with a vast array of toxic molecules for defense against predators, parasites, or pathogens. However, studies on egg toxins are rather scarce and the available information is scattered. The aim of this review is to provide an overview of animal egg toxins and to analyze the trends and patterns with respect to the chemistry and biosynthesis of these toxins. We analyzed their ecology, distribution, sources, occurrence, structure, function, relative toxicity, and mechanistic aspects and include a brief section on the aposematic coloration of toxic eggs. We propose criteria for a multiparametric classification that accounts for the complexity of analyzing the full set of toxins of animal eggs. Around 100 properly identified egg toxins are found in 188 species, distributed in 5 phyla: cnidarians (2) platyhelminths (2), mollusks (9), arthropods (125), and chordates (50). Their scattered pattern among animals suggests that species have evolved this strategy independently on numerous occasions. Alkaloids are the most abundant and widespread, among the 13 types of egg toxins recognized. Egg toxins are derived directly from the environment or are endogenously synthesized, and most of them are transferred by females inside the eggs. Their toxicity ranges from ρmol kg-1 to mmol kg-1, and for some species, experiments support their role in predation deterrence. There is still a huge gap in information to complete the whole picture of this field and the number of toxic eggs seems largely underestimated.
This study investigated the predation of eggs of the apple snail Pomacea dolioides in Itacoatiara, Amazonas, Brazil. Predation was compared between rural and urban areas, period of day, shaded and unshaded clutches. In addition, we evaluated clutch height and the behaviour of predators. Between April 2017 and July 2018, 962 egg clutches were observed at different times of the day: 492 in rural areas with predation of 68 egg clutches and 470 in urban areas with predation of only 9 egg clutches. Significant differences were recorded for predation rate and differences were recorded for egg clutch height in the areas. In the rural areas, the most frequent predators during the day were ants Solenopsis invicta, Crematogaster cf. carinata and the most abundant were S. invicta, Wasmannia aff. iheringi and C. cf. carinata. During the night, S. invicta was the most frequent and abundant predator. In urban areas, the frequency of predation and abundance of S. invicta were higher during the day and night than those of other predators. Populations of P. dolioides in rural areas are more affected by egg predation, mostly by S. invicta and C. cf. carinata, and predatory behaviour depended on the species of the predator.
This research aimed to examine on the hatching rate of golden apple snail. Five experimental conditions which based on what snail exposed; (a) water, (2) 1 ppm of glyphosate, (3) 0.26 ppm of cypermethrin, (4) 0.75 ppm of metaldehyde and (5) the control (without any exposure). The results shown that hatching rate of the snail was higher than 90% no statistical difference compared to the control (p≥0.05). The hatching rate in control group was 94±1.97% and the average rates found in exposure condition of water, glyphosate, cypermethrin and metaldehyde were 98.05±1.30%, 92.88±3.16%, 94.32±3.46% and 94.12±3.20%, respectively. For morphological study, the alterations could be classified into 5 groups; abnormal egg, egg shell deformed, fetus abnormal, decomposed tissue and the connection of tissue cells. The agrochemical causing highest alteration was cypermethrin. After the protein of snail egg in both stages; early spawning (dark pink) and early hatchery stage (light pink) were extracted, we found the difference in protein form were occurred. The important proteins found were 3 types comprising; Perivitellin 2 (Pv2) and Ovorubin (Ov). For Perivitellin 2 (Pv2) their molecular weight of protein were 98, 67 and 31 kDa while it was 28 kDa for Ovorubin. From our results, it indicated that golden apple snail highly tolerates to agrochemicals of high hatching rate. Therefore, this findings can be useful in assessing golden apple snail wild population that causing original habitat degradation.
Background
Species beloging to the genus Pomacea (Ampullariidae), often referred as apple snails, are freshwater, amphibious snails native to South, Central and North America. Some species such as P. canaliculata have become a driver of ecosystem changes in wetlands and an important rice and taro pest after its introduction to Asia and other parts of the world. Females deposit colored egg clutches above the waterline, a reproductive strategy that exposes the eggs to harsh conditions and terrestrial predation. However, eggs have no reported predators in their native range, probably because of the acquisition of unparalleled biochemical defenses provided by a set of proteins (perivitellins) that nourish embryos and protect them from predators and abiotic factors. Notably, ingestion of egg perivitelline fluid (PVF) decreases rat growth rate and alters their gastrointestinal morphology. The aim of the study is to determine the effect of apple snail egg PVF on mice gut digestive activity, morphology and nutrient absorption.
Methods
Carbohydrate digestion by intestinal disaccharidases (sucrase-isomaltase and maltase-glucoamylase) was evaluated ex vivo in mice gavaged with 1 or 4 doses of PVF. Changes in gut morphological and absorptive surface were measured. In addition, alteration on nutrient absorption rates, transport pathways and intestinal permeability was evaluated by luminal perfusions of small intestine with radiolabeled L-proline (absorbed by paracellular and transcellular pathways) and L-arabinose (absorbed exclusively by paracellular pathway).
Results
Perivitelline fluid affected mice displayed significant morphological changes in the small intestine epithelium inducing the appearance of shorter and wider villi as well as fused villi. This resulted in a diminished absorptive surface, notably in the proximal portion . Likewise, the activity of disaccharidases diminished in the proximal portion of the intestine. Total absorption of L-proline increased in treated mice in a dose-dependent manner. There were no differences neither in the ratio of paracellular-to-transcellular absorption of L-proline nor in gut permeability as revealed by the clearance of L-arabinose.
