Fourth Instars Research Articles

Detection and Characterization of Nosema bombycis Using TEM and SEM Techniques.

Pebrine disease is the most important and dangerous disease of silkworm caused by Nosema bombycis as an obligate intracellular parasitic fungus. It has caused tremendous economic losses in the silk industry in recent years. Given the fact that light microscopy method (with low accuracy) is the only method for diagnosing pebrine disease in the country, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) methods were adopted in this study for accurate morphological identification of the spores causing pebrine disease. Infected larvae and mother moth samples were collected from several farms (Parand, Parnian, Shaft, and Iran Silk Research Center in Gilan province, Iran). The spores were then purified using the sucrose gradient method. From each region, 20 and 10 samples were prepared for SEM and TEM analysis, respectively. In addition, an experiment was performed to evaluate the symptoms of pebrine disease by treating fourth instars with the spores purified for the present study, along with a control group. The results of SEM analysis showed that the mean±SD length and width of spores were 1.99±0.25 to 2.81±0.32 μm, respectively. Based on the obtained results, the size of spores was smaller than the Nosema bombycis (N. bombycis) as the classic species that cause pebrine disease. In addition, transmission electron microscopy (TEM) pictures showed that the grooves of the adult spores were deeper than those of other Nosema species, Vairomorpha, and Pleistophora, and resembled N. bombycis in other studies. Examination of pathogenicity of the studied spores indicated that the disease symptoms in controlled conditions were similar to those in the sampled farms. The most important symptom in fourth and fifth instrars were the small size and no growth in the treatment group compared with the control group. Findings of SEM and TEM analysis showed better morphological and structural details of parasite compared with light microscopy, and demonstrated that the studied species were a native strain of N. bombycis specific to Iran, whose size and other characteristics were unique and introduced for the first time in this study.

Lethal and Sublethal Toxicity Assessment of Cyclosporin C (a Fungal Toxin) against Plutella xylostella (L.).

Secondary metabolites/toxins produced by Purpeocillium lilacinum (Hypocreales; Phiocordycipitaceae), a well-known insect pathogen, can be used for the management of different insect pests. We report the lethal and sublethal effects of cyclosporin C (a toxin produced by P. lilacinum) against a major vegetable pest, Plutella xylostella, at specific organismal (feeding rate, larval growth, adult emergence, fecundity, and adult longevity) and sub-organismal levels (changes in antioxidant and neurophysiological enzyme activities). The toxicity of cyclosporin C against different larval instars of P. xylostella increased with increasing concentrations of the toxin and the maximum percent mortality rates for different P. xylostella larval instars at different times were observed for the 300 µg/mL cyclosporin C treatment, with an average mortality rate of 100% for all larval instars. The median lethal concentrations (LC50) of cyclosporin C against the first, second, third, and fourth larval instars of P. xylostella 72 h post-treatment were 78.05, 60.42, 50.83, and 83.05 μg/mL, respectively. Different concentrations of cyclosporin C caused a reduction in the average leaf consumption and average larval weight. Different life history parameters, such as the pupation rate (%), adult emergence (%), female fecundity, and female longevity were also inhibited when different concentrations of cyclosporin C were applied topically. The cyclosporin C concentrations inhibited the activities of different detoxifying (glutathione S-transferase, carboxylesterase, and acetylcholinesterase) and antioxidant enzyme (superoxide dismutase, catalase, and peroxidase) activities of P. xylostella when compared to the control. These findings can serve as baseline information for the development of cyclosporin C as an insect control agent, although further work on mass production, formulation, and field application is still required.

Biology of Pink Stem Borer <i>Sesamia Inferens</i> Walker on Barnyard Millet <i>Echinochloa Frumentacea</i>

