Structural and functional characterisation of the blood cells of the bivalve mollusc,Scrobicularia plana

Light and electron microscopical studies were carried out in order to characterise the blood cells of the bivalve mollusc, Scrobicularia plana. Three types of haemocytes were recognised: eosinophilic granular haemocytes, basophilic granular haemocytes and basophilic agranular haemocytes. The eosinophilic granulocytes were vesicular and contained large granules whereas the basophilic granulocytes were found to contain small granules and glycogen 'lakes'. The basophilic agranular haemocytes were significantly smaller than the granular haemocytes and had a high nucleus to cytoplasm ratio. Functional characterisation of the blood cells identified activity for the lysosomal enzymes: acid phosphatase, beta-glucuronidase, non-specific esterase and arylsulphatase. There was also a weak staining reaction for phenoloxidase and peroxidase activities. Phagocytosis of Gram-positive bacteria was demonstrated by the haemocytes and antibacterial activity was shown by cell-free haemolymph. Assays to determine release of reactive oxygen species from the haemocytes did not detect any reactive oxygen generation.     

Parasitization of Lacanobia oleracea (Lepidoptera: Noctuidae) by the ectoparasitc wasp,Eulophus pennicornis. Effects of parasitization,venom and starvation on host haemocytes

In contrast to the situation with endoparasitic wasps, little is known about the effects of ectoparasitoids and their secretions on the haemocytes of their insect hosts. To address this deficit, a study has been made of the ectoparasitic wasp, Eulophus pennicornis, and it's host, the tomato moth, Lacanobia oleracea. Using light microscopy, it was determined that L. oleracea has five main haemocyte types, namely, plasmatocytes, granular cells, spherule cells, oenocytoids and pro-haemocytes, representing 56%, 30%, 10%, 2% and 2% of the population, respectively. Parasitization by E. pennicornis, resulted in an increase in the number of circulating haemocytes up to day three, followed by a decrease towards day eight; the latter being associated with changes to the morphology and viability of the cells. For example, on day five after parasitization, plasmatocytes and granular cells had become more rounded and put out pseudopods less readily compared with those from non-parasitized controls, whilst from day seven onwards there was a significant decrease in haemocyte viability and by day nine, extensive haemocyte damage and disintegration was evident. These changes were not observed when larvae were injected with E. pennicornis venom, or when haemocytes were exposed directly to venom in vitro, neither did they occur in starved larvae. Thus, although the observed effects on L. oleracea haemocytes are definitely associated with parasitization they are not due to wasp venom components, nor are they a non-specific effect resulting from nutritional deprivation. The possibility that the feeding wasp larvae produce factors which perturb host haemocytes in order to help condition the host to ensure that successful parasitization occurs, is discussed.     

Morphological and cytoenzymatic characterization of haemocytes of the venus clam Chamelea gallina

A morphological and enzymatic characterization of Chamelea gallina haemocytes was carried out as a prerequisite for further studies on venus clam immunobiology. Two main types of circulating haemocytes were identified (1) hyalinocytes (79.2%), agranular cells with a central nucleus surrounded by a little cytoplasm, and (2) granulocytes (16.5%), smaller granular cells with smaller nuclei. Small cells with a strongly basophilic nucleus and a thin layer of peripheral cytoplasm, probably undifferentiated blast cells (4.3%), were also observed. Both granulocytes and hyalinocytes can assume a spreading or round morphology. The enzymatic activities of haemocytes were also investigated. Some of the granulocytes and hyalinocytes were positive for hydrolytic enzymes, suggesting a role for these cells in phagocytosis; no oxidative enzymes were detected in C. gallina haemocytes. Granulocytes and hyalinocytes can easily adhere to the substratum and exhibit a low phagocytosis activity towards foreign particles (6.3%), whereas the fraction of cells containing ingested material significantly increased after pre-incubation of test particles with cell-free haemolymph, which suggests the presence of opsonin(s) in the haemolymph.     

Experimental infection of white spot syndrome virus in freshwater crayfish Pacifastacus leniusculus.

The signal freshwater crayfish Pacifastacus leniusculus was found to be susceptible to infection with white spot syndrome virus (WSSV). Histopathological observations of various tissues of virus-injected crayfish showed similar symptoms to those from WSSV-infected penaeid shrimp, but no appearance of white spots on the cuticle or reddish body colour were observed, although these are the prominent gross signs of white spot disease in shrimp. A gene probe for detecting WSSV was developed in order to detect the virus in affected cells and tissues using in situ hybridisation. Strong signals were observed in cells of virus-injected crayfish, but not in control-injected crayfish. The number of granular haemocytes in virus-injected crayfish was significantly higher than in sham-injected and non-injected crayfish from Days 5 to 8 (p < or = 0.05) and Days 3 to 8 (p < 0.01) post-injection, respectively. The proportion of granular haemocytes in virus-injected crayfish was also significantly higher than in sham-injected controls from Days 3 to 8 (p < 0.01). These results indicate that WSSV has a significant effect on the proportion of different haemocyte types in the freshwater crayfish.     

