Post-larval morphology, growth, and development of Uca cumulanta Crane, 1943 (Crustacea, Decapoda, Ocypodidae) under laboratory conditions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Larval stages of Stenocionops furcatus (Olivier, 1791) (Decapoda : Brachyura : Majoidea) and a reappraisal of larval morphological characters for Mithracidae

Stenocionops furcatus is a spider crab found in the western Atlantic, from Georgia, USA to Rio Grande do Sul, Brazil, on sand, coral, rocks or mud bottoms from the intertidal zone to 180 m. We describe all laboratory-reared larval stages of S. furcatus obtained from the northern coast of São Paulo State, Brazil, and compare our data with existing larval descriptions for the genus and other mithracids. The larval development of S. furcatus consists of two zoeal stages and one megalopa. The durations of the first and second zoeal stage were similar to4 and 5 days respectively, the megalopa appearing 10-18 days after hatching. Our results show that the zoeae of S. furcatus differ from those of other Mithracidae by possessing four setae on the proximal lobe of the coxal endite of the maxilla, instead of five, and by the presence of mid-dorsal setae on the third abdominal somite in the second zoeal stage, which are lacking in other mithracids. Larval descriptions for Stenocionops in two previous publications were attributed to the subspecies S. furcatus coelatus from the Caribbean. Larvae from Brazilian waters closely resemble one of these accounts, suggesting that this taxon extends beyond the West Indies and that the other description represents larvae of S. furcatus furcatus. Additional morphological details, not available previously, are provided.

Larval development of Epialtus bituberculatus H. Milne Edwards, 1834 (Crustacea : Decapoda : Brachyura : Majidae) with comments on majid larvae from the southwestern Atlantic

The larval development of the spider crab Epialtus bituberculatus H. Milne Edwards which Lives on rocky shores with algae such as Sargassum and Hypneia, is described. Larvae were obtained from ovigerous females collected in Ubatuba, State of São Paulo, Brazil. Rearing was carried out at 24 +/- 1 degreesC, with an average salinity of 35 parts per thousand. Larval development consists of two zoeal stages and one megalopa. Zoeal development was completed in 9.5 days. Analysis indicated that zoeae of E. bituberculatus are very similar to those of E. brasiliensis Dana and Acanthonyx scutiformis (Dana). Differences noted between these species pertain to the setation of the carapace, maxillule and second maxilliped. The main morphological features useful for identification are presented together with a summary of features that characterize larvae of majid subfamilies in Brazil. A key for the identification of southwestern Atlantic majid zoeae to the family level is provided.

Larval stages of Paradasygyius depressus (Bell, 1835) (Crustacea : Decapoda : Brachyura : Majidae) and a phylogenetic analysis for 21 genera of Majidae

All larval stages and the first crab instar of Paradasygyius depressus (Bell) were obtained in laboratory culture. Larval development consists of two zoeal stages, followed by the megalopa. Each larval stage is described in detail. Beginning with the first zoea, the duration of each stage was 4--7 (4.5 +/- 0.7), 4-5 (4.5 +/- 0.5), and 7 days, the megalopa and first crab instar appearing 11 +/- 1 and 15 days after hatching, respectively. A phylogenetic analysis of 21 genera of Majidae is provided based on 34 zoeal and three megalopal characters. The phylogenetic analysis resulted in four equally parsimonious trees 173 steps long (CI = 0.66, RI = 0.71, and RC = 0.47) supporting the monophyly of Oregoniinae, Majinae, and Inachinae (with the exclusion of Macrocheira de Haan incertae sedis). Based on general agreement of sister-group hypotheses, we provide sets of larval characters that define Oregoniinae, Majinae, and Inachinae. Our phylogenetic hypothesis suggests that Oregoniinae is the most basal clade within the Majidae, and Majinae and the clade (Epialtus H. Milne Edwards + Inachinae [excluding Macrocheira incertae sedis]) are sister taxa. Within Inachinae, all trees suggest that Inachus Weber and Macropodia Leach are sister taxa nested as the most derived clade, followed by Achaeus Leach, Pyromaia Stimpson, Paradasygyius Garth, Anasimus A. Milne-Edwards, and the most basal Stenorhynchus Lamarck. The sister-group relationships of the clade (Pisa Leach (Taliepus A. Milne-Edwards + Libinia Leach)), Mithrax Latreille and Microphrys H. Milne Edwards remained unresolved.