Laboratory study was conducted to assess the biology of pink stem borer Sesamia inferens Walker on barnyard millet during February to April. The eggs were round and creamy white, and were laid inside the leaf sheath. The incubation period was 10.88± 0.41 days, and hatching % was 68.19± 1.16 and the larva had six instars. The weight of first, second, third, fourth, fifth and sixth instar were 0.21± 0.01, 6.86± 0.57, 36.29± 2.79, 63.75± 2.06, 135.97± 2.57 and 225.05± 4.65 mg, respectively; duration of these instars was 3.76± 0.23, 3.92± 0.17, 5.08± 0.21, 5.52± 0.28, 5.72± 0.23 and 6.68± 0.36 days, respectively. The total larval and prepupal duration were 30.68± 0.89 days and 1.48± 0.10 days, respectively, with a pupation of 86.4± 1.86% and pupal period of 9.96± 0.46 days. The pupal weights of males were 138.12± 3.05 mg and females were 196.63± 1.56 mg. The adult emergence was 84.4± 1.93%, with female being larger than males; with filiform antenna, while the had bipectinate antenna. The weight of male adult was 110.20± 4.73 mg and female adult was 189.30± 9.40 mg, and longevity was 4.92± 0.16 6.4± 0.21 days, in male and female, respectively. The fecundity ranged from 28 to 226 eggs/ female (118.4± 12.09) and the total lifecycle ranged from 41 to 63 days (51.2± 1.44 days) for male and from 47 to 70 days (58.12± 1.47 days) for female.

The age-stage specific functional response of a predator ladybird, Exochomus nigripennis (Erichson), to Gossyparia spuria Modeer at different temperatures

In this study, the age-stage specific functional response of fourth instars and adult females of Exochomus nigripennis (Erichson) was evaluated. Our results showed a type II functional response for predation toward adult Gossyparia spuria Modeer at four constant temperatures (23, 27, 31, and 35 °C). The estimated maximum a’ by a single E. nigripennis larva was 0.0317, 0.1404, 0.0656, and 0.0039 h−1, at 23, 27, 31, and 35 °C, respectively. Likewise, T h of E. nigripennis larvae significantly reduced as temperature elevated from 23 to 31 °C, and then increased at 35 °C. The a’ and T h of females at different temperatures followed the same pattern as larvae. Adult females and males consumed significantly more G. spuria than larvae at a prey density of 150 nymphs. Because of the large effect of temperature on the predation rate of G. spuria by E. nigripennis and the Type II functional response for fourth instar larvae and adults of this predator, temperature during inundative application of E. nigripennis against G. spuria in the field should be taken into account.

Evaluation of the in Vitro Efficacy of Capparis spinosa L. Alcoholic Extract in Controlling the Hairy Grain Beetle (khabra) Tragoderma granarium Everts (Coleoptera; Dermestidae)

This research was carried out to investigate the effects of the alcoholic extract of the leaves and roots of C. spinosa, at concentrations of 10, 20, and 30 mg/ml on the larvae of the second and fourth instars of the hairy grain beetle (Khabra) T. granarium. The results showed the superiority of the alcoholic extract of the leaves at the concentration of 30 mg/ml in killing the second and fourth larval instars, with mortality rates of 48.3 and 60.0%, respectively. As for the alcoholic extract of the roots, the concentration of 30 mg/ml caused the highest mortality rate in the second and fourth larval instars, with mortality rates of 23.3 and 28.3 %, respectively.

Infectivity of Entomopathogenic Fungal Isolates Against Tarnished Plant Bug Lygus lineolaris (Hemiptera: Miridae).

Twelve isolates of entomopathogenic fungi belonging to Metarhizium robertsii, M. pinghaense, M. brunneum, Beauveria bassiana, and Isaria fumosorosea were screened against tarnished plant bug. All isolates were pathogenic, causing mortality from 28.8 ± 3.4 to 96.3 ± 2.7%. The LT50 values ranged from 2.7 to 6.0 d while the LT90 values varied between 6.6 and 15.0 d. Metarhizium robertsii isolate CPD6 (will be under the trade name NoVil) was among the isolates that caused high mortality within shorter times and was selected for study on developmental stages and greenhouse trial. The third-, fourth-, and fifth-instar nymphs, and adults were inoculated with 106, 107, and 108 conidia per ml of NoVil. All the stages were susceptible to fungal infection. However, third and fourth instars were the most susceptible with no significant differences in mortality across the three concentrations. On the other hand, mortality was dose-dependent with fifth-instar nymph and adult stages. The LT50 and LT90 values were also dose-dependent, with higher concentrations having shorter lethal-time values as compared to the lower concentrations. In the greenhouse, pepper plants were sprayed with NoVil and chemical insecticide Flonicamid (as industrial standard), before releasing adult tarnished plant bug. Mortality of 37.3, 75.5, and 76.3% was recorded in the control, NoVil, and Flonicamid, respectively. This study has identified NoVil as a potential mycoinsecticide candidate for the control of tarnished plant bug under greenhouse conditions. Further field testing on juvenile and adults is needed to evaluate the potential for in-field control.