Discussion
Oral administration of apple snail PVF to mice adversely alters gut morphophysiology by reducing the intestinal absorptive surface, affecting enzymes of sugar metabolism and increasing the absorption rate of nutrients without affecting the relative contribution of the absorption pathways or gut permeability. These results further support the role of PVF in passive anti-predator defenses in Pomacea snail eggs that target the digestive system.
Apple snails (Ampullariidae) are among the largest and most ecologically important freshwater
snails. The introduction of multiple species has reinvigorated the field and spurred a
burgeoning body of research since the early 1990s, particularly regarding two species introduced
to Asian wetlands and elsewhere, where they have become serious agricultural pests.
This review places these recent advances in the context of previous work, across diverse fields
ranging from phylogenetics and biogeography through ecology and developmental biology,
and the more applied areas of environmental health and human disease. The review does not
deal with the role of ampullariids as pests, nor their control and management, as this has been
substantially reviewed elsewhere. Despite this large and diverse body of research, significant
gaps in knowledge of these important snails remain, particularly in a comparative framework.
The great majority of the work to date concerns a single species, Pomacea canaliculata, which
we see as having the potential to become a model organism in a wide range of fields. However,
additional comparative data are essential for understanding this diverse and potentially
informative group. With the rapid advances in genomic technologies, many questions, seemingly
intractable two decades ago, can be addressed, and ampullariids will provide valuable
insights to our understanding across diverse fields in integrative biology.
Background
The spread of the invasive snail Pomacea canaliculata is expanding the rat lungworm disease beyond its native range. Their toxic eggs have virtually no predators and unusual defenses including a neurotoxic lectin and a proteinase inhibitor, presumably advertised by a warning coloration. We explored the effect of egg perivitellin fluid (PVF) ingestion on the rat small intestine morphology and physiology.
Methodology/Principal Findings
Through a combination of biochemical, histochemical, histopathological, scanning electron microscopy, cell culture and feeding experiments, we analyzed intestinal morphology, growth rate, hemaglutinating activity, cytotoxicity and cell proliferation after oral administration of PVF to rats. PVF adversely affects small intestine metabolism and morphology and consequently the standard growth rate, presumably by lectin-like proteins, as suggested by PVF hemaglutinating activity and its cytotoxic effect on Caco-2 cell culture. Short-term effects of ingested PVF were studied in growing rats. PVF-supplemented diet induced the appearance of shorter and wider villi as well as fused villi. This was associated with changes in glycoconjugate expression, increased cell proliferation at crypt base, and hypertrophic mucosal growth. This resulted in a decreased absorptive surface after 3 days of treatment and a diminished rat growth rate that reverted to normal after the fourth day of treatment. Longer exposure to PVF induced a time-dependent lengthening of the small intestine while switching to a control diet restored intestine length and morphology after 4 days.
Conclusions/Significance
Ingestion of PVF rapidly limits the ability of potential predators to absorb nutrients by inducing large, reversible changes in intestinal morphology and growth rate. The occurrence of toxins that affect intestinal morphology and absorption is a strategy against predation not recognized among animals before. Remarkably, this defense is rather similar to the toxic effect of plant antipredator strategies. This defense mechanism may explain the near absence of predators of apple snail eggs.
The effects of food availability and age on the reproductive traits of Pomacea canaliculata were determined in snails fed at three food levels under laboratory conditions (14L:10D; 25°C). In males, copulation frequency tended to increase with age, while food availability did not affect copulation frequency. In females, spawn production was lower in food-limited conditions, but current or weight-specific reproductive effort (RE) did not differ among food levels. The weight-specific RE and spawn frequency of the well-fed females decreased with age, but their current RE increased with age.
A field survey and two experimental manipulations were made to investigate the extent of predation on eggs of the apple snail Pomacea canaliculata (Lamarck) by the fire ant Solenopsis geminata (Fabricius) in the Philippines. First, when Pomacea egg masses found along levees of paddy fields were observed, more than half of them had some damage. More fire ants were observed near egg masses with higher degrees of damage. Secondly, when egg masses were experimentally placed on levees, on average 50% of the eggs were lost (removed or damaged) within two days in March and 38% were lost within three days in August. Thirdly, egg masses were placed in cups with or without water on levees; no eggs were lost when ants were successfully excluded by water. The proportion of lost eggs was highly variable among egg masses, but there was no difference between day and night. Possible use of this ant as a biocontrol agent for the apple snail is considered.
