Separation of oyster hemocytes by density gradient centrifugation and identification of their surface receptors

Glutaraldehyde-fixed hemocytes of Crassostrea virginica were subjected to differential centrifugation on a 5, 10, 15, and 25% discontinous sucrose gradient. Five subpopulations of cells were separated by this technique. Subpopulation 1 coincides with the small granulocytes, subpopulation 2 is comprised of hyalinocytes, subpopulation 3 of medium-sized granulocytes, subpopulation 4 of large granulocytes, and subpopulation 5 of a mixture of very large granulocytes and aggregates of small cells. By using several plant and animal lectins, it was ascertained that cells of subpopulations 1, 3, and 4 were agglutinated with Con A and extracts of the albumin glands of Helix pomatia and Cepaea nemoralis while those of subpopulation 2 were agglutinated by the same three lectins as well as wheat germ agglutinin. By applying the Con A-peroxidase cytochemical technique, it was determined that approximately 20% of the granulocytes of subpopulations 1 and 3 do not possess Con A-binding sites and only 18% of the large cells comprising subpopulation 5 possess such sites. These results suggest that the subpopulations of C. virginica granulocytes distinguishable by their dimensions and densities may be further subdivided by differences in specific surface binding sites.     

Phagocytic ability declines with age in adult Drosophila hemocytes

Most multicellular organisms show a physiological decline in immune function with age. However, little is known about the mechanisms underlying these changes. We examined Drosophila melanogaster, an important model for identifying genes affecting innate immunity and senescence, to explore the role of phagocytosis in age‐related immune dysfunction. We characterized the localized response of immune cells at the dorsal vessel to bacterial infection in 1‐week‐ and 5‐week‐old flies. We developed a quantitative phagocytosis assay for adult Drosophila and utilized this to characterize the effect of age on phagocytosis in transgenic and natural variant lines. We showed that genes necessary for bacterial engulfment in other contexts are also required in adult flies. We found that blood cells from young and old flies initially engulf bacteria equally well, while cells from older flies accumulate phagocytic vesicles and thus are less capable of destroying pathogens. Our results have broad implications for understanding how the breakdown in cellular processes influences immune function with age.     

黑翅痂蝗血细胞形态学的研究

【目的】蝗虫血细胞的研究以及不同血细胞类型间的演化关系,是蝗虫细胞免疫学的依据,也是防蝗治蝗的重要依据。【方法】本研究通过Wright-Giemsa对黑翅痂蝗Bryodema nigroptera Zheng血细胞进行了混合染色,并使用光学显微镜进行了观察。【结果】发现黑翅痂蝗血细胞包括原血胞、浆血胞、粒血胞、类绛色血细胞、囊血胞、脂血胞、蠕形细胞和巨核血细胞8种类型。【结论】在成虫体内,原血胞占细胞总数的1%,浆血胞占32.2%,粒血胞占57.3%,类绛色血细胞占1%,囊血胞占6%,脂血胞占1%,蠕形细胞占1%,巨核细胞占细胞总数的0.5%,粒血胞数量最多,巨核细胞最少。     

Sterols and steroids in a freshwater crustacean (Proasellus meridianus): hormonal response to nutritional input

For crustaceans that eat shredded plant material in freshwater habitats, the amount and the composition of food greatly vary over time because of the seasonal succession of plant fragments and algal biomass. The acquisition of elements necessary for growth, immune defense, and reproduction depends largely on this variation in food type and availability. In particular, sterols that are required as cellular membrane components and as precursors of ecdysteroids (molting hormones) must be acquired through food because crustaceans do not synthesize the steroid nucleus de novo. The present study examined the possible link between nutrition, sterols, and ecdysteroids in an isopod, Proasellus meridianus. In a first step, quantitative and qualitative analyses of sterols of P. meridianus were performed by gas‐chromatography/mass spectrometry. The results suggested that members of P. meridianus are able to convert dietary plant sterols into cholesterol required for growth and reproduction. In a second step, by manipulating food availability and using an enzyme immuno‐assay, we showed that ecdysteroid content in males and females (ovigerous or not) of P. meridianus decreased significantly after a starvation period. A nutritional input following this starvation period triggered an increase in the ecdysteroid contents of these animals. The comparable ecdysteroid responses to food pulses in males and females suggested that a nutritional control on steroid hormones regulated growth or gametogenesis rather than egg maturation. Thus, it appears that P. meridianus possesses an efficient stop‐and‐go endocrine system that may have been selectively favored in response to seasonal pulses of food.     