Insect immunity-effects of factors produced by a nematobacterial complex on immunocompetent cells(1)

During in vitro incubations, the nematobacterial complex Steinernema carpocapsae-Xenorhabdus nematophilus produces different factors having toxic activities in vitro towards haemocytes, the insect cells responsible for cellular immune defense reactions. Among others, two effects were evident on haemocyte monolayers; one of them was a cytotoxic activity while the other was an unsticking effect. The factors responsible for cytotoxic activity and unsticking effect, were separated from each other by a single chromatography on anion exchange column. These two effects on haemocytes were lost after heat treatment at 57 degrees C for 1 h and 45 degrees C for 30 min, respectively. Both factors were recovered after dialysis in a 10(4) Da cut off membrane. The cytotoxic activity was susceptible to proteases. Cytotoxic and unsticking factors did not show any lipase or lecithinase activity but the unsticking factor had protease activity. Lipopolysaccharides, purified from the bacteria harvested after incubation of the complex, did not have cytotoxic or unsticking effect on the insect cells in vitro.     

The antioxidants dimethylsulfoxide and dimethylthiourea affect the immediate adhesion responses of larval haemocytes from 3 lepidopteran insect species

Antioxidants, dimethylsulfoxide (DMSO) and dimethylthiourea (DMTU), at concentrations not affecting the viability of blood cells (haemocytes) from the larval stage of 3 lepidopteran insects - Galleria mellonella, Lymantria dispar, and Malacosoma disstria - differed in their influence on the innate binding of haemocytes to glass, bacteria to haemocytes, and on humoral responses to alien materials. In vitro DMSO had little effect, whereas DMTU substantially impaired the adhesion of the haemocyte types, the plasmatocytes and granular cells, to slides as well as the attachment of Bacillus subtilis to these haemocytes. Although both antioxidants increased lysozyme and phenoloxidase activities, there was no correlation of enzyme activity and haemocyte adhesion responses, possibly reflecting sequestered radicals. Nitric oxide and hydroxyl radicals offset the DMTU effect. In the absence of antioxidants, inactivate protein kinases A (PKA) and C (PKC) enhanced haemocyte aggregation. In general, DMSO, as opposed to DMTU, did not alter the effects of PKA and PKC activators and inhibitors on haemocyte aggregation or of PKC and PKA activities. High concentrations of DMSO and all levels of DMTU, although inhibiting PKA and PKC, inhibited haemocyte adhesion to slides. Comparable results occurred for DMTU-treated haemocytes incubated with B. subtilis. In vivo DMSO, unlike DMTU, did not impair plasmatocyte or granular cell responses to foreign materials, including bacterial removal from the haemolymph and nodulation.     

Increased fecundity of malathion-specific resistant beetles in absence of insecticide pressure

Despite that resistance frequency is assumed to decline when selective pressure is relaxed, the stability of resistance frequency has been observed in some insects in the absence of insecticide. In the red flour beetle, Tribolium castaneum, the first case of malathion-resistance was reported in the early 1960s. The malathion-specific resistant phenotype has now almost completely replaced the susceptible one in red flour beetle populations. In the present study, several life-history traits that could influence the fitness of the insects were compared between insecticide-susceptible and malathion-specific resistant populations of the red flour beetle. On average, egg fertility and egg-to-adult development time did not differ between susceptible and resistant populations. However, the fecundity of resistant females was greater than that of susceptible ones. Generally, differences in development time between insecticide resistant and susceptible populations are considered as having more effect on fitness than do differences in fecundity. However, the observed increased female fecundity may participate, in combination with the previously observed increased male reproductive success, to the development and the stability of malathion-specific resistance in T. castaneum.     

Haemocytes in the repair of wounds in an insect (Rhodnius prolixus)

Four types of haemocytes may be distinguished in Rhodnius adults, based on their fine structure. The plasmatocytes are most active in the repair of an integumental wound. They contain dens homogeneous granules whose contents become less dense in response to wounding until microtubules about 150å in diameter are distinguishable. The significance of these changes is not yet known. Tight and intermediate junctions, and septate desmosomes appear between haemocytes which have accumulated in an excision.     