Although most eggs are intensely predated, the aerial egg clutches from the aquatic snail Pomacea canaliculata have only one reported predator due to unparalleled biochemical defenses. These include two storage-proteins: ovorubin that provides a conspicuous (presumably warning) coloration and has antinutritive and antidigestive properties, and PcPV2 a neurotoxin with lethal effect on rodents. We sequenced PcPV2 and studied whether it was able to withstand the gastrointestinal environment and reach circulation of a potential predator. Capacity to resist digestion was assayed using small-angle X-ray scattering (SAXS), fluorescence spectroscopy and simulated gastrointestinal proteolysis. PcPV2 oligomer is antinutritive, withstanding proteinase digestion and displaying structural stability between pH 4.0-10.0. cDNA sequencing and protein domain search showed that its two subunits share homology with membrane attack complex/perforin (MACPF)-like toxins and tachylectin-like lectins, a previously unknown structure that resembles plant Type-2 ribosome-inactivating proteins and bacterial botulinum toxins. The protomer has therefore a novel AB toxin combination of a MACPF-like chain linked by disulfide bonds to a lectin-like chain, indicating a delivery system for the former. This was further supported by observing PcPV2 binding to glycocalix of enterocytes in vivo and in culture, and by its hemaggutinating, but not hemolytic activity, which suggested an interaction with surface oligosaccharides. PcPV2 is able to get into predator's body as evidenced in rats and mice by the presence of circulating antibodies in response to sublethal oral doses. To our knowledge, a lectin-pore-forming toxin has not been reported before, providing the first evidence of a neurotoxic lectin in animals, and a novel function for ancient and widely distributed proteins. The acquisition of this unique neurotoxic/antinutritive/storage protein may confer the eggs a survival advantage, opening new perspectives in the study of the evolution of animal defensive strategies.
New developments in the program package
ATSAS
(version 2.4) for the processing and analysis of isotropic small-angle X-ray and neutron scattering data are described. They include (i) multiplatform data manipulation and display tools, (ii) programs for automated data processing and calculation of overall parameters, (iii) improved usage of high- and low-resolution models from other structural methods, (iv) new algorithms to build three-dimensional models from weakly interacting oligomeric systems and complexes, and (v) enhanced tools to analyse data from mixtures and flexible systems. The new
ATSAS
release includes installers for current major platforms (Windows, Linux and Mac OSX) and provides improved indexed user documentation. The web-related developments, including a user discussion forum and a widened online access to run
ATSAS
programs, are also presented.
While many invertebrates sequester toxic compounds to endow eggs with chemical defences, here we show, for the first time to our knowledge, the identification of a neurotoxin of proteinaceous nature localized inside an egg. Egg extracts from the freshwater apple snail Pomacea canaliculata displayed a neurotoxic effect in mice upon intraperitoneal injection (i.p.) (LD50, 96h 2.3mg/kg). Egg protein and total lipids were analysed separately and the only fraction displaying a highly toxic effect (LD50, 96h 0.25mg/kg, i.p.) was further purified to homogeneity as an oligomeric glyco-lipoprotein of 400kDa and two subunits biochemically and immunologically indistinguishable from the previously described perivitellin PV2. The neurotoxin was heat sensitive and there was evidence of circulating antibody response to sublethal i.p. doses on mice. Clinical signs, histopathological and immunocytochemical studies revealed damage mostly in mice spinal cord. Experiments showed chromatolysis and a decreased response to calbindin D-28K associated with a significant increase of TUNEL-positive cells in the dorsal horn neurons. These results suggest that calcium buffering and apoptosis may play a role in the neurological disorders induced by the toxin in mammalian central nervous system. This is the first report of a mollusc neurotoxin genetically encoded outside the cone-snail species.
Fieldwork has thoroughly established that most eggs are intensely predated. Among the few exceptions are the aerial egg clutches from the aquatic snail Pomacea canaliculata which have virtually no predators. Its defenses are advertised by the pigmented ovorubin perivitellin providing a conspicuous reddish coloration. The nature of the defense however, was not clear, except for a screening for defenses that identified a neurotoxic perivitellin with lethal effect on rodents. Ovorubin is a proteinase inhibitor (PI) whose role to protect against pathogens was taken for granted, according to the prevailing assumption. Through biochemical, biophysical and feeding experiments we studied the proteinase inhibitor function of ovorubin in egg defenses.
Mass spectrometry sequencing indicated ovorubin belongs to the Kunitz-type serine proteinase inhibitor family. It specifically binds trypsin as determined by small angle X-ray scattering (SAXS) and cross-linking studies but, in contrast to the classical assumption, it does not prevent bacterial growth. Ovorubin was found extremely resistant to in vitro gastrointestinal proteolysis. Moreover feeding studies showed that ovorubin ingestion diminishes growth rate in rats indicating that this highly stable PI is capable of surviving passage through the gastrointestinal tract in a biologically active form.
To our knowledge, this is the first direct evidence of the interaction of an egg PI with a digestive protease of potential predators, limiting predator's ability to digest egg nutrients. This role has not been reported in the animal kingdom but it is similar to plant defenses against herbivory. Further, this would be the only defense model with no trade-offs between conspicuousness and noxiousness by encoding into the same molecule both the aposematic warning signal and an antinutritive/antidigestive defense. These defenses, combined with a neurotoxin and probably unpalatable factors would explain the near absence of predators, opening new perspectives in the study of the evolution and ecology of egg defensive strategies.
