A morphometric study of the midgut in resistant and non-resistant Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) larvae to its nucleopolyhedrovirus (AgMNPV)

In this investigation, the anterior and posterior regions of the midgut of resistant (RL) and non-resistant (SL) Anticarsia gemmatalis larvae were analyzed morphometrically to characterize different regions along their length. Also, this investigation compares the results between SL and RL to improve the understanding of the resistance mechanisms to the virus. Histological sections were analyzed in a computerized system and the data were statistically analyzed by the Kruskal-Wallis test and by multivariate analysis. The midguts are morphometrically different in the two larval populations; we observed higher values in RL. The morphometric analysis of the epithelial cells showed that only columnar and goblet cells were distinct along the midgut, in both larvae, with the higher values found in the anterior region. Comparing the results between the two larval populations, all the epithelial cells presented significant differences, with RL showing the higher morphometric values. We concluded that there are regional differences along the length of midgut in SL and RL that confirm the idea of two morpho-functional distinct regions. The consistently morphometric superior values in RL indicate that this variability can be related with the resistance of A. gemmatalis to its AgMNPV.     

Chitin is only a minor component of the peritrophic matrix from larvae of Lucilia cuprina

The gut of most insects is lined with a peritrophic matrix that facilitates the digestive process and protects insects from invasion by micro-organisms and parasites. It is widely accepted that the matrix is composed of chitin, proteins and proteoglycans. Here we critically re-examine the chitin content of the typical type 2 peritrophic matrix from the larvae of the fly Lucilia cuprina using a range of techniques. Many of the histochemical and biochemical techniques indicate the presence of chitin, although they are often adversely influenced by the presence of highly glycosylated proteins, a principal component of the matrix. The alkali-stable fraction, which is used as an indicator of the maximum chitin content in a biological sample, is only 7.2% of the weight of the matrix. Larvae fed on the potent chitin synthase inhibitor polyoxin D or the chitin-binding agent Calcofluor White, showed strong concentration-dependent inhibition of larval weight and survival but no discernible effects on the matrix structure. A bacterial endochitinase fed to larvae had no effect on larval growth and no observable effect in vitro on the structure of isolated peritrophic matrix. RT–PCR did not detect a chitin synthase mRNA in cardia, the tissue from which PM originates. It is concluded that chitin is a minor structural component of the type 2 peritrophic matrix of this insect.     

Improved replication of the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) in vitro using proteins from Lonomia obliqua hemolymph

The baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV), a member of the family Baculoviridae, has been widely applied as a biopesticide for the control of the velvetbean caterpillar, a pest of soybean crop field. Baculoviruses are considered safe and efficient agents for this purpose, because they do not infect vertebrates, being safe for the health of humans and animals, as well as to the environment. The objective of this work was to identify proteins obtained from Lonomia obliqua hemolymph with potential application in the optimization of baculovirus AgMNPV replication in Sf9 insect cell culture. In this work the improvement of the cell culture and viral replication of the AgMNPV baculovirus was observed when Grace medium was supplemented with 10 % (v/v) Fetal Bovine Serum (FBS), 1 % (v/v) hemolymph extract, or 3 % (v/v) of hemolymph fractions or hemolymph sub-fractions obtained by purifying hemolymph through High Performance Liquid Chromatography. Hemolymph presented a positive effect on the synthesis of polyhedra and enhanced baculovirus replication in Spodoptera frugiperda (Sf9) cells (TCID₅₀/mL), and led to Sf9 cell culture improvement. Grace medium supplemented with 10 % (v/v) FBS and 1 % (v/v) hemolymph provided an increase of baculovirus replication, when the cells were infected with multiplicity of infection of 1. In this case, the baculovirus replication was 6,443.91 times greater than that obtained with the control: Grace medium supplemented with 10 % (v/v) FBS. In addition, this work suggests that hemolymph from L. obliqua could have an interesting application in biotechnology, due to an increase in the viability of the cells and virus replication.     

