Identification and expression analysis of microRNAs and targets in the biofuel crop sugarcane

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

Morphological aspects of cluster formation in the germarium of the sugarcane borer Diatraea saccharalis Fabricius (Lepidoptera : Pyralidae)

Diatraea saccharalis F. is one of the greatest pests of the sugar cane culture. This report aimed to characterize the germanium region of the sugarcane borer by light and transmission electron microscopy, emphasizing the morphological steps of the ovarian cluster formation. In the germanium of this insect, four zones could be morphologically identified during the cluster formation. In the most apical end of each ovariole - Zone I - the germ line stem cells undergo complete mitotic division, originating the cystoblasts. In the Zone II, each cystoblast produces a group of eight cells, the cystocytes, which are interconnected by the ring canals. Clusters containing all the cystocytes in the meiosis, characterizes the Zone III. Germ cells with ultrastructural features of apoptosis are also detected in this Zone. In the Zone IV the cystocytes differentiate, morphologically, into one oocyte and seven nurse cells. Interstitial somatic cells and pre-follicle cells exhibit, in their cytoplasm, heterogeneous vacuoles containing degenerated cellular fragments, characterized as apoptotic bodies. Our results pointed out to the morphological evidences related with important control mechanisms for new clusters/follicles production and for the cellular arrangement into the germanium, resulting from the programmed cell death. We believe that the morphological characterization of ovarian cluster formation in D. saccharalis provided valuable information for the understanding of the initial steps of oogenesis and contributed for the knowledge of the cellular mechanisms related with the oocyte production and with reproduction in insects.

Genetic monitoring of the Amazonian fish matrincha (Brycon cephalus) using RAPD markers: insights into supportive breeding and conservation programmes

The importance of genetic evaluations in aquaculture programmes has been increased significantly not only to improve effectiveness of hatchery production but also to maintain genetic diversity. In the present study, wild and captive populations of a commercially important neotropical freshwater fish, Brycon cephalus (Amazonian matrincha), were analyzed in order to evaluate the levels of genetic diversity in a breeding programme at a Brazilian research institute of tropical fish. Random Amplified Polymorphic DNA fingerprinting was used to access the genetic variability of a wild stock from the Amazon River and of three captive stocks that correspond to consecutive generations from the fishery culture. Although farmed stocks showed considerably lower genetic variation than the wild population, a significantly higher level of polymorphism was detected in the third hatchery generation. The results seem to reflect a common breeding practice on several hatchery fish programmes that use a small number of parents as broodstocks, obtaining reproductive success with few non-identified mating couples. The obtained data were useful for discussing suitable strategies for the genetic management and biodiversity conservation of this species.

Mitochondria in the midgut epithelial cells of sugarcane borer parasitized by Cotesia flavipes (Cameron, 1891)

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

Spherites in the midgut epithelial cells of the sugarcane borer parasitized by Cotesia flavipes

Diatraea saccharalis, the main pest of sugarcane, has been controlled by Cotesia flavipes. Very little is known about the effect of parasitism on the host organs, including the midgut. The Lepidoptera midgut epithelium is composed of columnar, goblet, regenerative, and endocrine cells. Spherites have been described in columnar and regenerative cells of several Lepidoptera species, and presented a lot of functional meaning. We identified spherites in the midgut epithelial cells of non-parasitized D. saccharalis larvae analyzed the effect of parasitism on spherite morphology and distribution along the length of the midgut. Midgut fragments of both non-parasitized and parasitized larvae were processed for transmission electron microscopy. All the midgut epithelial cells showed spherites, but they were not preferentially located in a particular part of the cells. Parasitized larvae had more spherites, mainly in the columnar cells, than non-parasitized larvae. This observation was associated with an ionic imbalance within the insect host. Spherites were more abundant in the anterior midgut region than in other regions, which suggests that this region is involved in ion transport by intracellular and/or paracellular route. The morphological variability of spherites in the cells of parasitized larvae was related to the developmental stages of these structures.

Possible causes of spatial variability in Diatraea saccharalis Fabr. (Lep., Crambidae) and its parasitoid populations in sugarcane fields in Brazil

Based on the large data set stored in some sugarcane mills regarding the biological control programme of Diatraea saccharalis using the parasitoids Cotesia flavipes and tachinid flies, the aim of the present study was to determine whether sugarcane varieties, number of cuts and plant ages are major determinants in promoting spatial variability in D. saccharalis, C. flavipes and tachinid fly populations in sugarcane fields in the state of São Paulo (Brazil). The data set used to support this study was provided by the Sao Joao and Barra sugarcane mills. Coefficient of variation was adopted as a measure of the spatial variability of population density. Spatial variability was estimated for the total density of D. saecharalis (parasitized + unparasitized larvae), and also for D. saccharalis larvae parasitized by tachinids and by C. flavipes. Statistical analysis revealed that the spatial variation in D. saccharalis populations was influenced by the number of cuts and by plant ages in the Barra and Sao Joao Mills, respectively. Similar results were obtained for D. saccharalis larvae parasitized by tachinids; however, in the Sao Joao Mill, the spatial variability of these populations was also influenced by the different number of varieties. Finally, considering D. saccharalis larvae parasitized by C. flavipes, no significant regressions with any of the three categories were observed for both sugarcane mills. The multitrophic implications for this agricultural system are also discussed.