The site of synthesis of molluscs lipoproteins is little known and was investigated for the egg lipoprotein perivitellin 1 (PV 1) or ovorubin in the freshwater snail Pomacea canaliculata. Tissues (albumen gland, gonad-digestive gland complex and muscle) of vitellogenic females were incubated in vitro at 25 degrees C for 12 h with 14C Leucine. After that, soluble proteins from tissue homogenates and medium samples were analysed for de novo protein synthesis by electrophoresis and HPLC, and radiolabelled proteins quantified by liquid scintillation. Gonad-digestive gland complex did not synthesise ovorubin, in spite its high protein synthesis levels. Three albumen gland radiolabelled proteins (35, 32 and 28 kDa) comigrated with the subunits of ovorubin and represented 1.3% of the total labelled protein of that tissue. Western blot analysis with polyclonal antibodies confirmed that these were ovorubin subunits. In vivo experiments where vitellogenic females were injected with 3H Leucine, revealed that ovorubin was not present in hemolymph. ELISA analysis confirmed ovorubin presence only in albumen gland and developing eggs with levels of 800 and 582 mg/g protein, which represent 30.3 and 28.4 mg ovorubin/g of tissue, respectively. Therefore, albumen gland is the single site of ovorubin synthesis as no extragland synthesis, circulation or accumulation could be detected in the apple snail.
The major 2S albumin allergen from Brazil nuts, Ber e 1, was subjected to gastrointestinal digestion using a physiologically relevant in vitro model system either before or after heating (100 degrees C for 20 min). Whilst the albumin was cleaved into peptides, these were held together in a much larger structure even when digested by using a simulated phase 1 (gastric) followed by a phase 2 (duodenal) digestion system. Neither prior heating of Ber e 1 nor the presence of the physiological surfactant phosphatidylcholine affected the pattern of proteolysis. After 2 h of gastric digestion, approximately 25% of the allergen remained intact, approximately 50% corresponded to a large fragment of M(r) 6400, and the remainder comprised smaller peptides. During duodenal digestion, residual intact 2S albumin disappeared quickly, but a modified form of the 'large fragment' remained, even after 2 h of digestion, with a mass of approximately 5000 Da. The 'large fragment' comprised several smaller peptides that were identified, by using different MS techniques, as deriving from the large subunit. In particular, sequences corresponding to the hypervariable region (Q37-M47) and to another peptide (P42-P69), spanning the main immunoglobulin E epitope region of 2S albumin allergens, were found to be largely intact following phase 1 (gastric) digestion. They also contained previously identified putative T-cell epitopes. These findings indicate that the characteristic conserved skeleton of cysteine residues of 2S albumin family and, particularly, the intrachain disulphide bond pattern of the large subunit, play a critical role in holding the core protein structure together even after extensive proteolysis, and the resulting structures still contain potentially active B- and T-cell epitopes.
Seasonal variations in the morphology of the parenchymal mass and function of the albumen gland/capsule gland complex have been studied in Pomacea canaliculata, together with the cellular types involved in the synthesis and secretion of perivitellin fluid components. The two major parenchymal cell types, albumen secretory cells (AS) and labyrinthic cells (LC), undergo seasonal variations throughout the annual reproductive cycle, which is divided into three periods. Both cellular types show maximal development and structural complexity during the reproductive period (spring and summer). AS cells have a well-developed Golgi complex and rough endoplasmic reticulum and their secretory granules show electron-dense particles of about 20 nm (probably galactogen). These cells are uniquely involved in ovorubin and PV2 perivitellin synthesis and their secretory granules are the single storage site for these two major perivitellins, as revealed by immunoelectron microscopy. AS also possess calcium deposits that infiltrate the cytoplasmic matrix. The luminal surfaces of LC exhibit long cilia intermingled with sparce short microvilli. Basally, the plasma membrane shows deep irregular folds that extend through the cytoplasm up to the subapical region. Calcium deposits infiltrate the cytoplasm and accumulate in the extracellular space of the basal labyrinth. Nerve terminals seem to be involved in the regulation of parenchymal cell secretion. At the post-reproductive period, AS markedly change their aspect following the release of most of the secretory granules into the acinar lumen. LC decrease in volume, the number of their cilia decreases, their cytoplasmic folds are much thinner and their extracellular spaces lack calcium particles. At the pre-reproductive period (winter), AS and LC recover and prepare for the subsequent period.
Snail Kites (Rostrhamus sociabilis sociabilis) observed along the lower Río Magdalena in northern Colombia fed heavily on Pomacea chemnitzi but also took Marisa cornuarietis, a smaller species. The kites captured Pomacea preferentially over Marisa and had difficulties extracting Marisa from its shell. They also failed to extract some large specimens of P. chemnitzi. Failures with both species were apparently related to problems in removing opercula. These observations indicate that R. sociabilis can no longer be considered a strict specialist on Pomacea.
The feeding habits of the Snail Kite (Rostrhamus sociabilis) were observed intermittently from 1967-1980 in Florida, USA. Approximately 97% of all observed foraging bouts were over marshes having sparse emergent vegetation. The visually-hunting kite was unable to forage over floating mats of exotic water hyacinth (Eichhornia crassipes). Male kites had shorter hunting bouts than females. For still-hunting, the birds' perches ranged from 0.15-4.6 m high and captures occurred an average of 5.8 m from perches. Females were significantly more successful (70%) for course-hunting than males (48%), but I found no difference for still-hunting. Birds tended to forage throughout the day, except for occasional inactive periods by some individuals during midday. On cooler days, foraging commenced slightly later in the morning than on warmer days. Kites probably capture freshwater apple snails (Pomacea paludosa) as deep as 16 cm. Capture rates for adults generally ranged from 1.7-3.4 snails per hour. Kites usually foraged over a common hunting area, and defense of foraging sites was rare. Handling of snails, from the kite's arrival at the feeding perch until consumption, averaged 2.7 min, with no significant difference between sexes. However, adult females were more efficient at the extraction portion of this process than were adult males. Snails were usually extracted before being brought to the nest, except in the latter part of the nestling period when some snails were extracted at or near the nest and some were brought intact. Adults feed small chicks bill to bill, and both parents generally shared equally in care of the young, except at two nests where the females did 67% or more of the feeding. Mean length of snails taken by kites was 42.8 mm (range 25.2-71.3, n=697) and mean diameter was 45.8 mm (range 27.4-82.4, n=697). The most common size classes taken were 30-60 mm in length and diameter. Nutritional and gross energy values were determined for apple snails. Female snails with albumen glands removed (versus males or mixed samples of both sexes of complete tissue or with viscera removed) had the highest caloric value (x̄=4.04 kcal/g, n=10). Kites cast pellets, a behavior documented here for the first time.
