Long-Term Evaluation of Poly(lactic acid) (PLA) Implants in a Horse: An Experimental Pilot Study.

In horses, there is an increasing interest in developing long-lasting drug formulations, with biopolymers as viable carrier alternatives in addition to their use as scaffolds, suture threads, screws, pins, and plates for orthopedic surgeries. This communication focuses on the prolonged biocompatibility and biodegradation of PLA, prepared by hot pressing at 180 °C. Six samples were implanted subcutaneously on the lateral surface of the neck of one horse. The polymers remained implanted for 24 to 57 weeks. Physical examination, plasma fibrinogen, and the mechanical nociceptive threshold (MNT) were performed. After 24, 28, 34, 38, and 57 weeks, the materials were removed for histochemical analysis using hematoxylin-eosin and scanning electron microscopy (SEM). There were no essential clinical changes. MNT decreased after the implantation procedure, returning to normal after 48 h. A foreign body response was observed by histopathologic evaluation up to 38 weeks. At 57 weeks, no polymer or fibrotic capsules were identified. SEM showed surface roughness suggesting a biodegradation process, with an increase in the median pore diameter. As in the histopathological evaluation, it was not possible to detect the polymer 57 weeks after implantation. PLA showed biocompatible degradation and these findings may contribute to future research in the biomedical area.

Assessing the ovarian development of Macrobrachium amazonicum (Heller, 1862) phenotypes by means of an integrative analysis.

Macrobrachium amazonicum is a species of economic interest with a wide distribution in the Americas and high morphological and reproductive variability. Three phenotypes can be observed in this species: i) large-size amphidromous, ii) large-size and iii) small-size hololimnetic prawns. In the present work, the morphological, histochemical and ultrastructural aspects of ovarian development in the three phenotypes were comparatively analyzed. In addition, the interaction between the ovary and the hepatopancreas was investigated in these phenotypes through the use of gonadosomatic (GSI) and hepatosomatic (HSI) indices. Despite the morphological differences and different reproductive strategies adopted by the females, the macroscopic, histochemical and ultrastructural patterns of ovarian development showed no differences between the phenotypes. The ovaries were macroscopically classified into five stages of development (I to V). In early stages (I and II), the ovaries are full of oogonia, previtellogenic oocytes and oocytes in primary or endogenous vitellogenesis. At these stages, the rough endoplasmic reticulum (RER) produces a granular electron-dense material and sends it to the Golgi apparatus, where it will be modified, compacted and transformed into immature yolk granules. From stage III, secondary or exogenous vitellogenesis begins (with no interruption of endogenous vitellogenesis), where follicular cells nourish the oocytes and extracellular material is absorbed by endocytic vesicles, which fuse with immature yolk granules (forming mature granules) or with existing mature yolk granules. In stages IV and V, secondary vitellogenesis continues and mature yolk granules progressively occupy the cytoplasm. In M. amazonicum, the patterns of increase in oocyte diameter are quite similar between phenotypes, being greater in the small-size phenotype. This is related to the formation of larger oocytes/eggs and the production of large lipid reserves for their larvae. Changes in GSI and HSI during ovarian development show strong similarity between phenotypes, supporting the results obtained by histology and ultrastructure. Females in stages III and IV mobilize hepatopancreas reserves for ovarian maturation, which justifies the higher HSI values recorded in these stages. On the other hand, females in stage V show higher GSI and lower HSI values, indicating a mobilization of resources for the end of ovarian development as the females are ready to spawn.

Is there a trade-off between sperm production and sexual weaponry in the Amazon River prawn Macrobrachium amazonicum (Heller, 1862)?

The Amazon River prawn Macrobrachium amazonicum shows populations with four well-defined morphotypes in males. Dominant males of morphotypes green claw 1 (GC1) and green claw 2 (GC2) have large bodies and chelipeds and a higher reproductive success in comparison with the submissive morphotypes - translucent claw (TC) and cinnamon claw (CC). However, recently, some populations of the species do not have dominant morphotypes. Here, we compared the patterns of spermatic production and concentration among morphotypes and populations with three different phenotypes: (i) large-size amphidromous prawns, and (ii) large-size ("i" and "ii" with dominant morphotypes) and (iii) small-size hololimnetic prawns (without morphotypes). We described the spermatogenesis and the histochemical features of vasa deferentia (VD) and evaluated the relationship between the investment in spermatic production and sexual weapons acquisition in males of different phenotypes. The spermatic production and concentration in populations with four morphotypes were similar between morphotypes. The exception was the CC morphotype in which males had the seminiferous tubules filled with spermatocytes and low spermatic concentration. The spermatogenesis, spermiogenesis, and VD structure were not different among the studied phenotypes and populations. The seminal fluid of M. amazonicum is comprised by glycoproteins and by concentric layers of secretions of types I, II (basophilic), and III (eosinophilic). We could infer that males of dominant morphotypes allocate a higher amount of energy to the development of strong sexual weapons at the expense of the energy allocated to the reproductive system during the sequential growth. Inversely, small-size males direct more energy toward the spermatic production and transference at the expense of sexual weapons. Therefore, there is a clear trade-off between the investment in the gonadal and sexual weapons development in males of M. amazonicum.

