Ni2+胁迫对斜纹夜蛾幼虫血淋巴中能量物质水平的适应性调节

为明确食物中重金属胁迫对昆虫血淋巴能量物质和生长的影响,本研究测定了饲料中不同浓度的Ni²⁺ （1~40 mg/kg）对连续3个世代斜纹夜蛾Spodoptera litura Fabricius 5龄始龄（0 h）至6龄末幼虫（分别记为0,24,48,72,96和120 h幼虫）血淋巴中能量物质总糖、蛋白质和脂肪含量,血淋巴中热量值及5龄和6龄幼虫体重的影响。结果表明：第1和2代48 h和96 h幼虫血淋巴的总糖含量均低于对照;而连续3代其他处理幼虫血淋巴中的总糖含量均在低浓度Ni²⁺（1~5 mg/kg）胁迫下增高,在高浓度Ni²⁺ （20~40 mg/kg）胁迫下降低。连续3个世代的Ni²⁺胁迫均显著降低了0~48 h幼虫血淋巴中的蛋白含量;低浓度Ni²⁺（1~5 mg/kg）胁迫增加了连续3代72~120 h幼虫血淋巴中的蛋白含量,而高浓度Ni²⁺（20~40 mg/kg）胁迫只降低了第1代72~120 h幼虫以及第2代72 h幼虫血淋巴中的蛋白含量。第1代0 h,48~72 h和120 h幼虫血淋巴中的脂肪含量在1~20 mg/kg Ni²⁺胁迫下均高于对照,但24 h和96 h幼虫血淋巴中的脂肪含量均低于对照中的含量。连续3代0~48 h幼虫血淋巴中的热量值均随饲料中Ni²⁺浓度的增加而降低;低浓度Ni²⁺胁迫（1~5 mg/kg）增加,而高浓度Ni²⁺（20~40 mg/kg）的胁迫降低第2和3代72 ~120 h幼虫血淋巴中热量值。同时,低浓度Ni2+胁迫增加而高浓度Ni²⁺胁迫降低了第3代5龄和6龄幼虫的体重。因此推测,Ni²⁺胁迫对S. litura幼虫血淋巴的能源物质含量以及热量值的影响与能量物质的种类、虫体的发育阶段和Ni²⁺的胁迫世代等都有一定的联系。     

The effects of host age and superparasitism by the parasitoid, Microplitis rufiventris on the cellular and humoral immune response of Spodoptera littoralis larvae

To study the dynamics of stage-dependent immune responses in Spodoptera littoralis (Boisd.) larvae (Lepidoptera: Noctuidae), single and superparasitism experiments were carried out using the parasitoid Microplitis rufiventris Kok. (Braconidae: Hymenoptera). Compared to younger (preferred) host larvae, the older (non-preferred) host larvae displayed a vigorous humoral response that often damaged and destroyed the single wasp egg or larva. Superparasitism and host age altered both the cellular and humoral immune responses. Younger host larvae showed a stronger encapsulation response compared to older host larvae. Moreover encapsulation rates in younger hosts (e.g., second instar) decreased with increasing numbers of parasitoid eggs deposited/larvae. In older larvae, the encapsulation rate was low in fourth, less in fifth and absent in sixth instar hosts. Conversely, the order and magnitude of the cellular immune response in S. littoralis hosts were highest in second instar larvae with the first instar larvae being a little lower. The immune response steadily decreased from the third through to the fifth instar and was least obvious in the sixth instar. In contrast, the general humoral immune response was most pronounced in sixth instar larvae and diminished towards younger stages. The results suggest that both cellular and humoral responses are stage-dependent. Wasp offspring in younger superparasitized host larvae fought for host supremacy with only one wasp surviving, while supernumerary wasp larvae generally survived in older superparasitized larvae, but were unable to complete development. Older instars seem to have a method for immobilizing/killing wasp larvae that is not operating in the younger instars.     