Effects of destruxins on free calcium and hydrogen ions in insect hemocytes

Destruxins, cyclohexadepsipeptidic mycotoxins isolated from the ento mopathogenic fungus Metarhizium anisopliae, inhibit innate insect immunity. However, their mechanism of action remains unclear. In this study, the effects ofdestruxins on changes in free calcium and hydrogen ions in the hemocytes ofExolontha serrulata, Bombyx mori and the Spodoptera litura SL1 cell line were detected using laser scanning confocal mi croscopy （LSCM）. An instant Ca2＋ influx of hemocytes induced by destruxins A and B （DA and DB） was recorded. The DA/DBdependent Ca2＋ influx was not influenced by the Ca2＋ channel inhibitors 2aminoethoxydiphenyl borane （2APB） and U73122. It also had an apparently different LSCM profile from that of the ionomycindependent Ca2＋ influx. However, the instant Ca2＋ influx was not seen in the SL1 cells; on the contrary, a slow, moderate enhancement of intracellular Ca2＋ was observed. Meanwhile, an instant intracellular free H＋ decrease aroused by DA and DB was found. DB at 20/zmol/L and DA at 690/zmol/L significantly reduced intracellular free H＋ levels. Furthermore, the vacuolar H＋ATPase （VATPase） inhibitor bafilomycin A1 had obvious effects on the decreases ofintracellular free H＋ in hemocytes. These results suggest that the mechanism of DA/DBdependent Ca2＋ influx is perhaps not related to Ca2＋ channels and ionophores; rather, the intracellular free H＋ decrease might be due to VATPase inhibition.     

Fine Structure of the Hemopoietic Tissues in the Mealworm Beetle, Tenebrio molitor

The fine structure of the hemopoietic tissue and its detailed reticular organization in the mealworm beetle, T. molitor were examined using light and scanning electron microscopes. The major hemopoietic tissues in the abdomen were located on the upper surface of the dorsal diaphragm which continuous over the ventral wall of the heart. Histologic characteristics of this hemopoietic tissues are dense clusters of cells. They are irregular in outline and are not surrounded by any connective tissue sheath. The hemopoietic tissue of this insect is consisted of three cellular components which are the reticular cells, hemocytic stem cells and several kinds of mature hemocytes. The reticular cells had numerous cytoplasmic processes and forming a complex network. The stem cells give rise to differentiating hemocytes of the different cell lineages. Mature hemocytes within this hemopoietic tissue are originated from the stem cells and differentiated into several types of hemocytes including prohemocytes, plasmatocytes, and granulocytes.     

Responses of seagrass to anthropogenic and natural disturbances do not equally translate to its consumers

Coastal communities are under threat from many and often co‐occurring local (e.g., pollution, eutrophication) and global stressors (e.g., climate change), yet understanding the interactive and cumulative impacts of multiple stressors in ecosystem function is far from being accomplished. Ecological redundancy may be key for ecosystem resilience, but there are still many gaps in our understanding of interspecific differences within a functional group, particularly regarding response diversity, that is, whether members of a functional group respond equally or differently to anthropogenic stressors. Herbivores are critical in determining plant community structure and the transfer of energy up the food web. Human disturbances may alter the ecological role of herbivory by modifying the defense strategies of plants and thus the feeding patterns and performance of herbivores. We conducted a suite of experiments to examine the independent and interactive effects of anthropogenic (nutrient and CO₂ additions) and natural (simulated herbivory) disturbances on a seagrass and its interaction with two common generalist consumers to understand how multiple disturbances can impact both a foundation species and a key ecological function (herbivory) and to assess the potential existence of response diversity to anthropogenic and natural changes in these systems. While all three disturbances modified seagrass defense traits, there were contrasting responses of herbivores to such plant changes. Both CO₂ and nutrient additions influenced herbivore feeding behavior, yet while sea urchins preferred nutrient‐enriched seagrass tissue (regardless of other experimental treatments), isopods were deterred by these same plant tissues. In contrast, carbon enrichment deterred sea urchins and attracted isopods, while simulated herbivory only influenced isopod feeding choice. These contrasting responses of herbivores to disturbance‐induced changes in seagrass help to better understand the ecological functioning of seagrass ecosystems in the face of human disturbances and may have important implications regarding the resilience and conservation of these threatened ecosystems.     