Consumer density can influence foraging patterns such as prey-size selection, but few studies have evaluated its effects in field conditions. Here we evaluate the hypothesis that habitat structure influences forager density, and that this in turn influences the size of prey consumed by two avian predators. The sizes of two apple snail species available to, and consumed by, snail kites and limpkins were determined at sites with high and low densities of snail kite foraging perches. Sites with more perches had higher densities of snail kites, but not of limpkins. Both predators consumed prey larger than those available in the marshes, but habitat structure influenced the probability of consumption of different prey sizes. Limpkins consumed larger prey at low-density sites when compared with high-density sites, in contrast to other studies that found no size selection. Thus, limpkins can present prey-size selectivity but the presence of other predators can influence the range of prey sizes consumed. When a wider range of prey sizes is available, limpkins can select larger prey; alternatively, higher densities of other predators can result in higher foraging risk, favoring the capture of smaller, easier to handle prey. Snail kites incorporated smaller prey to their diet at low-density sites than at high-density ones, probably due to the higher costs of carrying large prey, differential age distribution, or lower foraging risks. Thus, habitat structure can influence consumer density and foraging patterns in complex ways, influencing predator–prey interactions in natural systems.
A method is proposed for the determination of the optimum value of the regularization parameter (Lagrange multiplier) when applying indirect transform techniques in small-angle scattering data analysis. The method is based on perceptual criteria of what is the best solution. A set of simple criteria is used to construct a total estimate describing the quality of the solution. Maximization of the total estimate is straightforward. Model computations show the effectiveness of the technique. The method is implemented in the program GNOM [Svergun, Semenyuk & Feigin (1988). Acta Cryst. A44, 244–250].
Studying how invasive species respond to environmental stress at the molecular level can help us assess their impact and predict their range expansion. Development of markers of genetic polymorphism can help us reconstruct their invasive route. However, to conduct such studies requires the presence of substantial amount of genomic resources. This study aimed to generate and characterize genomic resources using high throughput transcriptome sequencing for Pomacea canaliculata, a nonmodel gastropod indigenous to Argentina that has invaded Asia, Hawaii and southern United States. De novo assembly of the transcriptome resulted in 128 436 unigenes with an average length of 419 bp (range: 150-8556 bp). Many of the unigenes (2439) contained transposable elements, showing the existence of a source of genetic variability in response to stressful conditions. A total of 3196 microsatellites were detected in the transcriptome; among 20 of the randomly tested microsatellites, 10 were validated to exhibit polymorphism. A total of 15 412 single-nucleotide polymorphisms (SNPs) were detected in the ORFs. LC-MS/MS analysis of the proteome of juveniles revealed 878 proteins, of which many are stress related. This study has demonstrated the great potential of high throughput DNA sequencing for rapid development of genomic resources for a nonmodel organism. Such resources can facilitate various molecular ecological studies, such as stress physiology and range expansion.
Energy partitioning within the developing eggs and early juveniles of the gastropod Pomacea canaliculata was investigated from the time of fertilization to newly hatched snails. The forms and locations of the energy stored as well as their changes while development proceeded were studied in six stages (from morula to 3-day hatchlings). The rates of utilization of different lipid classes, protein classes, and total carbohydrates were measured for the first time in embryos and perivitelline fluid. Perivitelline fluid is the primary energy store. Its biochemical composition at stage I is represented by carbohydrates, proteins, and lipids with 34.8%, 13.0%, and 1.5% dry weight, respectively. Dry weight of eggs showed no significant increase during development, indicating there was no important mass exchange with the medium. Carbohydrates and proteins were identified as the major energy-providing components, and lipids as the minor one, contributing 142, 56, and 12 cal/100 mg egg, dry weight, respectively. The overall conversion efficiency (CE) was 32.8% (calculated as percentage of perivitellus energy transformed into embryonic tissues). Carbohydrates were the most important energy reserve supplying virtually all the catabolic demand (CE 14.6%). Protein electrophoretic profiles during embryogenesis showed three distinct phases: An accumulation period (up to stage IV); a more active accumulation and selective utilization phase (stage V), and a selective consumption and protein synthesis period (hatchlings). Structural lipids from perivitellus (phospholipids and free sterols) were selectively conserved in embryos and juveniles, whereas triacylglycerols, hydrocarbons, and esterified sterols were preferentially depleted by hatchlings. Therefore, protein and lipid reserves in P. canaliculata perivitellus provide structural precursors during embryogenesis, while they partially contribute to the energy supplied by carbohydrates.