A temperature-dependent developmental model for a nucleopolyhedrosis virus of the velvetbean caterpillar,Anticarsia gemmatalis (Lepidoptera: Noctuidae)

Developmental times and rates of nucleopolyhedrosis virus infection in third-instar velvetbean caterpillar, Anticarsia gemmatalis, larvae were studied in the laboratory at a variety of constant and variable temperatures. Developmental time was considered to be time from inoculation until death. Viral infection exhibited a temperature optimum of ca. 30°C and was inhibited at 10 and 40°C. Mean developmental time of the virus ranged from 18.1 days (15°C) to 5.5 days (30°C). Means and standard deviations of viral developmental rates (= development time^?1) were used as inputs into a previously derived absolute reaction rate model designed to generate a set of kinetic constants usable in predicting developmental times. Actual distributions of viral cohort developmental times were compared to distributions generated by the model. Reasonable agreement between predicted and actual distributions was found at three of four temperatures tested.     

Cowpea (Vigna unguiculata) vicilins bind to the peritrophic membrane of larval sugarcane stalk borer (Diatraea saccharalis)

In this work, we show that vicilins from two Vigna unguiculata (cowpea) genotypes, Epace-10 and IT 81D-1045, which are susceptible and resistant to attack by the cowpea weevil Callosobruchus maculatus, respectively, associate with the peritrophic membrane (PM) from larvae of Diatraea saccharalis. Solutions with increasing concentrations of vicilins were incubated with PM of the larvae and subsequently analysed by electrophoresis with SDS. It was observed that the majority of the bands of approximately 50,000 Da (characteristic of vicilins) did not appear in the separating gel and only lower molecular weight polypeptides were seen. When vicilins were incubated with PM, and the solution was then heated after the incubation, the band pattern in the gel appeared completely different. It was observed that the vicilins were being hydrolysed by proteinases associated with the PM. When the incubated samples were heated after the reaction, the major bands reappeared, demonstrating that most of the vicilin molecules had bound to the PM of D. saccharalis. These results suggest that when the vicilins are in contact with the PM they are bound and also digested by the PM of this insect. The major and several minor proteinases from the PM were extracted with Triton X-100 and their activity and the inhibition of this activity were analysed by ingel assays. Based on the effects of proteinase inhibitors, the PM-associated activity is due to serine class proteinases. Larvae of D. saccharalis were fed on artificial diets containing purified vicilins from Epace-10 or IT 81D-1045 seeds. Vicilins from Epace-10 did not affect the larval development, while IT 81D-1045 vicilins reduced significantly the survival rate of the sugar cane borer.     

The physiological role of the peritrophic membrane and trehalase: Digestive enzymes in the midgut and excreta of starved larvae of Rhynchosciara

Amylase, cellulase, trehalase, aminopeptidase and trypsin were determined using the midgut and trehalose using the haemolymph of starved and of subsequently fed larvae of Rhynchosciara americana. Midgut trehalase activity decreases steadily during starvation and increases again on feeding, whereas haemolymph trehalose titres remain constant, suggesting that trehalase is a true digestive enzyme. The decrease in amylase, cellulase and trypsin activity in the midgut during starvation is of the same order as that recovered from the excreta. Since this finding is exactly what one would expect if enzyme production stops in response to starvation, this supports the hypothesis that synthesis that synthesis of these enzymes is controlled. The excretion rate of amylase, cellulase and trypsin is very low in comparison to their activity inside the peritrophic membrane and the travel time of the food bolus through the gut. It is proposed that the peritrophic membrane separates two extracellular sites for digestion as an adaptation to conserve secreted enzymes. This could be accomplished by the existence of an endo-ectoperitrophic circulation of the enzymes involved in the initial attack on the food and by restricting to the ectoperitrophic fluid the enzymes which participate only in intermediary digestion of food.     