Evidences of non-additive effects of multiple parasitoids on Diatraea saccharalis Fabr. (Lep., Crambidae) populations in sugarcane fields in Brazil

Biological control is a relatively benign method of pest control. However, considerable debate exists over whether multiple natural enemies often interact to produce additive or non-additive effects on their prey or host populations. Based on the large data set stored in the Sao Joao and Barra sugarcane mills (state of São Paulo, Brazil) regarding the programme of biological control of Diatraea saccharalis using the parasitoids Cotesia flavipes and tachinid flies, in the present study the author investigated whether the parasitoids released into sugarcane fields interfered significantly with the rate of parasitized D. saccharalis hosts. The author also observed whether there was an additive effect of releasing C. flavipes and tachinids on the rate of parasitized hosts, and looked for evidence of possible negative effects of the use of multiple parasitoid species in this biological control programme. Results showed that C. flavipes and the tachinids were concomitantly released in the Barra Mill, but not in the Sao Jao Mill. Furthermore, in the Barra Mill there was evidence that the parasitoids interacted because the percentage of parasitism did not increase after the release of either C. flavipes or tachinids. In the Sao Joao Mill, when both parasitoid species were released out of synchrony, both the percentage of parasitism by C. flavipes as well as that of the tachinids increased. When large numbers of tachinids were released in the Barra Mill, they caused a significant lower percentage of parasitism imposed by C. flavipes. The implications of the results as evidence of non-additive effects of C. flavipes plus tachinids on D. saccharalis populations are discussed.

Population structure and breeding season of the South Atlantic hermit crab, Loxopagurus loxochelis (Anomura, Diogenidae) from the Ubatuba region, Brazil

A population of Loxopagurus loxochelis was studied in terms of seasonal abundance, size frequency distribution, sex ratio, and reproductive period (percentage of ovigerous females). Specimens were collected monthly over a period of two years (from September 1995 to August 1997) in the non-consolidated areas of the Ubatuba, Mar Virado, and Ubatumirim bays (northern coast of São Paulo State, Brazil) with a double-rig trawl net. A total of 1,084 individuals were analysed. Animal size (minimum, maximum, and mean +/- SD shield length) was 2.8, 9.1, and 6.88 +/- 1.13 mm for 625 males; 2.8, 8.2, and 5.78 +/- 0.98 mm for 236 non-ovigerous females; and 4.6, 8.0, and 6.24 +/- 0.68 mm for 223 ovigerous females, respectively. Sexual dimorphism was recorded by the presence of males in the largest size classes. The sex ratio was 1.4 : 1 in favour of males. The highest incidence of ovigerous females occurred during winter and spring (June to October), with a low percentage in summer and fall (November to May), indicating continuity in the reproductive cycle. This strategy of reproduction is related to temperature. The occurrence of L. loxochelis in the Ubatuba region represents the final point of northern distribution of this species as a function of water mass influence, and could be its real limit on the Atlantic coast of South America.

Breeding season of the hermit crab petrochirus diogenes (anomura : diogenidae) in the north coast of São Paulo state, Brazil

Petrochirus diogenes, one of the largest hermit crabs in the western Atlantic, was studied in the north coast of the State of São Paulo, Brazil, to determine its breeding season through the analysis of gonad development at a macroscopic level. The analysis of 999 crabs collected from 1995 to 1999, based on four categories of gonad development, showed that males present gonads in the advanced stage during all seasons, and although females present developed ovaries all year as well, the peak incidence is in the summer. The peak of recruitment occurs in the winter.

Population structure and breeding period of Pachycheles monilifer (Dana) (Anomura, Porcellanidae) inhabiting sabellariid sand reefs from the littoral coast of São Paulo State, Brazil

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

Ultrastructural study of the salivary glands of the sugarcane spittlebug Mahanarva fimbriolata (Stal, 1854) (Euhemiptera : Cercopidae)

Spittlebugs are insects that suck sap from plants and regurgitate saliva containing toxic enzymes into the leaves. As a consequence, the conductive channels are blocked resulting in dry leaves, thus giving a burned aspect to the plantation. This work performed ultrastructural analyses of the salivary glands of the sugarcane spittlebug Mahanarva fimbriolata, since these organs produce the enzymes that are injected into the plants, thus being responsible for the economic losses in the production of sugarcane. Three kinds of secretory cells are found in principal gland, forming the lobules I-IV. The main differences among these cells relate to size, morphology and electron density of the secretory vesicles. The accessory glands contain different secretory vesicles to those in the principal gland. Muscular cells are found around the entire gland. The different secretory vesicles found in both principal and accessory glands indicate that the gland produces different substances or that the secretion in the interior of cells passes through a maturation process. (c) 2005 Elsevier Ltd. All rights reserved.