3‐acetylpyridine‐induced degeneration in the adult ascidian neural complex: Reactive and regenerative changes in glia and blood cells

Ascidians are interesting neurobiological models because of their evolutionary position as a sister‐group of vertebrates and the high regenerative capacity of their central nervous system (CNS). We investigated the degeneration and regeneration of the cerebral ganglion complex of the ascidian Styela plicata following injection of the niacinamide antagonist 3‐acetylpyridine (3AP), described as targeting the CNS of several vertebrates. For the analysis and establishment of a new model in ascidians, the ganglion complex was dissected and prepared for transmission electron microscopy (TEM), routine light microscopy (LM), immunohistochemistry and Western blotting, 1 or 10 days after injection of 3AP. The siphon stimulation test (SST) was used to quantify the functional response. One day after the injection of 3AP, CNS degeneration and recruitment of a non‐neural cell type to the site of injury was observed by both TEM and LM. Furthermore, weaker immunohistochemical reactions for astrocytic glial fibrillary acidic protein (GFAP) and neuronal βIII‐tubulin were observed. In contrast, the expression of caspase‐3, a protein involved in the apoptotic pathway, and the glycoprotein CD34, a marker for hematopoietic stem cells, increased. Ten days after the injection of 3AP, the expression of markers tended toward the original condition. The SST revealed attenuation and subsequent recovery of the reflexes from 1 to 10 days after 3AP. Therefore, we have developed a new method to study ascidian neural degeneration and regeneration, and identified the decreased expression of GFAP and recruitment of blood stem cells to the damaged ganglion as reasons for the success of neuroregeneration in ascidians. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 877–893, 2015     

粘虫和棉大卷叶螟幼虫体内肺结构的存在与功能验证

加拿大昆虫学家Locke提出鳞翅目幼虫体内有一个适应血细胞进行气体交换的肺结构。本文以Locke的研究为依据, 运用电镜及其他化学方法, 观察和研究了粘虫Leucania separata及棉大卷叶螟Sylepta derogata幼虫的肺结构和功能。结果表明: 肺存在于第8腹节的气管处, 该气管分支细短而丰富, 形成气管簇。亚甲基蓝标记血液发现, 血流方向为由前向侧后方向, 流经第8腹节和臀腔, 最后流回心脏。第8腹节气管簇的管壁比其他各节都薄, 且管壁内膜具有更多适合运动的细管, 这有利于气体的通透。正常情况下, 气管簇处有各种各样的血细胞聚集, 当缺氧胁迫时有大量的血细胞从组织中释放, 通过变形而紧贴气管簇, 以利于气体的交换。这些特征都与高等动物的肺功能相似, 因而判断此结构存在肺的功能。     

Widespread Wolbachia infection in terrestrial isopods and other crustaceans

Wolbachia bacteria are obligate intracellular alpha-Proteobacteria of arthropods and nematodes. Although widespread among isopod crustaceans, they have seldom been found in non-isopod crustacean species. Here, we report Wolbachia infection in fourteen new crustacean species. Our results extend the range of Wolbachia infections in terrestrial isopods and amphipods (class Malacostraca). We report the occurrence of two different Wolbachia strains in two host species (a terrestrial isopod and an amphipod). Moreover, the discovery of Wolbachia in the goose barnacle Lepas anatifera (subclass Thecostraca) establishes Wolbachia infection in class Maxillopoda. The new bacterial strains are closely related to B-supergroup Wolbachia strains previously reported from crustacean hosts. Our results suggest that Wolbachia infection may be much more widespread in crustaceans than previously thought. The presence of related Wolbachia strains in highly divergent crustacean hosts suggests that Wolbachia endosymbionts can naturally adapt to a wide range of crustacean hosts. Given the ability of isopod Wolbachia strains to induce feminization of genetic males or cytoplasmic incompatibility, we speculate that manipulation of crustacean-borne Wolbachia bacteria might represent potential tools for controlling crustacean species of commercial interest and crustacean or insect disease vectors.