Pomacea canaliculata is a freshwater snail that deposits eggs on solid substrates above the water surface. Previous studies have emphasized the nutritional and protective functions of the three most abundant perivitelline fluid (PVF) protein complexes (ovorubin, PV2, and PV3) during its embryonic development, but little is known about the structure and function of other less abundant proteins. Using 2-DE, SDS-PAGE, MALDI TOF/TOF, and LC-MS/MS, we identified 59 proteins from the PVF of P. canaliculata, among which 19 are novel. KEGG analysis showed that the functions of the majority of these proteins are "unknown" (n=34), "environmental information processing" (10), 9 of which are related to innate immunity, and "metabolism" (7). Suppressive subtractive hybridization revealed 21 PVF genes to be specific to the albumen gland, indicating this organ is the origin of many of the PVF proteins. Further, the 3 ovorubin subunits were identified with 30.2-35.0% identity among them, indicating their common origin but ancient duplications. Characterization of the PVF proteome has opened the gate for further studies aiming to understand the evolution of the novel proteins and their contribution to the switch to aerial oviposition.
The albumen gland is a female accessory sex gland that synthesizes and secretes perivitelline fluid around pulmonate eggs. The perivitelline fluid is composed of mainly galactogen and proteins, and is thought to provide nourishment to the embryos during development. We have previously identified the major secretory protein of the albumen gland of the freshwater snail Helisoma duryi as a native glycoprotein of ∼288 kDa, consisting of four 66-kDa subunits. In this study, the major albumen gland protein in H. duryi was purified, cloned, and the full-length cDNA sequence determined. Nucleotide sequence analysis revealed that the albumen gland protein (HdAGP) shared 83% identity with a partial cDNA sequence from a developmentally regulated albumen gland protein in Biomphalaria glabrata. The HdAGP mRNA was detected by RT-PCR in the albumen gland, ovotestis, mantle and digestive gland. SDS-PAGE analysis of the albumen gland protein in egg masses at different stages of development showed that the amount of HdAGP steadily decreased during embryogenesis, suggesting its possible catabolism by the developing embryos. Protein domain searches suggested that the HdAGP shared limited sequence identity, and adopted a similar three-dimensional conformation to the bactericidal, permeability increasing, protein family, raising the possibility of a potential bactericidal function for this important reproductive/developmental protein.
S ummary
The technique of disc electrophoresis has been presented, including a discussion of the technical variables with special reference to the separation of protein fractions of normal human serum.
There is little information on the egg proteins of gastropod mollusks. Here we focus on PV2, a novel neurotoxin from snail eggs, studying its size, shape, structure, and stability, using small angle X-ray scattering (SAXS), absorption and fluorescence spectroscopy, circular dichroism, electron microscopy and partial proteolysis. Results indicate that PV2 is a compact and well folded oligomer of 130x44 A. It is an octamer of four 98 kDa heterodimers composed of 67 and 31 kDa subunits. Subunits are held together by disulfide bonds. Dimers are assembled into native PV2 by non-covalent forces. The larger subunit is more susceptible to proteolysis, indicating it is less compactly folded and/or more exposed. Quenching of tryptophan fluorescence showed a single class of tryptophyl side chains occluded in hydrophobic regions. Native structure shows loss of secondary structure (alpha+beta) at 6 M urea or 60-70 degrees C; the effects on the quaternary structure suggest an unfolding without disassembling of the protein. The 3D model of PV2 presented here is the first for an egg proteinaceous neurotoxin in animals.
The macromolecules contributed by the freshwater gastropod Biomphalaria glabrata, intermediate host of Schistosoma mansoni, to developing offspring inside egg masses are poorly known. SDS-PAGE fractionated egg mass fluids (EMF) of M line and BB02 B. glabrata were analyzed by MALDI-TOF (MS and tandem MS). A MASCOT database was assembled with EST data from B. glabrata and other molluscs to aid in sequence characterization. Of approximately 20 major EMF polypeptides, 16 were identified as defense-related, including protease inhibitors, a hemocyanin-like factor and tyrosinase (each with possible phenoloxidase activity), extracellular Cu-Zn SOD, two categories of C-type lectins, Gram-negative bacteria-binding protein (GNBP), aplysianin/achacin-like protein, as well as versions of lipopolysaccharide binding protein/bacterial permeability-increasing proteins (LBP/BPI) that differed from those previously described from hemocytes. Along with two sequences that were encoded by "unknown" ESTs, EMF also yielded a compound containing a vWF domain that is likely involved in defense and a polypeptide with homology to the Aplysia pheromone temptin. Further study of B. glabrata pheromones is warranted as these could be useful in efforts to control these schistosome-transmitting snails. Several of the EMF polypeptides were contained in the albumen gland, the organ that produces most EMF. Thus, parental investment of B. glabrata in immunoprotection of its offspring is indicated to be considerable.