Ant diversity (Hymenoptera: Formicidae) and predation by ants on the different stages of the sugarcane borer life cycle Diatraea saccharalis (Lepidoptera: Crambidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ultrastructural study of the ovary of the sugarcane spittlebug Mahanarva fimbriolata (Hemiptera)

The present study describes the ultrastructure of meroistic telotrophic ovaries of the sugarcane spittlebug Mahanarva fimbriolata. In this type of ovary, nurse cells, oogonia, and prefollicular tissue are located at the terminal (distal) regions or tropharium of ovarioles. Oocytes in different developmental stages, classified from I to V, are observed in the vitellarium. Stage I oocytes do not exhibit intercellular spaces in the follicular epithelium, suggesting that synthesis and production of yolk during this stage occurs only through endogenous processes. Small yolk granules of different electron densities are present in the cytoplasm. Few lipid droplets are observed. Stage 11 oocytes exhibit small intercellular spaces in the follicular epithelium. More protein as well as lipid yolk granules are observed in the cytoplasm. In stage III oocytes, intercellular spaces in the follicular epithelium are larger than those observed in the previous stage. Electrondense protein granules of various sizes, larger than those observed in stage 11 oocytes predominate in the cytoplasm. Smaller lipid droplets are also present. In stage IV oocytes, the follicular epithelium exhibits large intercellular spaces. Our data clearly indicate that the opening of these spaces in the follicular epithelium of M. fimbriolata oocytes increases as the intake of exogenous proteins intensifies, that is, in stages IV and Voocytes. During these stages, granular yolk becomes viscous due to the lysis of granules. In stage Voocytes, viscous yolk predominates in the cytoplasm. This type of yolk, however, has not been described for other orders of insects. The chorion of M. fimbriolata oocytes consists of an external layer (exochorion) and an internal one (endochorion), which is in direct contact with the oocyte. Numerous small pores that probably facilitate oxygenation of the internal structures inside the eggs are observed in the exochorion. (c) 2006 Elsevier Ltd. All rights reserved.

Female reproductive system of the sugarcane spittlebug Mahanarva fimbriolata (Auchenorrhyncha): Vitellogenesis dynamics and protein quantification

The present study aimed describing the ovaries of the sugarcane spittlebug Mahanarva fimbriolata which are meroistic telotrophic with nurse cells and oocytes located in the tropharium. SEM revealed paired ovaries located dorsolaterally around the intestine, and oocytes exhibiting shapes ranging from round (less developed) to elliptic (more developed), suggesting a simultaneous, although, asynchronous development. Based on histological data we classified the oocytes in stages from I to V. Stage I oocytes exhibit follicular epithelium with cubic and/or prismatic cells, fine cytoplasmic granules. Stage II oocytes present intercellular spaces in the follicular epithelium due to the incorporation of yolk elements from the hemolymph. Small granules are present in the periphery of oocytes while larger granules are observed in the center. Stage III oocytes are larger and intercellular spaces in the follicular epithelium are evident, as well as the interface between follicular epithelium and oocyte. Yolk granules of different sizes are present in the cytoplasm. During this stage, chorion deposition initiates. Stage IV oocytes exhibit squamous follicular cells and larger intercellular spaces when compared to those observed in the previous stage. The oocyte cytoplasm present granular and viscous yolk, the latter is the result of the breakdown of granules. Stage V oocytes exhibit a follicular epithelium almost completely degenerated, smaller quantities of granular yolk and large amounts of viscous yolk. Based on our findings we established the sequence of yolk deposition in M. fimbriolata oocyte as follows: proteins and lipids, which are first produced by endogenous processes in stages I and II oocytes. Exogenous incorporation begins in stage III. In stages I and II oocytes, lipids are also produced by follicular epithelial cells. The third element to be deposited is polysaccharides, mainly found as complexes. Therefore, the yolk present in the oocytes of this species consists of glycolipoproteins. Molecular weights of proteins present in M. fimbriolata oocytes ranged from 10 to 92 KDa, differently from vitellogenin, the most common protein present in insect oocytes, weighing approximately 180 KDa. (c) 2006 Elsevier Ltd. All rights reserved.