Effects of the pathogenic Vibrio tapetis on defence factors of susceptible and non-susceptible bivalve species: I. Haemocyte changes following in vitro challenge

In microbial infections, the interaction between microorganisms and phagocytic cells is a crucial determinant in the outcome of the disease process. We used flow cytometry to study the in vitro interactions between Vibrio tapetis, the bacterium responsible for Brown Ring Disease (BRD) in the Manila clam Ruditapes philippinarum, and haemocytes from three bivalve species: the Manila clam (susceptible to BRD), the hard clam Mercenaria mercenaria and the eastern oyster Crassostrea virginica (both non-susceptible to BRD). Results demonstrated that V. tapetis cells and extracellular products elicit major changes in the haemocytes of R. philippinarum, including decreased viability and phagocytic activity, and altered size and internal structure. V. tapetis was able to kill haemocytes from M. mercenaria and C. virginica but to a far lesser extent than those of R. philippinarum. These results suggest that disease resistance is not solely dependent on a host activity against the pathogen, but is also a function and magnitude of the injury to the host cell by a given pathogen.     

Impact of insecticides on the haemogram of Rhynocoris kumarii Ambrose and Livingstone (Hem., Reduviidae)

Abstract:  The haemogram of Rhynocoris kumarii Ambrose and Livingstone comprises prohaemocytes, plasmatocytes, granular haemocytes, cystocytes and oenocytoids. The impact of five insecticides, viz. monocrotophos, dimethoate, methylparathion, quinalphos and endosulfan on the total haemocyte count (THC) and differential haemocyte counts (DHC) was studied. All of the insecticides except endosulfan initially reduced both prohaemocytes and plasmatocytes, increased the granular haemocytes, altered the percentage of cystocytes and oenocytoids and increased the total haemocyte count (THC). On the contrary, endosulfan initially increased the prohaemocytes and plasmatocytes, decreased the granular haemocytes and also the THC. The highest impact on the DHC and THC was caused by methylparathion and monocrotophos and the least impact by endosulfan. Hence, endosulfan is considered as the safest insecticide followed by dimethoate and quinalphos among these five insecticides to use with R. kumarii .     

Quantitative trait locus mapping of pyrethroid resistance in Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)

Quantitative trait locus (QTL) mapping is a valuable new tool for locating genomic regions that underlie variation in important traits such as insecticide resistance. Because QTL mapping complements a candidate gene strategy for understanding the genetic architecture of important traits, it may also facilitate the identification of genes causing important variation. After mapping the QTL locations, markers closely linked to QTL can be used for genetic analysis of population structure and to measure the spread and increase of resistance-causing QTL alleles. In this study, QTL influencing resistance to the pyrethroid insecticide esfenvalerate were mapped in the Colorado potato beetle Leptinotarsa decemlineata (Say) (CPB). Three QTL contributing to esfenvalerate resistance were identified from a mapping population of 79 individuals analyzed at 90 marker loci. One QTL had a large effect and two QTL had smaller effects. The major QTL occurs on the X chromosome, overlapping the position of a candidate gene (Leptinotarsa decemlineata Voltage sensitive sodium channel [LdVssc1]) previously implicated in pyrethroid resistance. Resistance-increasing alleles at the two minor-effect QTL originated with the susceptible parent, suggesting that alleles of small effect may be segregating in susceptible populations. Comparison of the New York population from which the susceptible parent originated with a more-susceptible population from North Carolina suggests that the minor-effect loci identified here may explain some of the variation in tolerance observed among susceptible populations. DNA sequencing of a portion of LdVssc1 shows that the resistance-conferring allele from the resistant parent does not contain the kdr mutation previously found in CPB and typically observed in other insects that are resistant to pyrethroid insecticides because of changes in this gene.     

