Oral delivery of dsHvlwr is a feasible method for managing the pest Henosepilachna vigintioctopunctata(Coleoptera:Coccinellidae)

RNA interference(RNAi)techniques have emerged as powerful tools that facilitate development of novel management strategies for insect pests,such as Henosepilachna vigintioctopunctata(Coleoptera:Coccinellidae),which is a major pest of solanaceous plants in Asia.In this study,the potential of oral delivery of in vvYro-synthesized and bacterially expressed double-stranded H.vigintioctopunctata lesswright(Iwr)gene(dsHvlwr)to manage of H.vigintioctopunctata was investigated.Our results showed that the gene Hvlwr had a 480-bp open reading frame and encoded a 160-amino acid protein.Hvlwr expression levels were greater in the fat body than other tissue types.Hvlwr silencing led to greater H.vigintioctopunctata mortality rates and appeared to be time-and partially dose-dependent,likely as a result of the number of hemocytes increasing with dsRNA concentration,but decreasing with time.Bacterially expressed dsHvlwr that was applied to leaf discs caused 88%,66%,and 36%mortality in 1st instars,3rd instars,and adults after 10,10,and 14 d,respectively;when applied to living plants,there was greater mortality in 1 st and 3rd instars,but there was no effect on adults.Furthermore,dsHvlwr led to improved plant protection against H.vigintioctopunctata.Our study shows an effective dietary RNAi response in H.vigintioctopunctata and that Hvlwr is a promising RNAi target gene for control of this pest species.     

A solanum beetle on a fabaceous weed: Possible rapid progress of adaptation to a novel host-plant in a geographical context

Our knowledge on how the local distribution pattern of ordinary and novel hosts promotes or hinders the progress of adaptation to the novel hosts by phytophagous insects is limited. The herbivorous ladybird beetle Henosepilachna vigintioctopunctata (Coleoptera: Coccinellidae) depends mainly on solanaceous plants as hosts; the major wild host of this beetle species in Java, Indonesia, is Solanum torvum. However, in several regions of Southeast Asia, including Java, H. vigintioctopunctata also occurs on the introduced fabaceous weed, Centrosema molle. Circumstantial evidence indicates that the use of C. molle by beetles became frequent in the very early 2000s in East Java. In the present study, based on laboratory and field data obtained from 2003 to 2005, we evaluated the degree of adaptation to C. molle by H. vigintioctopunctata populations from East Java and documented the geographic pattern of host-plant distribution in East Java. Laboratory experiments revealed that the beetles from East Java possessed the highest degree of adaptation to C. molle among the beetle populations thus far investigated, suggesting that the adaptation to C. molle by beetles proceeded quite rapidly in East Java in the early 2000s. Meanwhile, field surveys showed that the habitats in East Java consisted of mosaics with sites where only C. molle was distributed and sites where C. molle and solanaceous plants co-occurred. We discussed the role of such geographical structure of habitats in promoting the rapid adaptation of H. vigintioctopunctata to C. molle in East Java.     

A solanum beetle on a fabaceous weed: Host plant generalization and specialization are two sides of the same coin

Investigations of the ongoing evolutionary change of host specificity, especially of that in the initial phase, contribute largely to our understanding of the mechanisms responsible for the diversification of phytophagous insects. However, empirical studies of this aspect in natural systems are very scanty. In the present study, we document the evolutionary change of the degree of adaptation to an introduced legume centro by adults and larvae of the herbivorous ladybird beetle Henosepilachna vigintioctopunctata (Fabricius), which depends normally on various solanaceous plants. Results obtained through experiments conducted in seven successive years revealed a fluctuating degree of adaptation, but with a gradual increase, to centro by H. vigintioctopunctata, showing a tendency towards host plant generalization. Of particular importance, our results suggest that both host plant specialization and generalization are possible evolutionary outcomes of a dynamic initial phase of ongoing host range expansion. In addition, results of quantitative genetic analyses on larval development and other circumstantial evidence suggested that the evolutionary trajectories to specialization/generalization are largely determined by ecological conditions rather than by the insects' intrinsic genetic architecture. We also discuss some special aspects of acquisition of, and adaptation to, novel hosts by H. vigintioctopunctata and other herbivorous beetles, of which adults also feed on plant leaves.     