Much more than hooked: Setal adaptations for camouflage in Macrocoeloma trispinosum (Latreille, 1825) (Crustacea: Decapoda: Brachyura).

Several majoid crabs are known to adhere exogenous materials to their bodies, a behaviour called decoration. Until now, the adhesion of exogenous materials to the body is most attributed to the well-known hooked setae. Here, we analysed the carapace of Macrocoeloma trispinosum (Latreille, 1825) under light and electron microscopy to study the different mechanisms allowing majoid crabs to decorate themselves. Five setal types are described here, of which four for the first time: velvet type I, velvet type II, depressa and cattail seta. These setae are morphologically and histologically detailed, and new hypotheses about the fixation of exogenous material on the carapace are explored. M. trispinosum has a complex setal apparatus for the adhesion of the decoration, with tegumental ducts along the shaft of most setae. These tegumental ducts are connected to glands formed by large cells arranged radially (rosette or acini) at the base of the setae, in the connective tissue, just below the epithelium. We could observe these glands in different stages of maturation, and no valve-like structure was observed, which may indicate a continuous flow of protein secretion that could serve as an adhesive substance found in the apex of most setae. This is the first record indicating a potential chemical adhesion mechanism aiding the masking process in decorator crabs.

From copula to fertilization: The reproduction of primitive crab Hypoconcha parasitica (Linnaeus, 1763) (Podotremata: Dromiidae).

This study describes the mating behavior of Hypoconcha parasitica under laboratory conditions highlighting the spermathecal morphology and focusing on the seminal fluid storage and release of spermatozoa. The pairs were kept in aquaria where the mating behavior was recorded and described. The spermathecae of the female were analyzed using scanning electron microscopy, X-ray micro-CT, histology, and histochemistry. No pre- or post-copulatory mate guarding was observed in H. parasitica. The sperm transfer occurred with each pair maintaining the protection shield (bivalve shell) on their dorsum. The pair of spermathecae is covered exclusively by the cuticle, following the Podotremata pattern. Many muscle fiber bundles are attached to the cuticular wall facing toward the cephalothorax cavity, especially covering the lateral and slightly dorso-anterior region toward the apodeme of Sternite 7. The spermathecal organization indicates that the process of sperm release during fertilization occurs through muscular action exerted by the female on the wall of the chamber. Thus, the musculature distribution in Hypoconchinae distinguishes them from described for other Podotremata such as Homolidae, which shows the musculature associated with the spermathecae aperture. Like the Homolidae, the first pleopod in H. parasitica seems to take part in the transfer/ movement of spermatozoa and oocytes between the sternum and abdomen, which forms a temporary chamber where fertilization occurs. In conclusion, the spermathecal morphology and associated structures bring new insights to the mechanisms involved in the sperm storage and fertilization of primitive crabs and how the Dromiidae spermatheca perform a new pattern among the poorly studied Podotremata.

The extract of the jellyfish Phyllorhiza punctata promotes neurotoxic effects.

Phyllorhiza punctata (P. punctata) is a jellyfish native to the southwestern Pacific. Herewith we present the biochemical and pharmacological characterization of an extract of the tentacles of P. punctata. The tentacles were subjected to three freeze-thaw cycles, homogenized, ultrafiltered, precipitated, centrifuged and lyophilized to obtain a crude extract (PHY-N). Paralytic shellfish poisoning compounds such as saxitoxin, gonyautoxin-4, tetrodotoxin and brevetoxin-2, as well as several secretory phospholipase A(2) were identified. PHY-N was tested on autonomic and somatic neuromuscular preparations. In mouse vas deferens, PHY-N induced phasic contractions that reached a peak of 234 ± 34.7% of control twitch height, which were blocked with either 100 µ m of phentolamine or 1 m m of lidocaine. In mouse corpora cavernosa, PHY-N evoked a relaxation response, which was blocked with either L-N(G) -Nitroarginine methyl ester (0.5 m m) or 1 m m of lidocaine. PHY-N (1, 3 and 10 µg ml(-1) ) induced an increase in tonus of the biventer-cervicis neuromuscular preparation that was blocked with pre-treatment of galamine (10 µ m). Administration of 6 mg kg(-1) PHY-N intramuscularly produced death in broilers by spastic paralysis. In conclusion, PHY-N induces nerve depolarization and nonspecifically increases neurotransmitter release.