Effect of starvation upon baculovirus replication in larval Bombyxmori and Heliothisvirescens

The progression of baculovirus (BmNPV, BmCysPD, AcMNPV or AcAaIT) infection in larval Bombyx mori and Heliothis virescens (1st, 3rd or 5th instar) was investigated following various starvation regimes. When the larvae were starved for 12 or 24 h immediately following inoculation, the median lethal time to death (LT₅₀) was delayed by 9.5-19.2 h in comparison to non-starved controls. This corresponded to a delay of 10-23% depending upon the larval stage and virus that was used for inoculation. When a 24 h-long starvation period was initiated at 1 or 2 days post inoculation (p.i.), a statistically significant difference in LT₅₀ was not found indicating that the early stages of infection are more sensitive to the effects of starvation. Viral titers in the hemolymph of 5th instar B. mori that were starved for 24 h immediately following inoculation were 10-fold lower (p < 0.01) than that found in non-starved control larvae. Histochemical analyses indicated that virus transmission was reduced in 5th instar B. mori that were starved for 24 h immediately following inoculation in comparison to non-starved control larvae. In general, the mass of larvae that were starved immediately after inoculation was 30% lower than that of non-starved control insects. Our findings indicate that starvation of the larval host at the time of baculovirus exposure has a negative effect on the rate baculovirus transmission and pathogenesis.     

Molecular characterization and expression pattern of Spodoptera litura (Lepidoptera: Noctuidae) vitellogenin, and its response to lead stress

Vitellogenin (Vg) cDNA from Spodoptera litura Fabricius was cloned and sequenced. The open reading frame (ORF) of Vg cDNA was 5247 nucleotides in length (GenBank Accession no. EU095334), which encoded for a protein of 1748 amino acids. S. litura Vg comprised three conserved regions (Vitellogenin-N domain, DUF1943 and von Willebrand factor type D domain (VWD)), a 17 amino-acid signal peptide and a RXXR cleavage signal (RTIR). The highly conserved GL/ICG motif, the DGXR motif and cysteine residues were found in the C-terminus of the Vg. Vg mRNA was found specifically in the female fat body. Vg expression was first transcribed in 6th day female pupae and levels increased with insect development. The maximum level of Vg mRNA appeared in 24-h-old adults. When S. litura larvae were exposed to lead (Pb) (25-200 mg Pb/kg), there was a significant inhibition in Vg of female adults. The start of Vg expression was advanced ahead by Pb, from 6th day pupae to 3rd day or 4th day pupae. Low levels of Vg in male adults were also induced by low concentrations of Pb (12.5 and 25 mg Pb/kg). These data show that Pb stress elicits an important Vg response in S. litura.     

Contributions of immune responses to developmental resistance in Lymantria dispar challenged with baculovirus

How the innate immune system functions to defend insects from viruses is an emerging field of study. We examined the impact of melanized encapsulation, a component of innate immunity that integrates both cellular and humoral immune responses, on the success of the baculovirus Lymantria dispar multiple nucleocapsid nucleopolyhedrovirus (LdMNPV) in its host L. dispar. L. dispar exhibits midgut-based and systemic, age-dependent resistance to LdMNPV within the fourth instar; the LD₅₀ in newly molted larvae is approximately 18-fold lower than in mid-instar larvae (48-72 h post-molt). We examined the role of the immune system in systemic resistance by measuring differences in hemocyte immunoresponsiveness to foreign targets, hemolymph phenoloxidase (PO) and FAD-glucose dehydrogenase (GLD) activities, and melanization of infected tissue culture cells. Mid-instar larvae showed a higher degree of hemocyte immunoresponsiveness, greater potential PO activity (pro-PO) at the time the virus is escaping the midgut to enter the hemocoel (72 h post-inoculation), greater GLD activity, and more targeted melanization of infected tissue, which correlate with reduced viral success in the host. These findings support the hypothesis that innate immune responses can play an important role in anti-viral defenses against baculoviruses and that the success of these defenses can be age-dependent.     