Genome size increase in the phytophagous ladybird beetle Henosepilachna vigintioctomaculata species complex (Coleoptera: Coccinellidae)

This study reports genome size (C‐value) estimates for seven species of ladybird beetles (Coleoptera: Coccinellidae) in Japan using flow cytometry. The results demonstrated genome sizes of 1.0–1.4 Gb in four closely related phytophagous ladybird beetles belonging to the Henosepilachna vigintioctomaculata species complex. These values were approximately two times larger than that of a congeneric phytophagous ladybird beetle H. vigintioctopunctata (0.66 Gb), and of two very distantly related common carnivorous ladybird beetles, Harmonia axyridis (0.46 Gb) and Coccinella septempunctata (0.42 Gb). These lines of evidence suggest that rapid and large genome size increase occurred just after the branching of the common ancestor of the H. vigintioctomaculata species complex from other ladybird species.     

Differences in the susceptibility of five herbivore species and developmental stages to tomato resistance induced by methyl jasmonate treatment

We compared the susceptibility of five herbivores to tomato resistance induced by methyl jasmonate (MeJA) treatment. We tested for lethal effects against five herbivores (Spodoptera litura, Mamestra brassicae, Frankliniella occidentalis, Tetranychus urticae, and Henosepilachna vigintioctopunctata) at various MeJA concentrations. The mortality of all five herbivores increased significantly with increasing MeJA concentration. The 25 % lethal concentration was 0.03 μM for both first-instar larvae of S. litura and third-instar larvae of M. brassicae, 0.51 μM for third-instar larvae of S. litura, 0.76 μM for adult T. urticae, 2.4 μM for first-instar larvae of F. occidentalis, and 5.7 μM for first-instar larvae of H. vigintioctopunctata. Thus, the degree of susceptibility to MeJA-induced resistance of tomato was first-instar larvae of S. litura = third-instar larvae of M. brassicae > third-instar larvae of S. litura ≈ adult T. urticae > first-instar larvae of F. occidentalis > first-instar larvae of H. vigintioctopunctata. Mortality of first-instar larvae of M. brassicae was >90 % at all concentrations. Mortality of fourth-instar larvae of H. vigintioctopunctata (<7 %) was similar to that of the control at all MeJA concentrations. We also detected statistically significant weight loss of the surviving lepidopteran larvae, increased larval duration of F. occidentalis and H. vigintioctopunctata, and reduced egg production by T. urticae grown on MeJA-treated tomato, suggesting that the MeJA-induced resistance can control these herbivores, but effectiveness is different on different species and growth stage. Feeding by both M. brassicae and H. vigintioctopunctata larvae activated JA-inducible genes in tomato.     

Roles of transient receptor potential channels in eclosion and movement in the red flour beetle Tribolium castaneum

Transient receptor potential channels (TRPs) are a family of cation channels involved in various sensory mechanisms in Drosophila melanogaster, including mechanosensing. Phylogenetic analysis of mechanosensory TRPs in seven members of the TRPV, TRPN and TRPA subfamilies reveals a unique TcTrpA5 in Tribolium castaneum that is likely lost in D. melanogaster. Because mechanosensors are implicated in various key physiology, we investigate the roles of eight candidate mechanosensory TRPs in survival, walking behaviour and tonic immobility in T. castaneum using the RNA interference technique. Double‐stranded RNA treatment of nompC (dsNompC) and trpA5 (dsTrpA5) results in eclosion failure, causing 93% mortality of beetles subjected to each of these treatments. The beetles that survive the dsNompC treatment show defects during sclerotization of the elytra. Adult beetles treated with dsNanchung and dsInactive show defects in the folding of the joint between the femur and tibia segments of the hind legs, resulting in abnormal walking behaviour and reduced walking speed. Regarding tonic immobility induced by mechanical stimulations on the ventral surface, knockout responses (death feignings) are significantly extended with dsNanchung, dsInactive and dsWaterwitch treatments. These data suggest that both nompC and trpA5 are required for adult eclosion. The functions of nanchung and inactive are involved in the folding the hind leg segments. In addition, nanchung, inactive, and waterwitch are likely the receptors involved in recovery from tonic immobility.     