Is Potimirim potimirim (Crustacea, Decapoda, Atyidae) a protandric hermaphrodite species? Behavioral and morphological aspects of the reproductive system.

We evaluated the mating behavior of Potimirim potimirim in the laboratory based on the assumption that this caridean might be hermaphroditic. We also performed histology and scanning electron microscopy analyses of the reproductive system of females, males, and differentiated individuals. The mating experiments produced three behavioral stages, namely, interaction, lateral positioning, and copulation (which did not necessarily occur in this sequence). The hypothesis of a random pure searching mating was corroborated by the lack of male courtship, postcopulatory guarding, and the high aggregation of individuals. Three macroscopic ovarian stages were recorded in adult females: rudimentary, developing and mature. Secondary vitellogenesis begins at the developing stage, producing mature yolk. The male reproductive system is formed by testes and the vasa deferentia (VD), that is divided into three regions: proximal, middle, and distal. The proximal VD has a typhlosole that produces a thin layer of type II secretion around the central spermatozoa mass immersed in a type I secretion. External and adherent type III secretion is produced from the MVD to DVD, and both compound the primordial spermatophore. Potimirim potimirim has a gonochoric reproductive system and mating behavior and its sexual system does not fit into any of the previously described protandric systems.

Myzobdella platensis (Hirundinida: Piscicolidae) is true parasite of blue crabs (Crustacea: Portunidae).

Leeches exhibit a marked scope of diversity, including different kinds of symbiosis. The aim of the present study was to demonstrate through biochemical and histological analysis that a species of piscicolid leech, Myzobdella platensis, is a true parasite of blue crabs, feeding on their hemolymph and using them as a site for cocoon deposition. In a total of 48 blue crabs collected on October 2007 at 3 sites of the São Vicente Estuary, 12 specimens were infested with leeches. Callinectes bocourti (n = 7) was the most infested species with leeches and cocoons; it was chosen for biochemical and histological assays. The immunoblotting assays showed a positive reaction of the proteins in the intestinal samples of leeches collected from crabs using antihemocyanin polyclonal antibody of Ampullaria canaliculata. In addition, leech intestinal samples were recognized by antihemolymph polyclonal antibody of nonparasitized blue crabs. Histological sections of leech gut showed hemocytes and a granular matrix similar to those found in crab blood vessels. Collectively, this evidence strongly suggests a parasitic interaction between the leech M. platensis and the blue crab C. bocourti, in which the former utilizes the latter as a site for cocoon deposition and possibly for dispersal similar to that proposed for Myzobdella lugubris in Callinectes sapidus in North America.

Seminal fluid production and sperm packaging in dromiid crabs (Brachyura, Podotremata).

Reproductive anatomy, including sperm storage structures and sperm transfer, is an important feature used to analyze phylogenetic relationships among taxa. We describe the male reproductive anatomy, seminal fluid production and packaging of spermatozoa in the vas deferens of primitive crabs. In all species of Dromiidae, the testes were tubular type and the vas deferens is a tube with a simple epithelium. The spermatozoa are in a central mass immersed in type I secretion, forming a large spermatic cord. In Moreiradromia antillensis and Dromia erythropus the spermatic cord is surrounded by a more complex secretion layer composed by protein granules and polysaccharides that seem to be responsible to form the sperm plaque which was absent in Hypoconcha. The sperm plaque was found in all Dromiinae species, which may result from the mating and probably act as a barrier to subsequent copulations. These patterns of elongated coenospermic spermatophore found here are unique and different from the spermatophores of all true crabs and may be considered a plesiomorphic state to spermatophoric structure in Brachyura. Our results show a novel type of spermatozoa packaging and new insights into how sperm might be transferred in Podotremata.

Leucism in the marbled swamp eel Synbranchus marmoratus Bloch, 1795 (Synbranchiformes: Synbranchidae) / Leucismo em muçum Synbranchus marmoratus Bloch, 1795 (Synbranchiformes: Synbranchidae)

Abstract: Leucism is an anomaly recorded in fish species as well as in many other groups of animals in natural environments. An individual with leucism is characterized by the loss of brown or black pigmentation over most of the body. Here, we recorded this condition for an individual of marbled swamp eel Synbranchus marmoratus (Synbranchiformes: Synbranchidae) captured in the Furnas megareservoir, Grande River basin, in Minas Gerais state, southeastern Brazil. The individual had a general color of the body with shades ranging from pale to intense yellowish, but the eyes were black. We also present a discussion on the influence of leucism on the survival of the individual in this study.