Disintegration of the peritrophic membrane of silkworm larvae due to spindles of an entomopoxvirus

Mode of action by which entomopoxvirus (EPV) spindles enhance nucleopolyhedrovirus (NPV) infection remains unclear. Spindles of Anomala cuprea entomopoxvirus (AcEPV), a coleoptran EPV, are known to enhance Bombyx mori NPV (BmNPV) infection in silkworm larvae. AcEPV spindles were orally administered to silkworm larvae with or without BmNPV polyhedra, and the peritrophic membranes (PMs) were observed using a binocular microscope. Soon after the larvae's access to spindles with or without the polyhedra had been terminated, some PMs disappeared wholly and some were observed in partial form. Some of the partial PMs observed were very fragile. The disintegration of the PM due to spindles also was observed by the histological sectioning of the midgut. However, a day after the larvae had terminated their access to the spindles, the PM regenerated partially or wholly. In contrast, the administration of AcEPV spheroids caused neither the disintegration of PMs nor the enhancement of BmNPV infection in silkworm larvae. These findings strongly suggest that the enhancement of NPV infection occurs due to that a greater number of NPV virions reaching the microvilli of midgut susceptible to NPV, since spindles lead to the disintegration of the PM as a barrier against NPV virions.     

Plant resistance and its effect on the peritrophic membrane of southwestern corn borer (Lepidoptera: Crambidae) larvae

The southwestern corn borer, Diatraea grandiosella Dyar (Lepidoptera: Crambidae), is a serious pest of corn, Zea mays L., in the southern United States. Corn germplasm lines with conventional genetic leaf-feeding resistance to this pest, the fall armyworm, Spodoptera frugiperda (J.E. Smith), and other lepidopterans have been released to the public by USDA-ARS scientists located in Mississippi. Recent studies suggest the insect resistant lines disrupt the integrity of the peritrophic membrane of the fall armyworm. The objectives of this study were to investigate any morphological differences in the structure of the peritrophic membrane of southwestern corn borer larvae feeding on resistant and susceptible corn hybrids and to quantify the damage. Larvae were reared under field and laboratory conditions on three corn hybrids (two resistant and one susceptible). Scanning electron microscopy was used to examine the peritrophic membrane for abnormalities such as holes or tears and to count the holes or tears in the membrane. Differences in the degree of damage to peritrophic membrane of larvae fed on resistant and susceptible plants were not detected. Up to five distinct layers of the membrane were observed in each larva. Variation in the amounts of damage to the peritrophic membrane observed from larvae feeding on all plant material was high. Plant resistance adversely affects growth and development of southwestern corn borer larvae, and further investigations are needed to explain the role of plant resistance and its relation to peritrophic membrane in southwestern corn borer larvae.     

Fluorescent Brighteners Improve Anticarsia gemmatalis (Lepidoptera: Noctuidae) Nucleopolyhedrovirus (AgMNPV) Activity on AgMNPV-Susceptible and Resistant Strains of the Insect