Neospora caninum: Early immune response of rat mixed glial cultures after tachyzoites infection

Neospora caninum causes neurologic disease in dogs and abortion in cattle. Little is known about the immune response of the CNS against this protozoan. The aim of this study was to evaluate production of IL-6, IL-10, TNF-α, IFN-γ, and NO in rat mixed glial cell cultures infected by N. caninum. IFN-γ was not observed. The mean cytokine released after 24 and 72 h of infection were 3.8 ± 0.6 and 3.7 ± 0.6 pg TNF-α/mg protein and 2.7 ± 0.69 and 4.1 ± 0.64 pg IL-10/mg protein, respectively, and more than 8.0 pg IL-6/mg protein for both time points. NO levels increased 24 h post-infection (2.3 ± 0.8 pg/mg protein) until 72 h (4.2 ± 1.1 pg/mg protein) and the number of tachyzoites reduced with the time. Our results show high levels of regulatory cytokines that may suppress the harmful effects of IFN-γ; high levels of TNF-α and NO may represent an effective response by infected glial cells against N. caninum.     

Response of Lymantria dispar L. (Lepidoptera: Lymantriidae) to Bacillus thuringiensis subsp. kurstaki at different ingested doses and temperatures

We examined mortality and feeding inhibition response of Lymantria dispar L. (Lepidoptera: Lymantriidae) larvae to ingested doses of Bacillus thuringiensis subsp. kurstaki as a function of dose, instar and temperature. We developed generalized (logistic) linear mixed models and a mixture survival model, commonly used in medical statistics, to analyze the complex data set. We conducted bioassays of Foray 48B with larvae from the NJSS laboratory stock, using droplet imbibing or force-feeding to ensure dose ingestion. The dose causing mortality in 50% of the test population (LD₅₀) under standard test conditions (22 °C) ranged from 0.019 International Units (IU)/larva for first instar larvae (L₁) to 1.6 IU/larva for L₄. Temperature affected larval mortality in two ways. Mortality occurred sooner and progressed more rapidly with increasing temperature (13-25 °C) at each dose level and instar, while the maximum level of mortality attained by each instar decreased with increasing rearing temperature. The mechanisms underlying this effect are being investigated. Larvae that survived exposure to B. thuringiensis resumed feeding after a period that was dependent on instar, dose, and temperature. The equations describing observed mortality and feeding recovery responses were used to construct a simulation model, which was able to predict both processes, and which forms the basis for a process-oriented model that can be used as a decision support tool in aerial sprays.     

Helicoverpa armigera (Lepidoptera: Noctuidae) larvae that survive sublethal doses of nucleopolyhedrovirus exhibit high metabolic rates

To determine the effect of sublethal doses of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearSNPV) on the metabolic rate of H. armigera, the respiration rates of third instar H. armigera larvae inoculated with sublethal doses of HearSNPV were evaluated. Respiration rates, measured as the rate of CO₂ production (VCO₂), were recorded daily using closed-system respirometry. By 4 days post-inoculation (dpi), the metabolic rates of LD₂₅ or LD₇₅ survivors were significantly higher than that of uninoculated controls. When dose data were pooled, the VCO₂ values of larvae that survived inoculation (0.0288 ml h⁻¹), the uninoculated controls (0.0250 ml h⁻¹), and the larvae that did not survive inoculation (0.0199 ml h⁻¹) differed significantly from one another. At 4 dpi, the VCO₂ of the uninoculated controls were significantly lower than the VCO₂ of inoculation survivors, but significantly higher than the VCO₂ of inoculation non-survivors. Inoculation survivors may have had high metabolic rates due to a combination of viral replication, organ damage, and an energy-intensive induced cellular immune response. The high 4 dpi metabolic rate of inoculation survivors may reflect an effective immune response and may be seen as the metabolic signature of larvae that are in the process of surviving inoculation with HearSNPV.     