Disruption of kynurenine pathway reveals physiological importance of tryptophan catabolism in Henosepilachna vigintioctopunctata

Kynurenine pathway is critically important to catabolize tryptophan, to produce eye chromes, and to protect nervous system in insects. However, several issues related to tryptophan degradation remain to be clarified. In the present paper, we identified three genes (karmoisin, vermilion and cardinal) involved in kynurenine pathway in Henosepilachna vigintioctopunctata. The karmoisin and cardinal were highly expressed in the pupae and adults having compound eyes. Consistently, high-performance liquid chromatography result showed that three ommochrome peaks were present in adult heads rather than bodies (thoraces, legs, wings and abdomens). RNA interference (RNAi)-aided knockdown of vermilion caused accumulation of tryptophan in both adult heads and bodies, disappearance of ommochromes in the heads and a complete loss of eye color in both pupae and adults. Depletion of cardinal brought about excess of 3-hydroxykynurenine and insufficient ommochromes in the heads and decolored eyes. RNAi of karmoisin resulted in a decrease in ommochromes in the heads, and a partial loss of eye color. Moreover, a portion of karmoisin-, vermilion- or cardinal-silenced adults exhibited negative phototaxis, whereas control beetles showed positive phototaxis. Furthermore, dysfunctions of tryptophan catabolism impaired climbing ability. Our findings clearly illustrated several issues related to kynurenine pathway and provided a new insight into the physiological importance of tryptophan catabolism in H. vigintioctopunctata.

     

Geographical variation of elytral spot patterns in the phytophagous ladybird,Epilachna vigintioctopunctata (Coleoptera: Coccinellidae) in the Province of Sumatera Barat,Indonesia

Geographical variation in the elytral spot patterns of a phytophagous ladybird, Epilachna vigintioctopunctata, was studied in the Province of Sumatera Barat, Indonesia. Populations of E. vigintioctopunctata were divided into four major groups (I–IV) by the incidence of spot pattern variations. Group I, occurring in the coastal plains and inland lowlands, and Group IV, confined to the highlands, were the extremes of the spot pattern variations, the latter had many more non-persistent spots and confluences with larger body size and advanced melanism than the former. These two groups were connected with each other via the intermediate groups. A positive relationship was detected between the elevations of sample sites and the average number of non-persistent spots per elytron. Consequently, present results favor the view that the two previously recognized forms of E. vigintioctopunctata (formae A and B inKatakura et al., 1988) represent a complicated intraspecific variation rather than two distinct sibling species. Elytral spot pattern variations were not different between the sexes or between the beetles collected from different kinds of host plants.     

Seasonal Occurrence of Henosepilachna vigintioctopunctata (F.) (Coleoptera: Coccinellidae) and Its Parasitoid on Ashwagandha in India

The outbreak of Henosepilachna vigintioctopunctata (F.) was noticed on a medicinal plant, Withania somnifera Dunal during 2004-05. The population level of the pest was reached its peak in August. The parasitoid Pediobius foveolatus caused 51.94%±12.20% parasitism. The pest completed its life cycle in 20.15±1.50 days on W. somnifera. The longevity of the male and female adults of the pest was 22.07±3.71 and 31.07±4.38 days, respectively. The gravid females of H. vigintioctopunctata laid an average of 287.64±33.38 eggs during their oviposition period of 10.40±2.80 days.     

Rearing the 28-spotted ladybird beetle,Henosepilachna vigintioctopunctata (Coleoptera: Coccinelidae), with a switchover from host plant leaves to artificial diet

In this study, I tested an artificial diet, Insecta LFS, for rearing the 28-spotted ladybird beetle, Henosepilachna vigintioctopunctata (Fabricius). H. vigintioctopunctata larvae could not be reared through all of the larval stages on the artificial diet. However, initially they could be reared on tomato leaves up to the second or third instar, and thereafter exclusively on the artificial diet. The larval and pupal periods were not significantly different from those reared only on tomato leaves. For females reared by the diet-switching method, the preoviposition period was significantly longer and the number of eggs laid significantly lower than for those on tomato leaves. However, these differences did not become a hindrance to laboratory rearing. This rearing method saves labor involving obtaining fresh plant leaves and produces a reliable supply of the insects.     