Fluorescent (optical) brighteners are known for their characteristics of protecting baculoviruses against deactivation by ultraviolet (UV) light and enhancing the activity of these agents as microbial insecticides on hosts and semipermissive hosts. These substances were evaluated in combination with the velvetbean caterpillar, Anticarsia gemmatalis Hübner, multiple-embedded nucleopolyhedrovirus (AgMNPV). The first trial involved 4 fluorescent brighteners (Blankophor BBH, Blankophor HRS, Blankophor RKH, and Tinopal LPW) obtained from the United States. The second trial was conducted with 11 fluorescent brighteners (Tinopal UNPA-GX, Tinopal DMS, Tinopal CBS, Leukophor DUB, Leukophor BSBB, Hostalux KS-N, Hostalux ETBN, BRY 10 D2 100, BRY 10 D2 150, Uvitex BHT, and Uvitex NFW) available in Brazil in combination with the AgMNPV to determine the degree of enhancement of viral activity. These brighteners were also evaluated with regard to AgMNPV protection against deactivation by UV light. Combinations of the virus with selected fluorescent brighteners were tested against both AgMNPV-susceptible and resistant strains of A. gemmatalis. In the first trial, brighteners obtained from the United States promoted increases in AgMNPV activity from 5.2-fold (Blankophor HRS) to 76.6-fold (Blankophor RKH) and reduced the mean time to death by 2.8 to 3.5 days. In the second trial, the most effective brightener (Tinopal UNPA-GX) reduced the LC₅₀ in A. gemmatalis larvae from 7083 occlusion bodies (OBs)/ml (virus alone) to 77.8 OBs/ml (≈90-fold). When 4 selected brighteners were tested in combination with the AgMNPV in resistant insects, the LC₅₀ was reduced by ca. 10,000-fold (Leukophor DUB) to ca. 62,000-fold (Tinopal UNPA-GX), in comparison to the LC₅₀ of 3.7 × 10⁷ OBs/ml observed for the virus alone. Therefore, mortality of highly resistant A. gemmatalis larvae to the AgMNPV increased dramatically when the virus was combined with some fluorescent brighteners. UV protection measured by original activity remaining (OAR) varied from <30% OAR (Uvitex NFW) to >90% OAR (Tinopal UNPA-GX and BRY 10 D2 100). All efficacious brighteners were stilbene disulfonic acid derivatives and, when used alone, none showed negative effects against A. gemmatalis larvae.     

Resistance to a nuclear polyhedrosis virus in the light-brown apple moth Epiphyas postvittana (Lepidoptera: Tortricidae)

Measurements were made of the relative susceptibility to a nuclear polyhedrosis virus of three populations of light-brown apple moth Epiphyas postvittana: a resistant laboratory strain (CAN), a susceptible laboratory strain (BAR), and a field population. CAN was found to be 50 times more resistant than BAR and 160 times more resistant than the field line. Experiments on hybrid crosses of resistant and susceptible strains showed that resistance is genetically determined. This serves as a warning of the possible selection of virus-resistant strains of insect pests, where viral insecticides are being used in the field.     

Acylamines enhance the infection of a baculovirus of the armyworm, Pseudaletia unipuncta (Noctuidae,Lepidoptera)

The acylamines (cationic detergents) enhance the infection of a nuclear polyhedrosis virus (NPV) in larvae of the armyworm, Pseudaletia unipuncta. Those with 6 carbons (hexylamine) or 12 carbons (dodecylamine) in the acyl group are more effective in enhancing the NPV than those with 4, 8, or 10 carbons. Hexylamine and dodecylamine increase the rate of infection beyond the 50% level (ID₅₀) by 10 to 100 times when based on the concentration of the virus polyhedra (inclusion bodies).     

Dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by the digestive fluid of Trichoplusia ni (Lepidoptera:Noctuidae) larvae

The dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus by digestive fluid collected from 5th stage Trichoplusia ni larvae was studied in vitro. Observations were made at timed intervals using phase contrast microscopy, and scanning and transmission electron microscopy. Dissolution occurred rapidly and in a detectable sequence. Under phase contrast, most polyhedra lost their refringence by 0.5 min. The polyhedra became rounded in appearance with small protuberances on the surface and Brownian movement was observed within. After 1 min, the envelope of most polyhedra had ruptured, releasing the enclosed virions. The protuberances were also observed under the scanning electron microscope after digestion for 0.5 min. Many shell fragments devoid of internal contents were seen after more lengthy digestion. Internal structural changes were revealed by electron microscopy. After 1 min of exposure, polyhedra were observed in all stages of dissolution. By 3 min, only virions, scattered about in heterogeneous material, could be distinguished.     