Characterization and transcriptional regulation of thioredoxin reductase 1 on exposure to oxidative stress inducing environmental pollutants in Chironomus riparius

We characterized thioredoxin reductase 1 (TrxR1) from Chironomus riparius (CrTrxR1) and studied its expression under oxidative stress. The full-length cDNA is 1820 bp long and contains an open reading frame (ORF) of 1488 bp. The deduced CrTrxR1 protein has 495 amino acids and a calculated molecular mass of 54.41 kDa and an isoelectric point of 6.15. There was a 71 bp 5’ and a 261 bp 3' untranslated region with a polyadenylation signal site (AATAAA). Homologous alignments showed the presence of conserved catalytic domain Cys-Val-Asn-Val-Gly-Cys (CVNVGC), the C-terminal amino acids ‘CCS’ and conserved amino acids required in catalysis. The expression of CrTrxR1 is measured using quantitative real-time PCR after exposure to 50 and 100 mg/L of paraquat (PQ) and 2, 10 and 20 mg/L of cadmium chloride (Cd). CrTrxR1 mRNA was upregulated after PQ exposure at all conditions tested. The highest level of CrTrxR1 expression was observed after exposure to 10 mg/L of Cd for 24 h followed by 20 mg/L for 48 h. Significant downregulation of CrTrxR1 was observed after exposure to 10 and 20 mg/L of Cd for 72 h. This study shows that the CrTrxR1 could be potentially used as a biomarker of oxidative stress inducing environmental contaminants.     

Developmental changes in urea concentrations in the haemolymph of Daizo (T), an original strain of the silkworm,Bombyx mori reared on an artificial diet and on fresh mulberry leaves

Urea concentrations in the haemolymph of Daizo (T), an original strain of the silkworm, Bombyx mori, reared on an artificial diet and on fresh mulberry leaves were determined by a urease-indophenol method during the larval-pupal-adult development. Urea concentrations in the fourth and fifth instar larvae reared on an artificial diet (diet I) were between 0.10 and 0.15 mg urea N/ml haemolymph, and increased during the larval-pupal transformation to reach 0.33 mg/ml at the larval-pupal ecdysis. A further increase was observed during pupal-adult development and finally reached 0.48 mg/ml at day 7 pharate adult. In the fourth and fifth instar larvae reared on fresh mulberry leaves, the concentrations were low (0.05 mg/ml). From the larval-pupal ecdysis until day 8 pharate adult, further low urea concentrations (0.04 mg/ml) were observed. By starvation from 72 hr of the fifth instar larvae reared on another artificial diet (diet II), the elevation of urea concentrations (between 1.4- and 3.0-fold against the controls) was observed from just after starvation until day 1 spinning. From day 5 pupae, both the starved and the control insects showed a marked elevation of urea concentrations in the haemolymph, which was never observed on animals reared on diet I.     

In-vitro assessment of food consumption,utilization indices and losses promises of leafworm,Spodoptera litura (Fab.), on okra crop

The lepidopteran insect pests have significant importance in vegetable production. The present study was performed to investigate the baseline studies about the assessment of feeding and consumption potential, utilization indices and losses promises of leafworm, Spodoptera litura (Fab.) on Okra. The data regarding feeding potential, food utilization and consumption indices as well as losses of different larval instars were recorded and subjected to appropriate statistical analysis. The results showed that, in the beginning, the approximate digestibility of various instars was increase, e.g. third instar (51.36%–64.03%), fourth instar (63.42%–69.45%) and fifth instar (70.25%–76.10%). However, after a certain period, the digestibility was decreased and efficiency to convert the ingested food into biomass varied significantly. The consumption index values increased with an increase in time but the consumption and growth rate was declined of fourth instar larvae. The ingestion and digestion increased of third (10.01–13.06, 8.32–11.91 mg), fourth (11.27–17.28, 10.96–14.03 mg) and fifth (12.60–19.40, 11.93–15.28 mg) larval instars. The corrected weight of consumed leaves increased with a gain in body weight. However, in the third instar, a decline was observed on the last day of feeding. Maximum leaf area was consumed by fifth instar larvae (44.66 cm²) followed by fourth (35.41 cm²) and third (27.98 cm²) instars. In conclusion, all the dependent parameters, including food utilization potential, consumption indices and losses were higher for fifth instar larvae than others. These results emphasized the re-establishment of fundamental (economic threshold level: ETL, economic injury level: EIL) integrated pest management concepts.     