Resumo: Leucismo é uma anomalia registrada em espécies de peixes assim como em muitos outros grupos de animais em ambientes naturais. Um indivíduo com leucismo é caracterizado pela perda de pigmentação marrom ou preta na maior parte do corpo. Neste trabalho, nós registramos essa condição para um indivíduo de muçum Synbranchus marmoratus (Synbranchiformes: Synbranchidae) capturado no megareservatório de Furnas, bacia do Rio Grande, no estado de Minas Gerais, sudeste brasileiro. O indivíduo apresentava coloração geral do corpo com tonalidades que variavam do amarelado pálido ao intenso, mas os olhos eram pretos. Nós também apresentamos uma discussão sobre a influência do leucismo na sobrevivência do indivíduo deste estudo.

Comparative spermatozoal ultrastructure and molecular analysis in dromiid crabs and their phylogenetic implications for Dromiidae and Podotremata (Decapoda: Brachyura).

We described the spermatozoal ultrastructure and conducted a molecular analysis of Dromiidae Hypoconcha parasitica, Hypoconcha arcuata, Moreiradromia antillensis and Dromia erythropus. To elucidate the relationship between the different species of this brachyuran group, we also compared the spermatozoal morphologies and phylogenetic positioning among species of Dromiidae, Dromioidea and Podotremata. Specimens were collected from the northern coast of São Paulo, Brazil and were fixed and processed followed by transmission electron microscopy and molecular analysis routines. The Dromiidae spermatozoa studied are characterized by a discoidal acrosome, with three or four concentric zones, which are centrally separated by a bilaterally capitate perforatorial chamber, with a "mushroom"-shaped apex in the Hypoconchinae and a "T-shape" in Dromiinae. Above the perforatorial chamber, there is an apical protuberance, continuous with the subopercular region and the operculum, which forms a low, centrally perforated dome. Under differential interference contrast microscopy, the spermatozoa show 3 to 4 radial arms. The spermatozoal characters in Hypoconchinae and Dromiinae do not separate these subfamilies from the Dromiidae and Dromioidea. Ultrastructural differentiation was only found between representative Dromioidea and other Podotremata. Thus, the spermiotaxonomy of these Hypoconcha, Moreiradromia and Dromia species corroborated previous morphological and molecular studies, supporting the monophyly of Dromiidae and Dynomenidae in relation to Homolidae and Latreilliidae.

Sperm ultrastructure of shrimps from the family Penaeidae (Crustacea: Dendrobranchiata) in a phylogenetic context.

We describe the sperm ultrastructure of six penaeid species, including at least one member of each tribe (Penaeini, Parapenaeini and Trachypenaeini). Fragments of the vas deferens of the Penaeidae Farfantepenaeus brasiliensis, Farfantepenaeus paulensis, Litopenaeus schmitti, Parapenaeus americanus, Rimapenaeus constrictus and Xiphopenaeus kroyeri were fixed and processed according to the routine for transmission electron microscopy. The morphological results were contextualized in an evolutionary perspective using molecular markers for the phylogenetic reconstruction of this group. A phylogram was proposed by Bayesian inference based on 1007 bp of 33 sequences of the combined genes (16S rDNA and COI mtDNA) from 27 dendrobranchiate specimens. Our findings show that morphological differences in the sperm ultrastructures of members among the tribes of Penaeidae can be used as a baseline to understand their evolutionary relationships. Individuals from the Penaeini tribe show plesiomorphic characteristics in the sperm ultrastructure compared to the Trachypenaeini tribe from which they were derived, such as shrimp from family Sicyoniidae. The morphological complexity of the sperm of the different penaeid members corroborated with the genetic phylogeny, which showed different clades for each tribe and the close relationship with Sicyoniidae. The sperm features of the selected species studied here reflected their evolutionary history. These features confirm the previous phylogenetic hypothesis and question the monophyly of Penaeidae, which should be verified in the future with a more complete set of representative members of each tribe.

Morphological changes in the seminal receptacle during ovarian development in the speckled swimming crab Arenaeus cribrarius.