An infection with Angiostrongylus cantonensis, the main causative agent for human eosinophilic encephalitis, can be acquired through the consumption of the freshwater snail Pomacea canaliculata. This snail also provides a suitable model to study the developmental morphology and behavior of A. cantonensis larvae, facilitated by the snail's distinct lung structure. We used microanatomy for studying the natural appearance and behavior of A. cantonensis larvae while developing within P. canaliculata. The distribution of refractile granules in the larval body and characteristic head structures changed during the developmental cycle. Two well-developed, rod-like structures with expanded knob-like tips at the anterior part were observed under the buccal cavity as early as the late second developmental stage. A "T"-shaped structure at the anterior end and its tenacity distinguished the outer sheath from that shed during the second molting. Early first-stage larvae obtained from fresh rat feces are free moving and characterized by a coiled tail, whereas a mellifluous "Q"-movement was the behavioral trait of third-stage A. cantonensis larvae outside the host tissue. In combination, the distribution of refractive granules, distinct head features, variations in sheaths, and behavioral characteristics can be utilized for differentiation of larval stages, and for distinguishing A. cantonensis larvae from those of other free-living nematodes.
Ovorubin, a 300-kDa thermostable oligomer, is the major egg protein from the perivitellin fluid that surrounds the embryos of the apple snail Pomacea canaliculata. It plays essential roles in embryo development, including transport and protection of carotenoids, protease inhibition, photoprotection, storage, and nourishment. Here, we report ovorubin dimensions and global shape, and test the role of electrostatic interactions in conformational stability by analyzing the effects of pH, using small-angle X-ray scattering (SAXS), transmission electron microscopy, CD, and fluorescence and absorption spectroscopy. Analysis of SAXS data shows that ovorubin is an anisometric particle with a major axis of 130 A and a minor one varying between 63 and 76 A. The particle shape was not significantly affected by the absence of the cofactor astaxanthin. The 3D model presented here is the first for an invertebrate egg carotenoprotein. The quaternary structure is stable over a wide pH range (4.5-12.0). At a pH between 2.0 and 4.0, a reduction in the gyration radius and a loss of tertiary structure are observed, although astaxanthin binding is not affected and only minor alterations in secondary structure are observed. In vitro pepsin digestion indicates that ovorubin is resistant to this protease action. The high stability over a considerable pH range and against pepsin, together with the capacity to bear temperatures > 95 degrees C, reinforces the idea that ovorubin is tailored to withstand a wide variety of conditions in order to play its key role in embryo protection during development.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
A simple procedure is described by which the principal protein component of the eggs and mature ovaries of six decapod crustaceans was isolated in relatively pure form and in large amounts. The component, in all cases, was a lipoprotein (30% lipid) which lacked protein-bound phosphorus and had an average molecular weight of 3.5 × 105. The relatively intense chromatic properties of the crustacean lipoproteins were due to the presence of a carotenoid noncovalently bonded to the lipid and/or protein. Those proteins displaying colors (purple, blue, andgreen) associated with shorter wavelengths of the visible spectrum undergo spectral changes dependent upon solvent conditions. Although these crustacean proteins may not be homologous to the vertebrate lipovitellins, they appear to serve in the same capacity, and generic use of the term "lipovitellin" is thus suggested for the major high-density lipoprotein found within animal eggs. The crustacean lipovitellins appear to offer several advantages for studies of lipoproteins in general and lipoprotein synthesis in particular.
Aplysianin-A, an antibacterial glycoprotein in the albumen gland of the sea hare Aplysia kurodai, inhibited the growth of both Gram-positive and Gram-negative bacteria. Aplysianin-A cDNA clones were isolated from an albumen gland cDNA library. Sequence analysis reveals that aplysianin-A is produced as a precursor protein of 556 amino acid residues with a signal peptide of 19 amino acid residues and contains 6 potential N-glycosylation sites. Aplysianin-A mRNA was expressed tissue-specifically in the albumen gland. Homology search reveals that aplysianin-A has a 50% overall amino acid sequence homology to achacin, an antibacterial glycoprotein of the giant African snail Achatina fulica.
The lipid and protein composition of the perivitelline fluid of the eggs of Pomacea canaliculata was investigated. Two lipoproteins (PV 1 and PV 2) and one lipoprotein fraction (PV 3) were detected for the first time in gastropods. They represent 57.0, 7.5, and 35.5% of the egg total proteins, respectively. PV 1 is a glyco-carotene-protein complex with characteristics of a very high-density lipoprotein (VHDL). It has 0.33% lipids, mainly free sterols and phospholipids. The particle has a MW of 300 Kd and is composed of three subunits of 35, 32, and 28 Kd, respectively. PV 2 particle is a VHDL of 400 Kd and 3.75% lipids. The major lipid classes are free sterols and phospholipids and also have significant quantities of energy-providing triacylglycerides and free fatty acids. It is composed of two apoproteins of 67 and 31 Kd. PV 3 density corresponds to a high-density lipoprotein (HDL). It was fractionated into two subfractions "h" and "p". Fraction "h" contains 5.16% lipids, mainly free sterols, phospholipids, and free fatty acids, and two particles of 100 and 64 Kd. Dissociating electrophoresis showed two subunits of 34 and 29 Kd. Fraction "p" is composed of a single particle of 26 Kd that contains 9.5% lipids, which represents 30% of the total egg lipids. It has high levels of a carotenoid pigment. Besides it contains free fatty acids, hydrocarbons, sterified sterols, and triacylglycerides. These three fractions are probably the major supply of lipids and amino acids for the developing embryo.