On the larval stages of Macrocoeloma diplacanthum (Decapoda : Brachyura : Majidae), and a review of mithracine phylogenetic aspects

Larval development of Macrocoeloma diplacanthum (Stimpson) consists of two zoeal stages, followed by the megalopa. Each larval stage is described in detail. The duration of the zoeal stages was 2-3 (2.4 +/- 0.5) and 3-4 (3.5 +/- 0.5) days for the first and second zoea, respectively, the megalopa phase appearing 6-8 (7.0 +/- 0.5) days after hatching. Unlike for other majids, zoeal stages of M. diplacanthum can be readily distinguished by their distended forehead with strong underlying muscle bands, undercut dorsal carapace spine, and spine on the terminal endopod segment of the first maxilliped. No other known mithracine or majid zoeae exhibit this combination of features. Our zoeal account of M. diplacanthum from Mexico is remarkably consistent with Floridian specimens previously described. However, we have found some differences between descriptions, which could be attributed to natural variation or inadequate description. Previous attempts to evaluate the relationships within Mithacinae have been based on larval characters widely distributed throughout Majidae and therefore are considered inadequate to infer sister-group relationships. The phylogenetic analysis of majids suggested that the position of Mithracinae is still uncertain, as is its monophyletic status. We recommend that additional characters, particularly of the megalopa phase, be sought for a better resolution of majid evolutionary history.

Searching for larval support for majoid families (Crustacea : Brachyura) with particular reference to Inachoididae Dana, 1851

We re-evaluated the larval support for families within majoids using the Wilcoxon signed-rank test with emphasis on Inachoididae. To accomplish our objectives, we added 10 new taxa, two of which are traditionally assigned to the family of special interest, to a previous larval database for majoids, and re-appraised the larval characters used in earlier studies. Phylogenetic analysis was performed with PAUP* using the heuristic search with 50 replicates or the branch-and-bound algorithm when possible. Multi-state transformation series were considered unordered; initially characters were equally weighted followed by successive weighting, and trees were rooted at the Oregoniidae node. Ten different topological constraints were enforced for families to evaluate tree length under the assumption of monophyly for each taxonomic entity. Our results showed that the tree length of most constrained topologies was not considerably greater than that of unconstrained analysis in which most families nested as paraphyletic taxa. This may indicate that the present larval database does not provide strong support for paraphyly of the taxa in question. For Inachoididae, although the Wilcoxon signed-rank test rejected a significant difference between unconstrained and constrained cladograms, we were unable to provide a single synapomorphy for this clade. Except for the conflicting position of Leurocyclus and Stenorhynchus, the two clades correspond to the traditional taxonomic arrangement. Among inachoidids, the clade (Anasimus (Paradasygyius (Collodes + Pyromaia))) is supported, whereas for inachids, the clade (Inachus (Macropodia + Achaeus)) is one of the most supported clades within majids. As often stated, only additional characters will provide a better test for the monophyly of Inachoididae and other families within Majoidea.

Larval development of Apiomithrax violaceus (A. Milne Edwards, 1868) (Decapoda : Brachyura : Majoidea : Pisidae) reared in laboratory conditions, and a review of larval characters of Pisidae

Apiomithrax violaceus (A. Milne Edwards, 1868 ) is a pisid majoid crab occurring in tropical and subtropical coastal waters of the eastern and western South Atlantic. Larval development consists of two zoeal stages and a megalopa. Beginning with the first zoea, the duration of each larval stage at 24degreesC was 3-8 (5+/-1), 3-5 (4+/-0.5) and 9-15 (11+/-2) days, the megalopa and first crab instar appearing 9-11 (10+/-1) and 20-27 (23+/-2) days after hatching, respectively. Larval characters agree with those proposed for the Majoidea, in having nine or more setae on the scaphognathite in the first zoea and well-developed pleopods in the second zoea. However, larvae of A . violaceus do not fit larval pisid features. Zoeal stages differ from most other Pisidae in having lateral spines, a long rostral spine extending beyond the antenna, two spines per telson fork and a dorsolateral process on the third abdominal somite. The megalopa differs in having a spine dorsally on the carapace and on the basial segment of the second pereiopod. Two characters that are potentially unique to Apiomithrax include a zoeal antenna with an exopod that is much longer than the protopod, and a rostral spine that is longer than the dorsal spine. These characters should facilitate the identification of this taxon and could also be useful for phylogenetic studies. A review of larvae of 28 species among 14 genera indicated that there is no apparent single larval character that differentiates the Pisidae, with more limited phylogenetic analyses suggesting that this is a paraphyletic group. Apiomithrax , Eurynolambrus , Pisoides , Rochinia and Scyra have the most divergent morphological characters within the family. The analysis and inclusion of additional taxa is likely to shed more light on the sister-group relationships of the Pisidae. However, based on the extent of morphological interspecific variability of known larvae it is likely that the group, as presently defined by adult morphology, is not monophyletic.