To understand sperm plug dissolution and spermatophore dehiscence in Portunidae, histological and ultrastructural changes in the seminal receptacle (SR) of Arenaeus cribrarius were investigated during ovarian development. In juvenile females the SR was filled with acid polysaccharides and the dorsal epithelium was stratified. Mated females with rudimentary ovaries showed a large SR filled by a glycoprotein sperm plug. This plug was present until the developing-ovary stage, when spermatophore dehiscence and intense holocrine secretions in the dorsal dense layer occurred. The plug was absent after the intermediate stage, and the SR became flaccid. The secretion produced moved the spermatophores into the ventral region. The modified dorsal epithelium in the transition between the dorsal and ventral regions released acid polysaccharides, which were found among the sperm, by exocytosis. The morphological changes of the SR in A. cribrarius, including the presence of the sperm plug, followed the macroscopic pattern observed in other members of Portunidae, such as blue crabs. However, in this species dissolution of the sperm plug was synchronized with ovarian development and occurred simultaneously with spermatophore dehiscence, showing the evolutionary relationship of the seminal receptacle and the female reproductive system to the storage of spermatophores and spermatozoa.

Effects of tributyltin exposure in hermit crabs: Clibanarius vittatus as a model.

Tributyltin (TBT) contamination affects the reproductive system of many species of invertebrates worldwide. The present study was designed to evaluate the effects of exposure to TBT pollution on the reproduction of the hermit crab Clibanarius vittatus. An orthogonal experiment was designed with two treatments: contamination (with or without TBT in the food) and crab sex (males and females). The animals were reared in the laboratory for nine months, and macroscopic and histological analyses of reproductive organs were carried out after the end of the experiment. Tributyltin was recorded in exposed crabs, but no morphological alterations were detected in the gonads of males, regardless of whether they were exposed to TBT. In contrast, females exposed to TBT displayed disorganization and atrophy of their ovaries, thus directly affecting reproduction in this hermit crab species. This effect observed in female hermit crabs may harm populations located in harbor regions, where TBT concentration is high, even after the worldwide TBT ban.

Antibacterial and antiparasitic effects of Bothrops marajoensis venom and its fractions: Phospholipase A2 and L-amino acid oxidase.

Some proteins present in snake venom possess enzymatic activities, such as phospholipase A(2) and l-amino acid oxidase. In this study, we verify the action of the Bothrops marajoensis venom (BmarTV), PLA(2) (BmarPLA(2)) and LAAO (BmarLAAO) on strains of bacteria, yeast, and Leishmania sp. The BmarTV was isolated by Protein Pack 5PW, and several fractions were obtained. Reverse phase HPLC showed that BmarPLA(2) was isolated from the venom, and N-terminal amino acid sequencing of sPLA(2) showed high amino acid identity with other lysine K49 sPLA(2)s isolated from Bothrops snakes. The BmarLAAO was purified to high molecular homogeneity and its N-terminal amino acid sequence demonstrated a high degree of amino acid conservation with others LAAOs. BmarLAAO was able to inhibit the growth of P. aeruginosa, C. albicans and S. aureus in a dose-dependent manner. The inhibitory effect was more significant on S. aureus, with a MIC=50 microg/mL and MLC=200 microg/mL. However, the BmarTV and BmarPLA(2) did not demonstrate inhibitory capacity. BmarLAAO was able to inhibit the growth of promastigote forms of L. chagasi and L. amazonensis, with an IC(50)=2.55 microg/mL and 2.86 microg/mL for L. amazonensis and L. chagasi, respectively. BmarTV also provided significant inhibition of parasitic growth, with an IC(50) of 86.56 microg/mL for L. amazonensis and 79.02 microg/mL for L. chagasi. BmarPLA(2) did not promote any inhibition of the growth of these parasites. The BmarLAAO and BmarTV presented low toxicity at the concentrations studied. In conclusion, whole venom as well as the l-amino acid oxidase from Bothrops marajoensis was able to inhibit the growth of several microorganisms, including S. aureus, Candida albicans, Pseudomonas aeruginosa, and Leishmania sp.

Ultra morphology of the digestive system of Anastrepha fraterculus AND Ceratitis capitata (DIPTERA TEPHRITIDAE)

Anastrepha fraterculus and Ceratitis capitata are widely distributed fruit flies that cause significant damageto fruit crops in tropical and temperate regions. The economic importance of these flies has resulted innumerous studies of their biology, with particular emphasis on their control and management. However,various aspects of the biology of these species are still poorly understood. In this work, we used scanningelectron microscopy (SEM) to examine the external anatomy and organization of the digestive system inthese two species. Adult males and females of A. fraterculus and females of C. capitata were dissected inphysiological saline solution, and the digestive tracts were removed and prepared for microscopy. SEMshowed that the crop was covered by a strong muscular layer that consisted of circular fibers connected bylongitudinal fibers; this arrangement was probably related to the post-feeding behavior of these flies in whichthe crop contents are regurgitated and reingested. The size of the rectum varied and was probably related tothe different body sizes of the two species.