Comparative study on the susceptibility of cutworms (Lepidoptera: Noctuidae) to Agrotis segetum nucleopolyhedrovirus and Agrotis ipsilon nucleopolyhedrovirus

The common cutworm (Agrotis segetum) and the black cutworm (Agrotis ipsilon) are serious soil pests of many vegetable and field crops all over the world. We have demonstrated the cross-infectivity of two baculoviruses, A. segetum nucleopolyhedrovirus (AgseNPV) and A. ipsilon nucleopolyhedrovirus (AgipNPV) for these two insect pests. The susceptibility of A. segetum to AgipNPV was confirmed by DNA restriction endonuclease analyses of DNA isolated from virus harvested from infected A. segetum larvae. For an initial comparison of both viruses, partial polyhedrin sequences were amplified by PCR, cloned, and sequenced. Both viruses shared a very similar polyhedrin gene sequence resulting in only three amino acid substitutions. Phylogenetic analyses clearly demonstrated that both viruses belong to NPV group II and are most closely related to a clade consisting of Spodoptera exigua NPV, Spodoptera frugiperda NPV, and Spodoptera littoralis NPV. Since AgipNPV shows high virulence for both cutworm species, it appears to be a suitable candidate as a single biological control agent of A. segetum and A. ipsilon.     

Susceptibility of Hoplia philanthus (Coleoptera: Scarabaeidae) larvae and pupae to entomopathogenic nematodes (Rhabditida: Steinernematidae, Heterorhabditidae)

Biological control potential of nine entomopathogenic nematodes, Heterorhabditis bacteriophora CLO51 strain (HbCLO51), H. megidis VBM30 strain (HmVBM30), H. indica, Steinernema scarabaei, S. feltiae, S. arenarium, S. carpocapsae Belgian strain (ScBE), S. glaseri Belgian strain (SgBE) and S. glaseri NC strain (SgNC), was tested against second-, and third-instar larvae and pupae of Hoplia philanthus in laboratory and greenhouse experiments. The susceptibility of the developmental stages of H. philanthus differed greatly among tested nematode species/strains. In the laboratory experiments, SgBE, SgNC, HbCLO51 and HmVBM30 were highly virulent to third-instar larvae and pupae while SgBE was only virulent to second-instar larvae. Pupae were highly susceptible to HbCLO51, HmVBM30, SgBE and SgNC (57–100%) followed by H. indica and S. scarabaei (57–76%). In pot experiments, HbCLO51, SgBE and S. scarabaei were highly virulent to the third-instar larvae compared to the second-instar larvae. Our observations, combined with those of previous studies on other nematode and white grub species, show that nematode virulence against white grub developmental stages varies with white grub and nematode species.     

Proteomic analysis of nucleopolyhedrovirus infection resistance in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

Silkworm hemolymph is an important defense tissue to resist bacteria and virus infections. To study the response of silkworm hemolymph in the resistance of Bombyx mori L. nucleopolyhedrovirus (BmNPV), we constructed a near-isogenic silkworm line with BmNPV resistance using highly resistant and highly susceptible parental strains. In this paper, two-dimensional gel electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization (MALDI)-mass spectrometry were employed to investigate the differences of protein patterns in the hemolymph of the highly resistant, highly susceptible and near-isogenic silkworm strains after BmNPV was administrated to the larvae. A comparison between the proteomes of these three silkworm strains led us to identify two differentially expressed proteins, beta-N-acetylglucosaminidase 2 and aminoacylase. The expression levels of these proteins were higher in the BmNPV resistant strains.     

Two Bacillus sphaericus binary toxins share the midgut receptor binding site: implications for resistance of Culex pipiens complex (Diptera: Culicidae) larvae

This work demonstrates that Bin1 and Bin2 toxins, produced by Bacillus sphaericus strains IAB59 and 2362, respectively, share a binding site in midgut brush border membranes (BBMF) from Culex pipiens complex larvae. However, a colony selected with strain IAB59, displaying a resistance ratio of only 42-fold to IAB59, but a 162,000-fold resistance to strain 2362, was found to miss receptors for Bin2 in the BBMF. This correlates with results showing that Bin1, produced in strain IAB59, failed to bind specifically to BBMF from other colony highly resistant to strain 2362. Data indicate the loss of the BBMF bound receptor as a general mechanism of resistance to binary toxins in mosquito.     