Larval cannibalism rates in the mangrove crab Ucides cordatus (Decapoda : Ocypodidae) under laboratory conditions

Predation of zoeas by megalopae of Ucides cordatus is frequently observed in the laboratory during larval rearing, a phenomenon that could considerably reduce the output of larviculture. Experiments were carried out in the present study to assess how the survivorship of larvae at the end of the larviculture is influenced by cannibalism by megalopae on the larvae of earlier stages, as well as on other megalopae. In addition, tests were performed to assess whether the adoption of different feeding protocols can decrease cannibalism rates. Experiments were carried out in plastic vials containing ocean water (salinity 25 g L-1) under controlled environmental conditions (26 degrees C and 16:8 h LD photoperiod). An ensemble analysis of all the developmental stages indicated that zoeal mortality rates were significantly higher in the presence of megalopae, a result that is consistent with cannibalism by megalopae. However, separate analysis for each developmental stage indicated that only zoea IV, V and VI show reduced survivorship. No cannibalism was detected among megalopae. Food supplementation using Artemia sp. at a density of 6 nauplii mL(-1) proved to be successful in reducing cannibalism rates, whereas supplementation at a lower density (0.3 nauplii mL(-1)) failed to show such an effect. The implications of these results for the larviculture of U. cordatus are discussed.

FIRST ZOEAL STAGE and MEGALOPA of UCA (UCA) MARACOANI (DECAPODA: BRACHYURA), WITH COMMENTS on THE LARVAL MORPHOLOGY of SOUTH-AMERICAN SPECIES of OCYPODIDAE

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ultrastructure of last larval instar fat body cells of Pachycondyla (= Neoponera) villosa (Formicidae : Ponerinae): cytochemical and chemical analysis

The ultrastructure of the fat body cells (trophocytes) of the last larval instar of Pachycondyla (= Neoponera) villosa is presented. The cytoplasm is restricted to the cell periphery and to the smaller strips among the vacuoles, protein granules, lipid droplets, and around the nucleus. Cytochemically, the presence of basic amino acids in the protein granules and in the nuclei was observed by using the ethanolic phosphotungstic acid technique (EPTA). The lipid droplets stained for unsaturated lipids. This result was further confirmed by gas chromatography and mass spectrometry, where the unsaturated fatty acids were identified as oleic and linoleic acids together with saturated fatty acids such as palmitic and stearic acid. Carbohydrates (glycogen) were also detected in the fat body. The glycogen is present as beta particles distributed among the lipid droplets and sometimes attached to them.

Programmed cell death in the larval salivary glands of Apis mellifera (Hymenoptera, Apidae)