Identification of a fatty acid synthase gene (FAS1) from Laodelphax striatellus planthoppers contributing to fecundity

Fatty acid synthase (FAS) is a multifunctional enzyme that plays an important role in the formation of fatty acids. The fatty acids take part in many processes, such as cell signaling and energy metabolism, and in insects they are important in both cuticular hydrocarbon (CHC) formation and reproduction. Here we characterized the sequence structure and function of an FAS from the small brown planthopper (SBPH), Laodelphax striatellus. The full-length open reading frame (ORF) sequence of LsFAS1 was 7122 bp, encoding a predicted protein of 2373 amino acid residues. There were 7 functional domains in the LsFAS1 protein sequence. Gene expression screening by real-time quantitative polymerase chain reaction (RT-qPCR) showed that LsFAS1 was expressed in all developmental stages. Relative expression was highest at the 4th-instar and female adult stages. Among different tissues, the expression level of LsFAS1 in the ovary was the highest. Phylogenetic analysis showed that LsFAS1 clustered in a clade with 2 FASs from Nilaparvata lugens. Furthermore, these 3 FASs are related to cockroach BgFAS and locust LmFAS. After RNA interference-mediated knock-down, most treated insects died at eclosion. In addition, the lifespan of dsFAS1-treated female adults was shorter than that of the dsGFP-injected control, and offspring production decreased. Also, the expression of vitellogenin (Vg) and vitellogenin receptor (VgR) genes decreased. Virgin females dissected at days 2 and 4 post-eclosion showed many matured oocytes in planthoppers treated with dsGFP but not with dsFAS1. These data highlight the importance of LsFAS1 in SBPH, including a role in reproduction.     

RNA interference of a trehalose-6-phosphate synthase gene reveals its roles in the biosynthesis of chitin and lipids in Heortia vitessoides(Lepidoptera:Crambidae)

Trehalose 6-phosphate synthase(TPS),an enzyme that hydrolyzes two glucose molecules to yield trchalose,plays a pivotal role in various physiological processes.In this study,we cloned the trehalose-6-phosphate synthase gene(HvTPS)and investigated its expression patterns in various tssues and d:velopmental stages in Heortia vitessoides Moore(Lepidoptera:Crambidac).HvTPS was highly expressed in the fat body and after pupation or before molting.We knocked down TPS in H.vitessoides by RNA interference and found that 3.0μg of dsHvTPS resulted in optimal interference at 24 h and 36 h post-injection and caused a sharp decline in the survival rate during the 5th instar larval-pupal stage and obviously abnormal or lethal phenotypes.Additionally.compared to the controls,TPS activity and trehalose contents were significantly lower and the glucose content was significantly higher 24 h or 36 h after injection with 3.0μg of dsHIvTPS.Furthermore,the silencing of HvTPS suppressed the cxpression of six key genecs in the chitin biosynthesis pathway and one key gene related to lipid catabolism.The expression levels of two genes associated with lipid biosynthesis were upregulated.These results strongly suggest that HvTPS is essential for the normal growth and development of H.vitessoides and provide a reference for further studies of the utility of key genes involved in chitin and lipid biosynthesis for controlling insect development.     

Studies on the population dynamics of a thistle-feeding lady beetle,Henosepilachna pustulosa (Kôno) in a cool temperature climax forest

1. A field study was carried out on the population dynamics of a thistle-feeding lady beetle, Henosepilachna pustulosa (Kôno ) living in a cool temperature climax forest in northern Kyoto Prefecture, central Japan.
2. Intensive marking, release and recapture program was carried out to estimate the adult population parameters by usingJolly-Seber method.
3. Sampling ratio was around 50%. Marking ratio rapidly rised as the census progressed and approached to 100%.
4. Sex ratio (% ♀) was 63–69% in both overwintered and new adults.
5. Daily survival rate was as high as 0.95 or more and constant throughout the season. Adult longevity in the spring was longer than 40 days.
6. Reproductive rate i. e., the ratio of the number of newly emerged adults in a given generation to that of overwintered adults in the preceding generation, is very small, ranging 1–3, whereas winter survival is higher than 50%, consequently the size of populations in the study area remain in a remarkably constant size and it never reached a level where intraspesific competition occurred.
7. The population characteristics of H. pustulosa are compared with those of the two closely related species, H. vigintioctopunctata and H. vigintioctomaculata, which are the pests of Solanaceous crops. Hp is more K-strategic than the two pest species.