Molecular characterization of an ecdysteroid inducible carboxylesterase with GQSCG motif in the corn borer, Sesamia nonagrioides

We obtained a full-length cDNA encoding a carboxylesterase in Sesamia nonagrioides. The complete cDNA sequence is comprised of 1838 bp with an open reading frame encoding 576 amino acid residues with predicted molecular mass of 64.24 kDa. The deduced amino acid sequence showed high identity to JHE-Related of Trichoplusia ni (65% amino acid identity) and 49-46% amino acid identity to JHEs of other lepidopterans and contained all five functional motifs of insect JHEs. The gene has been termed as SnJHE-Related (SnJHER) to denote its similarity to other insect JHE genes and the occurrence of an unusual cysteine residue immediately adjacent to the catalytic serine, instead of the conventional alanine residue. Phylogenetic analyses localised SnJHER together with TnJHER in a branch of the lepidopteran's JHEs group, with other carboxylesterases (COEs) occuring in separated groups. The JH analog methoprene did not affect the expression of SnJHER in contrast to other insect JHEs. Additionally, ecdysteroid analogs induced SnJHER gene expression. The SnJHER mRNA levels were higher in long-day non-diapausing larvae than in short-day diapausing ones. In the fifth instar of non-diapausing and ninth instar of diapausing larvae, the SnJHER mRNAs reached higher expression levels on the days close to each larval molt. In the last (sixth) non-diapausing larval instar, SnJHER mRNA levels peaked in the intermolt period but were lower than during the fifth instar.     

Histopathology and effect on development of the PhopGV on larvae of the potato tubermoth, Phthorimaea operculella (Lepidoptera: Gelechiidae)

Larvae of the potato tubermoth (PTM), Phthorimaea operculella, feed on potato plants and tubers and are a major pest in the tropics and subtropics worldwide, causing up to 100% damage. The PTM granulovirus (PhopGV) provides significant potato protection, but little is known about its effect on larval development or its histopathology. Here we show that only 10% of larvae exited from PhopGV-treated tubers (1.4 × 10⁸ granule/ml), lagging significantly behind controls, and most of these died by 72 h after emergence. Histopathology studies showed the fat body and epidermis were the principal tissues infected. PhopGV morphogenesis was similar to other GVs, the exception being small vesicles between mature granules.     

Diapause induction, maintenance and termination in the rice stem borer Chilo suppressalis (Walker)

The rice stem borer, Chilo suppressalis, enters facultative diapause as fully grown larvae in response to short-day conditions during the autumn. Our results showed that the critical night length for diapause induction in C. suppressalis was between 10 h 22 min and 10 h 45 min at 22, 25 and 28 °C, 11 h 18 min at 31 °C, and between 10 h 5 min and 10 h 20 min under field conditions (average temperature ranged from 27.2 to 30.7 °C). The diapause incidence declined in ultra-long nights (18-22 h scotophases) and DD, and increased in ultra-short nights (2-6 h scotophases) and LL. Moreover, we found that the third instar was the stage most sensitive to the photoperiod, and night length played an essential role in the initiation of diapause. Night-interruption experiments with a 1-h light pulse at LD 12:12 (light 12:dark 12) exhibited two troughs of diapause inhibition, with one occurring in early scotophase and the other in late scotophase. Field observations for six years showed that most larvae entered winter diapause in August in response to declining day lengths, despite the high temperatures prevailing during August. By periodically transferring the field-collected overwintering larvae to different photoperiods and temperatures, the results showed that photoperiod had a significant influence on diapause development during the early phase of diapause, while high temperature significantly accelerated the termination of larval diapause.     