The morphological and histochemical features of degeneration in honeybee (Apis mellifera) salivary glands were investigated in 5th instar larvae and in the pre-pupal period. The distribution and activity patterns of acid phosphatase enzyme were also analysed. As a routine, the larval salivary glands were fixed and processed for light microscopy and transmission electron microscopy. Tissue sections were subsequently stained with haematoxylin-eosin, bromophenol blue, silver, or a variant of the critical electrolyte concentration (CEC) method. Ultrathin sections were contrasted with uranyl acetate and lead citrate. Glands were processed for the histochemical and cytochemical localization of acid phosphatase, as well as biochemical assay to detect its activity pattern. Acid phosphatase activity was histochemically detected in all the salivary glands analysed. The cytochemical results showed acid phosphatase in vesicles, Golgi apparatus and lysosomes during the secretory phase and, additionally, in autophagic structures and luminal secretion during the degenerative phase. These findings were in agreement with the biochemical assay. At the end of the 5th instar, the glandular cells had a vacuolated cytoplasm and pyknotic nuclei, and epithelial cells were shed into the glandular lumen. The transition phase from the 5th instar to the pre-pupal period was characterized by intense vacuolation of the basal cytoplasm and release of parts of the cytoplasm into the lumen by apical blebbing; these blebs contained cytoplasmic RNA, rough endoplasmic reticule and, occasionally, nuclear material. In the pre-pupal phase, the glandular epithelium showed progressive degeneration so that at the end of this phase only nuclei and remnants of the cytoplasm were observed. The nuclei were pyknotic, with peripheral chromatin and blebs. The gland remained in the haemolymph and was recycled during metamorphosis. The programmed cell death in this gland represented a morphological form intermediate between apoptosis and autophagy.

Determination of the antimutagenicity of an aqueous extract of Rhizophora mangle L. (Rhizophoraceae), using in vivo and in vitro test systems

An aqueous extract of Rhizophora mangle L. bark is used as raw material in pottery making in the State of Espirito Santo, Brazil. This extract presents large quantities of tannins, compounds possessing antioxidant properties. Tannin antioxidant activity, as a plant chemical defense mechanism in the process of stabilizing free radicals, has been an incentive to studies on anti-mutagenicity. The present work aimed to evaluate possible antimutagenic activity of a R. mangle aqueous extract, using the Allium cepa test-system and micronuclear (MN) assay with blockage of cytokinesis in Chinese hamster ovary cells (CHO-K1). The Allium cepa test-system indicated antimutagenic activity against the damage induced by the mutagenic agent methyl methanesulfonate. A reduction in both MN cell frequency and chromosome breaks occurred in both the pre and post-treatment protocols. The MN testing of CHO-K1 cells revealed anti-mutagenic activity of the R. mangle extract against methyl methanesulfonate and doxorubicin in pre, simultaneous and post-treatment protocols. These results suggest the presence of phyto-constituents in the extract presenting demutagenic and bio-antimutagenic activities. Since the chemical constitution of Rhizophora mangle species presents elevated tannin content, it is highly probable that these compounds are the antimutagenic promoters themselves.

Ultramorphology and histology of the larval salivary gland of Pachycondyla villosa (Fabricius) (Hymenoptera: Formicidae, Ponerinae)

A glândula salivar apresenta-se com um duto anterior único, formado por um epitélio colunar, dois dutos laterais curtos, os quais apresentam-se com epitélio cúbico simples e que na sua porção mais proximal torna-se colunar. Posterior a estes, encontram-se os dois reservatórios, os quais possuem o epitélio bastante delgado e é neste reservatório que a região secretora da glândula se abre. Os ramos dorsal e ventral da região secretora da glândula conectam-se por meio de comissuras transversais, sendo que, posteriormente, a região secretora termina em forma de alça. A região secretora é uniforme, não apresenta tipos celulares distintos e é formada por um epitélio cúbico simples. Neste trabalho é apresentada, também, a revisão sobre a morfologia da glândula salivar larval em insetos, principalmente com relação aos Hymenoptera-Aculeata.

Inhibitory Action of Neem Aqueous Extract (Azadirachta indica A. Juss) on the Vitellogenesis of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) Ticks

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Nuclear alterations associated to programmed cell death in larval salivary glands of Apis mellifera (Hymenoptera : Apidae)

The silk glands of bees are a good model for the study of cell death in insects. With the objective to detect the nuclear features during glandular regression stage, larvae at the last instar and pre-pupae were collected and their silk glands were dissected and processed for ultrastructural analysis and histologically for cytochemical and imunocytochemical analysis. The results showed that the cellular nuclei exhibited characteristics of death by atypical apoptosis as well as autophagic cell death. Among the apoptosis characteristic were: nuclear strangulation with bleb formation in some nuclei, DNA fragmentation in most of the nuclei and nucleolar fragmentation. Centripetal chromatin compaction was observed in many nuclei, forming a perichromatin halo differing from typical apoptotic nuclei. With regards to the characteristics of autophagic-programmed cell death, most relevant was the delay in the collapse of many nuclei. (C) 2006 Elsevier Ltd. All rights reserved.