内脏团插核术刺激对三角帆蚌血细胞的影响

为了探讨三角帆蚌(Hyriopsis cumingii Lea)血细胞的类型及内脏团插核手术刺激对血细胞形态结构和数量的影响,研究利用相差显微镜、光学显微镜、透射电子显微镜和流式细胞仪对三角帆蚌血细胞进行了形态学研究。流式细胞术光散射图谱显示血细胞被分两类,一类为颗粒度高的大细胞,另外一类为颗粒度低的小细胞;相差显微镜观察显示,血细胞可分为胞体暗、折光性差和胞体明亮、折光性强的两类;Giemsa和H.E染色显示细胞分为胞质染色不均一、胞内颗粒明显和胞质染色均一、胞内颗粒不明显的两类;透射电镜超薄切片观察显示,颗粒明显的细胞胞质内线粒体、高尔基体等细胞器较丰富,颗粒不明显的细胞胞质内细胞器较少;负染结果表明血细胞主要分为表面不光滑、突起明显和细胞表面光滑、突起较不明显的两类。综合上述实验结果可见,三角帆蚌血细胞分为颗粒明显的细胞和颗粒不明显的透明细胞两大类。内脏团插核术刺激后,血细胞的形态和比例均发生显著变化。血细胞形态更多样,伪足状突起更明显,细胞内囊泡状物质增多,血细胞密度显著增高(PP<0.01)。研究表明,作为三角帆蚌免疫系统重要组成部分的血细胞,在插核手术后,其类型、形态结构和数量均产生明显变化,这是机体对外界刺激产生的免疫防御反应,其中颗粒细胞担负着主要的免疫功能。       

Haemocyte changes in resistant and susceptible strains of D. melanogaster caused by virulent and avirulent strains of the parasitic wasp Leptopilina boulardi

Two strains of Drosophila melanogaster (resistant and susceptible) were parasitized by a virulent or avirulent strain of the parasitoid wasp Leptopilina boulardi. The success of encapsulation depends on both the genetic status of the host strain and the genetic status of the parasitoid strain: the immune cellular reaction (capsule) is observed only with the resistant strain-avirulent strain combination. The total numbers of host haemocytes increased in all 4 combinations, suggesting that an immune reaction was triggered in all hosts. Resistant host larvae infected with the virulent or avirulent strains of parasitoid wasp had slightly more haemocytes per mm(3) than did susceptible host larvae at the beginning of the reaction (less than 15 h post-parasitization). This difference disappeared later. Only the virulent parasitoid strain caused the production of a high percentage of altered lamellocytes (from a discoid shape to a bipolar shape), half the total number of lamellocytes are altered. This suggests that the alteration of lamellocyte shape alone is not sufficient to explain the lack of capsule formation seen in resistant hosts parasitized by the virulent strain. Lastly, there were very few altered lamellocytes in resistant or susceptible hosts parasitized by the avirulent parasitoid strain, two combinations in which no capsule was formed. As is now established for Drosophila-parasitoid interactions, virus-like particles contained in the long gland of the female wasp affect the morphology of the lamellocytes. The results presented here are further proof of the action (direct or indirect) of virus like particles of the virulent strain on lamellocytes.     

Antigenic cross-reactivity of crustacean haemocytes using monoclonal antibodies produced against haemocytes of shrimp (Litopenaeus vannamei)

Eight monoclonal antibodies produced against haemocytes of shrimp (Litopenaeus vannamei) were used to research the antigenic cross-reactivity of crustacean haemocytes. 2C3 cross-reacted with the haemocytes of all the experimental animals, while 1H8 and 2C11 did not cross-react with the experimental animals. The other five monoclonal antibodies cross-reacted with some of the experimental animals.     

Parasitization of Lacanobia oleracea (Lepidoptera) by the ectoparasitic wasp,Eulophus pennicornis,suppresses haemocyte-mediated recognition of non-self and phagocytosis

Although many endoparasitic wasps suppress the haemocyte-mediated immune defences of their insect hosts, the effects of ectoparasitoids are virtually unknown. In view of this, a study has been made of the ectoparasitic wasp, Eulophus pennicornis, and its host, the tomato moth, Lacanobia oleracea. For unparasitized insects, in vitro assays indicated that less than 3.0% of L. oleracea haemocytes on a monolayer formed rosettes with yeast cells or fresh rabbit erythrocytes (rbc), and virtually no phagocytosis of these particles occurred. In addition, although fixed rbc formed rosettes with 51.21% of haemocytes, only about 3.0% of the haemocytes ingested one or more of these particles. In contrast to this, B. cereus and E. coli were readily phagocytosed by 14.75% and 53.70% of haemocytes, respectively. These results indicate that L. oleracea haemocytes can recognise different types of non-self particles and demonstrate that ingestion does not necessarily follow attachment. When rosetting and phagocytosis assays were performed with fixed rbc and FITC-labelled E. coli, and haemocytes from starved L. oleracea, PBS injected L. oleracea, and experimentally envenomated insects on day five of treatment, there was no significant difference in the percentage of rosetting or phagocytosis occurring. When haemocytes from parasitized insects on day five of treatment were utilised, however, rosetting and phagocytosis were reduced by 31.41% and 34.94%, respectively. Thus, the effects of parasitization and experimental envenomation are not the same. In addition, suppression of host haemocyte-mediated recognition and phagocytosis was not a secondary effect of nutritional deprivation and was not due to ectoparasitoid venom components, rather it was a direct result of parasitization of L. oleracea by E. pennicornis. The putative nature and source of the immunosuppressive factor(s) involved is discussed with reference to those produced by endoparasitic wasps.     