A method is proposed to restore ab initio low resolution shape and internal structure of chaotically oriented particles (e.g., biological macromolecules in solution) from isotropic scattering. A multiphase model of a particle built from densely packed dummy atoms is characterized by a configuration vector assigning the atom to a specific phase or to the solvent. Simulated annealing is employed to find a configuration that fits the data while minimizing the interfacial area. Application of the method is illustrated by the restoration of a ribosome-like model structure and more realistically by the determination of the shape of several proteins from experimental x-ray scattering data.
Eggs and egg masses of the freshwater gastropod mollusc Lymnaea provide a microenvironment for developing embryos. Secretions of the exocrine albumen gland of Lymnaea are packaged in the eggs of an egg mass before the eggs are laid externally. The perivitelline fluid that directly surrounds individual oocytes is the main source of nutrition for developing embryos. During early stages of development, the perivitelline fluid is initially internalized by pinocytosis and degraded by lysosomes; in later stages, the embryo ingests the fluid. We previously found that the albumen gland produces large amounts of Lymnaea epidermal growth factor. The albumen gland also appears to produce significant amounts of a novel Lymnaea trypsin inhibitor (LTI), a second peptide that was purified and characterized from Lymnaea albumen gland extracts. The primary structure was determined by microsequence analysis, mass spectrometry, and C-terminal sequence analysis, and showed that LTI is a 57-residue glycosylated peptide. Comparison of the LTI sequence with other known serine protease inhibitors indicates that LTI is a member of the bovine pancreatic trypsin inhibitor family. Reverse phase-high performance liquid chromatography, microsequence analysis, mass spectrometry, and immunocytochemistry demonstrated that abundant amounts of intact LTI are packaged in egg masses. The presence of a trypsin inhibitor in the perivitelline fluid compartment of the egg mass may minimize digestion of peptides and proteins in the perivitelline fluid that are important for the development of the embryo, for example, Lymnaea epidermal growth factor.
The site of synthesis of mollusc lipoproteins is hitherto unknown and was investigated for perivitellin 2 (PV2), an egg lipoprotein found in the freshwater snail Pomacea canaliculata. Tissues (albumen gland, gonad-digestive gland complex, and muscle) from vitellogenic females were incubated in vitro with 14C-leucine at 25 degrees C for 12 hr. At the end of incubation, soluble proteins from tissue homogenates and medium were analyzed for de novo protein synthesis by electrophoresis and HPLC, and radiolabeled proteins were quantified by liquid scintillation. Two albumen gland radiolabeled proteins (67 and 31 kDa) co-migrated with the subunits of PV2, and they represented 6.0% of the total labeled protein in that tissue. Western blot analysis confirmed the presence of PV2 only in the albumen gland. In vivo experiments where adult females were injected with 3H-leucine revealed that PV2 was not present in hemolymph. ELISA analysis in all tissues of the snail confirmed the PV2 presence only in the albumen gland and developing eggs with levels of 26 and 98 mg/g protein, respectively. Therefore, the albumen gland is the only site for PV2 synthesis, and no extra-gland synthesis, circulation, or accumulation could be found. PV2 subunits were further characterized analyzing N-terminal sequences which showed no homology with other proteins.
A novel role of ovorubin as a protection system against oxidative damage in eggs from Pomacea canaliculata was investigated. Carotenoid composition, and their antioxidant capacity, as well as the carotenoid-apoprotein interaction, were studied for this lipoglycocarotenoprotein. Carotenoid extracts from ovorubin were analysed by TLC and spectrophotometry. The major carotenoid was astaxanthin in its free (40%), monoester (24%), and diester (35%) forms, mainly esterified with 16:0 fatty acid. The antioxidant capacity of ovorubin carotenoids was studied by the inhibition of microsomal oxidation in a non-enzymatic system, showing strong protection against oxidative damage (IC50=3.9 nmol/mg protein). The carotenoid-apoprotein interaction was studied by spectrophotometry and electrophoresis using reconstituted ovorubin. Astaxanthin does not seem to affect the structural characteristics of ovorubin, however the carotenoid-protein association significantly protected astaxanthin against oxidation. Ovorubin therefore, besides its role in providing energy and structural precursors during embryogenesis, would be an antioxidant carrier, protecting at the same time this pigment from oxidation in the perivitellin fluid environment of the egg.
Helix pomatia agglutinin (HPA) is a N-acetylgalactosamine (GalNAc) binding lectin found in the albumen gland of the roman snail. As a constituent of perivitelline fluid, HPA protects fertilized eggs from bacteria and is part of the innate immunity system of the snail. The peptide sequence deduced from gene cloning demonstrates that HPA belongs to a family of carbohydrate-binding proteins recently identified in several invertebrates. This domain is also present in discoidin from the slime mold Dictyostelium discoideum. Investigation of the lectin specificity was performed with the use of glycan arrays, demonstrating that several GalNAc-containing oligosaccharides are bound and rationalizing the use of this lectin as a cancer marker. Titration microcalorimetry performed on the interaction between HPA and GalNAc indicates an affinity in the 10(-4) M range with an enthalpy-driven binding mechanism. The crystal structure of HPA demonstrates the occurrence of a new beta-sandwich lectin fold. The hexameric quaternary state was never observed previously for a lectin. The high resolution structure complex of HPA with GalNAc characterizes a new carbohydrate binding site and rationalizes the observed preference for alphaGalNAc-containing oligosaccharides.