     

Feeding behaviour of Epilachna vigintioctopunctata (Coleoptera: Coccinellidae) on Luffa aegyptiaca

Observations on adult Epilachna vigintioctopunctata Fabr. on Luffa aegyptiaca show that these insects prefer flowers in light and leaves and flowers in dark. Probably physical and chemical stimuli, originating from the flowers, guide beetles to them in light. In darkness the insects' comparatively greater preference to leaves and flowers suggests that the attractants and arrestants present in both these parts of the plant are mainly in the form of chemical stimuli bearing equal intensity. These insects prefer to feed on the corolla of the flower of L. aegyptiaca. This preference is affected by stimulants in these parts of the plant. The nature and characteristics of these stimuli are unknown.

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Comportement alimentaire d'Epilachna vigintioctopunctata (Coleoptera, coccinellidae) sur Luffa aegyptiaca

Résumé Cette étude expose les résultats d'observations sur l'orientation et sur le préférendum alimentaire de la coccinelle Epilachna vigintioctopunctata Fabr. à l'égard des diverses parties de sa plante hôte: Luffa aegyptiaca. Les expériences réalisées sont décrites et les données numériques obtenues ont été soumises au calcul statistique. Il a été établi que dans des conditions d'obscurité les insectes se portent en nombre presque égal sur les fleurs et sur les feuilles, alors qu'à la lumière, les fleurs sont nettement plus attractives. En ce qui concerne leur alimentation, les insectes préfèrent se nourrir aux dépens de la corolle de la fleur.Les divers facteurs, chimiques ou visuels, pouvant intervenir dans l'orientation de l'insecte et dans son comportement alimentaire, sont discutés.

     

Ultradeformable Archaeosomes for Needle Free Nanovaccination with Leishmania braziliensis Antigens

Total antigens from Leishmania braziliensis promastigotes, solubilized with sodium cholate (dsLp), were formulated within ultradeformable nanovesicles (dsLp-ultradeformable archaeosomes, (dsLp-UDA), and dsLp-ultradeformable liposomes (dsLp-UDL)) and topically administered to Balb/c mice. Ultradeformable nanovesicles can penetrate the intact stratum corneum up to the viable epidermis, with no aid of classical permeation enhancers that can damage the barrier function of the skin. Briefly, 100 nm unilamellar dsLp-UDA (soybean phosphatidylcholine: Halorubrum tebenquichense total polar lipids (TPL): sodium cholate, 3:3:1 w:w) of -31.45 mV Z potential, containing 4.84 ± 0.53% w/w protein/lipid dsLp, 235 KPa Young modulus were prepared. In vitro, dsLp-UDA was extensively taken up by J774A1 and bone marrow derive cells, and the only that induced an immediate secretion of IL-6, IL-12p40 and TNF-α, followed by IL-1β, by J774A1 cells. Such extensive uptake is a key feature of UDA ascribed to the highly negatively charged archaeolipids of the TPL, which are recognized by a receptor specialized in uptake and not involved in downstream signaling. Despite dsLp alone was also immunostimulatory on J774A1 cells, applied twice a week on consecutive days along 7 weeks on Balb/c mice, it raised no measurable response unless associated to UDL or UDA. The highest systemic response, IgGa2 mediated, 1 log lower than im dsLp Al₂O₃, was elicited by dsLp-UDA. Such findings suggest that in vivo, UDL and UDA acted as penetration enhancers for dsLp, but only dsLp-UDA, owed to its pronounced uptake by APC, succeeded as topical adjuvants. The actual TPL composition, fully made of sn2,3 ether linked saturated archaeolipids, gives the UDA bilayer resistance against chemical, physical and enzymatic attacks that destroy ordinary phospholipids bilayers. Together, these properties make UDA a promising platform for topical drug targeted delivery and vaccination, that may be of help for countries with a deficient healthcare system.