Insecticide susceptible/resistance status in Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus (Diptera: Culicidae) in Thailand during 2003-2005

Susceptibility baselines and diagnostic doses of the technical grade insecticides deltamethrin, permethrin, fenitrothion, and propoxur were established based on Aedes aegypti (L.), Bora (French Polynesia), a reference susceptible strain. Field-collected Aedes mosquitoes from each part of Thailand were subjected to bioassay for their susceptibility to the diagnostic doses of each insecticide. Almost all Ae. aegypti collected were incipient resistant or resistant to deltamethrin and permethrin, except those from some areas of Songkhla (southern) and Phan district of Chiang Rai (northern) province. Susceptibility to fenitrothion was found in mosquitoes from Bangkok (central), Chonburi (eastern), Chiang Rai, Kanchanaburi (western), and Songkhla, whereas they were resistant in almost all areas of Nakhon Sawan (north central) and Nakhon Ratchasima (northeastern) provinces. Most of Ae. aegypti were susceptible to propoxur except those from Mae Wong, Nakhon Sawan province. Various levels of insecticide resistance and susceptibility in adjacent areas revealed a focal susceptible/resistance profile in the country. It could be noted that almost all of Ae. albopictus were susceptible to the insecticides tested at the same diagnostic doses. In conclusion, resistance to pyrethroids (permethrin and deltamethrin) has developed in Ae. aegypti in most of the collected areas, suggesting that an alternative choice of insecticide or other control measures should be applied.     

Insecticide mixtures for mosquito net impregnation against malaria vectors

Insecticides belonging to the pyrethroid family are the only compounds currently available for the treatment of mosquito nets. Unfortunately, some malaria vector species have developed resistance to pyrethroids and the lack of alternative chemical categories is a great concern. One strategy for resistance management would be to treat mosquito nets with a mixture associating two insecticides having different modes of action. This study presents the results obtained with insecticide mixtures containing several proportions of bifenthrin (a pyrethroid insecticide) and carbosulfan (a carbamate insecticide). The mixtures were sprayed on mosquito net samples and their efficacy were tested against a susceptible strain of Anopheles gambiae, the major malaria vector in Africa. A significant synergism was observed with a mixture containing 25 mg/m2 of bifenthrin (half the recommended dosage for treated nets) and 6.25 mg/m2 of carbosulfan (about 2% of the recommended dosage). The observed mortality was significantly more than expected in the absence of any interaction (80% vs 41%) and the knock-down effect was maintained, providing an effective barrier against susceptible mosquitoes.     

Fitness traits of insecticide resistant and susceptible strains of tea mosquito bug Helopeltis theivora Waterhouse (Heteroptera: Miridae)

We compared the biological traits of insecticide resistant and susceptible field populations of tea mosquito bug Helopeltis theivora Waterhouse. The insecticide resistant population of the conventional Tea Estate “Chuapara” of the Dooars, Jalpaiguri, differed significantly from the susceptible strain of the organic Tea Estate “Makibari” of Darjeeling. Both these tea plantation areas are located in the northern part of West Bengal, India. Adverse changes in biological and developmental traits were observed mainly in: (i) reduction in oviposition period; (ii) fecundity; and (iii) prolongation of nymphal and total developmental period. However, all other parameters such as pre- and post oviposition periods, egg incubation period, hatchability and adult longevity were not significantly different. These results clearly demonstrated that only certain fitness components in the resistant strains appear to be adaptively changed and lowered.     

Haemocytes of the pink bollworm, Pectinophora gossypiella, during larval development and diapause

Seven types of haemocytes were observed in the last larval instar of the pink bollworm, Pectinophora gossypiella (Saunders): prohaemocytes, plasmatocytes, granular haemocytes, spherule cells, adipohaemocytes, oenocytoids, and podocytes. Total and differential haemocyte counts made from diapausing and non-diapausing larvae showed that during diapause there was a significant reduction in the numbers of all haemocyte types. Upon termination of diapause, the haemocyte level increased. There were no significant differences in the level of haemocytes in the pharate pupae that developed from diapause or non-diapause type larvae, except in the case of adipohaemocytes, which were three times as prevalent in pharate pupae from diapausing larvae. Functional aspects of various types of haemocytes are discussed, and it is suggested that the lower haemocyte level observed during diapause is the result of lower metabolic activity.