Inheritance of resistance to Bacillus thuringiensis Cry1Ab protein in the sugarcane borer (Lepidoptera: Crambidae)

Inheritance traits of a Cry1Ab-resistant strain of the sugarcane borer, Diatraea saccharalis (F.) were analyzed using various genetic crosses. Reciprocal parental crosses between Cry1Ab-susceptible and Cry1Ab-resistant populations, F₁ by F₁ crosses, and backcrosses of F₁ with the Cry1Ab-resistant population were successfully completed. Larval mortality of the parental and cross-populations were assayed on Cry1Ab diet and Bacillus thuringiensis (Bt)-corn leaf tissue. Maternal effects and sex linkage were examined by comparing the larval mortality between the two F₁ populations. Dominance levels of resistance were measured by comparing the larval mortality of the Cry1Ab-resistant, -susceptible, and -heterozygous populations. Number of genes associated with the resistance was evaluated by fitting the observed mortality of F₂ and backcross populations with a Mendelian monogenic inheritance model. Cry1Ab resistance in D. saccharalis was likely inherited as a single or a few tightly linked autosomal genes. The resistance was incompletely recessive on Bt corn leaf tissue, while the effective dominance levels (D_ML) of resistance increased as Cry1Ab concentrations decreased with Cry1Ab-treated diet. D_ML estimated based on larval mortality on intact Bt corn plants reported in a previous study ranged from 0.08 to 0.26. This variability in D_ML levels of Cry1Ab resistance in D. saccharalis suggests that Bt corn hybrids must express a sufficient dose of Bt proteins to make the resistance genes functionally recessive. Thus, Bt resistant heterozygous individuals can be killed as desired in the “high/dose refuge” resistance management strategy for Bt corn.     

Comparative binding of Cry1Ab and Cry1F Bacillus thuringiensis toxins to brush border membrane proteins from Ostrinia nubilalis, Ostrinia furnacalis and Diatraea saccharalis (Lepidoptera: Crambidae) midgut tissue

The European (Ostrinia nubilalis Hübner) and Asian corn borers (Ostrinia furnacalis Guenée) are closely related and display similar sensitivity to Cry1 toxins. In this study, we compared the binding patterns of Cry1Ab and Cry1F toxins between both Ostrinia spp., as well as the expression of putative cadherin- and aminopeptidase-N (APN)-like protein receptors. Additionally, cDNA sequences of these putative toxin receptors from both Ostrinia species were compared. Ligand blots for both species indicated a similar binding pattern for Cry1Ab with the strongest immunoreactive band at 260 kDa in both species. In addition, similar expression of the putative cadherin- and APN-like protein receptors were observed at 260 and 135 kDa, respectively. A high degree of similarity (98% amino acid sequence identity) of cDNA sequences for both putative receptor sequences was observed. The Cry1F ligand blot revealed that O. furnacalis and O. nubilalis BBMV exhibited slightly different binding patterns, with strong binding to putative proteins at 150 and 140 kDa, respectively. Both proteins appeared to also bind Cry1Ab, although the signal intensity was much reduced with Cry1Ab. O. furnacalis showed an additional but weaker band at 210 kDa relative to the 150 kDa band. Diatraea saccharalis (Fabricius), which was used as an outgroup species, exhibited different binding patterns than either Ostrinia species, with both Cry1Ab and Cry1F toxins binding to a 210 kDa protein. These results support the previous experiments indicating that O. nubilalis and O. furnacalis share similar patterns of susceptibility to Cry toxins.