A circadian system is involved in photoperiodic entrainment of the circannual rhythm of Anthrenus verbasci

In the circannual pupation rhythm of the varied carpet beetle, Anthrenus verbasci, entrainment to annual cycles is achieved by phase resetting of the circannual oscillator in response to photoperiodic changes. In order to examine whether a circadian system is involved in expression of the periodic pattern and phase resetting of the circannual rhythm as photoperiodic responses, we exposed larvae to light-dark cycles with a short photophase followed by a variable scotophase (the Nanda-Hamner protocol). When the cycle length (T) was a multiple of 24 h, i.e., 24, 48, or 72 h, short-day effects were clearer than when T was far from a multiple of 24 h, i.e., 36 or 60 h. Exposure to light-dark cycles of T = 36 h had effects similar to exposure to long-day cycles of T = 24 h. The magnitude of phase shifts depended on the duration and the phase of exposure to the cycles of T = 36 or 60 h. It was therefore concluded that a circadian system is involved in photoperiodic time measurement for phase resetting of the circannual oscillator of A. verbasci.     

Bursera fagaroides, effect of an ethanolic extract on ornithine decarboxylase (ODC) activity in vitro and on the growth of Entamoeba histolytica

The effect of an ethanolic extract from the stem bark of Bursera fagaroides on ornithine decarboxylase (ODC) activity in vitro and on the growth of Entamoeba histolytica was evaluated. For this purpose, increasing concentrations of the extract, up to 8.0 mg/mL, were added to amoeba cultures or ODC reaction mixtures, which were incubated at 37 °C. Metronidazole and G418 were added as controls. After 1.5 and 72 h, the ODC activity in vitro and growth, respectively, were determined. Results revealed a strong inhibition of growth with IC₅₀ values on the order of 0.05 mg/mL. ODC activity, on the other hand, was inhibited by 12% and 50% at concentrations of 4.0 and 8.0 mg/mL, respectively.     

Parnassius apollo last-instar larvae development prediction by analysis of weather condition as a tool in the species’ conservation

Parnassius apollo (Lepidoptera: Papilionidae) has already disappeared or is under threat of extinction in many of its former habitats. It has been documented that weather conditions – anomalies in particular – contributed to this process. In this study, we combined developmental data obtained previously for the last-instar Apollo larvae (collected in 1996, 1997, and 2003) with corresponding meteorological data to assess the effects of ambient temperature and rainfall episodes on the duration and the completion of the instar. For comparing the temperature effect, we applied the degree-day concept. We found significant positive correlation between the number of rainy days during the instar development (x) and its duration time (y): y = 8.293 + 0.936x (± 2.813) (r = 0.662, P < 10⁻⁷). Logarithmic transformation of the growth curves of the last-instar Apollo larvae revealed that there was no difference in growth among females; however, there was slower growth of males in 2003 in comparison to 1996. Growth (y) of female Apollo larvae as a function of instar duration (x) can by described by one common equation, irrespectively of the year: y = 317.6 + 502.3 lnx (± 263.3) (r = 0.82, P < 10⁻⁴).     

Development of Microplitis bicoloratus on Spodoptera litura and implications for biological control

Microplitis bicoloratus Chen (Hymenoptera:Braconidae:Microgastrinae), a new species of Microplitis Förster from China, is a solitary endoparasitoid of the larvae of the cotton leafworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae). This parasitoid is the first to be successfully reared and evaluated in the laboratory as a potential agent for the biological control of S. litura in China. Oviposition, immature development, and the effects of parasitism on the development of S. litura were studied. In long-term oviposition trials, females laid eggs on S. litura larvae for up to 10 days; oviposition was heavily skewed toward the first few days, with approximately one third of the eggs laid on day 1 and over 50% laid by day 3. This rapid oviposition rate increases the potential for biological suppression of host populations because the likelihood of mortality for the parasites from exposure to detrimental environmental factors or generalist predators increases with time. Immature development of the parasitoid in its host only required 7 days: eggs hatched within 24 h, the first instar larva required 2 days, the second instar larva needed 3 days, and the third instar larvae exited the host and pupated in 1 day, at 27±1°C, 60–80% relative humidity and a 12:12-h (long day) photoperiod. The development of the parasitized hosts was disrupted. When the parasitoid larvae finished development, the body weights of host larvae were significantly reduced regardless of which host instar was parasitized. Our results suggest that M. bicoloratus has considerable potential as a biological control agent for S. litura.