Detection, characterization and virulence analysis of nucleopolyhedrovirus isolated from the cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae)

BackgroundIn the present study, detection, characterization and virulence analysis of a field collected nucleopolyhedrovirus isolated from the cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) was carried out. The obtained isolate, named SpliNPV-YW, was collected from diseased S. littoralis larvae in El-Menoufia governorate, Egypt.ResultsTransmission electron microscopy showed the presence of typical occlusion bodies with average size of (1.06 × 1.19 µm). Upon digestion using two different endonucleases, PstI and ScaI, no clear difference was detected in the collected isolate (SpliNPV-YW) DNA genome pattern compared to the reference strain SpliNPV-AN1956. The evolutionary analysis of the polyhedrin gene's partial nucleotide sequence revealed that SpliNPV-YW isolate was closed and had a genetic origin with the NPV isolate SpliMNPV-A26-5 that belongs to group II NPVs with identity of 99.7%. The median lethal concentration (LC50) and the median lethal time (LT50) values were estimated for second and fourth larval instars of S. littoralis. The LC50 values were 2.8 × 104 OB/ml for second larval instar and 5.2 × 105 OB/ml for fourth larval instar after 10 days of treatment. Regarding the speed of killing of the viral isolate, the results showed that the LT50 value for the second instar larvae (LT50 = 5.5 days) was lower than that of the fourth instar larvae (LT50 = 6.2 days) at concentrations of 4.3 × 1010 (ob/ml) and 1.2 × 1011 (ob/ml) for second and fourth instar larvae, respectively.ConclusionsHost specificity and virulence characteristics make SpliNPV-YW isolate a good potential to be utilized as a candidate biopesticide for the control of S. littoralis population in Egypt.

Sensitivity of wMel and wAlbB Wolbachia infections in Aedes aegypti Puducherry (Indian) strains to heat stress during larval development

BackgroundICMR-Vector Control Research Centre, Puducherry, India, developed two colonies of Aedes aegypti infected with wMel and wAlbB Wolbacia strains called Ae. aegypti (Pud) lines for dengue control. The sensitivity of wMel and wAlbB strains in Ae. aegypti (Pud) lines to heat stress was studied.MethodswMel and wAlbB infected and uninfected Ae. aegypti larvae (first to fourth instars) were reared in the laboratory to adults at 26 °C, 30 °C, 36 °C and 40 °C constant temperatures and also 26–30 °C, 26–36 °C and 26–40 °C diurnal cyclic temperatures. The adults were tested for Wolbachia infection. Experiments were also carried out rearing the larvae under simulated field conditions in summer (April and June) under sunlight using fully open and half open bowls and also under sunlight and natural shade.ResultsAt 36 °C and 40 °C constant temperatures, complete larval mortality was observed. At 30 °C and 26 °C, no larval mortality occurred, but Wolbachia density was relatively low in wMel infected males compared to control (maintained at 26 ± 1 °C). At diurnal cyclic temperature of 26–40 °C, Wolbachia density was reduced in males of both the (Pud) lines, but not in females. At 26–36 °C, reduction in Wolbachia density was observed in wMel males but not in wAlbB males. At 26–30 °C, no significant reduction in Wolbachia density was observed with wMel and wAlbB strains. In simulated field conditions (April), under sunlight, the daytime water temperature reached a maximum of 35.7 °C in both full and half open bowls. No larval mortality occurred. Wolbachia frequency and density was reduced in wMel-infected Ae. aegypti (Pud) males from both type of bowls and in females from full open bowls, and in wAlbB males from half open bowls. In June, rearing of larvae under sunlight, the first-instar larvae experienced a maximum daytime water temperature of > 38 °C that caused complete mortality. No larval mortality was observed in bowls kept under shade (< 32 °C).ConclusionsExposure of larvae to higher rearing temperatures in the laboratory and simulated-field conditions reduced the densities of wMel and wAlbB strains particularly in males, but the impact was more pronounced for wMel strain. The actual effect of heat stress on the stability of these two Wolbachia strains needs to be tested under natural field conditions.Graphical

Mortality of Emerald Ash Borer Larvae in Small Regenerating Ash in New York Forests

Despite a robust biocontrol program against emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), parasitoid populations cannot build quickly enough to save mature ash trees. The future of ash as a viable component of North American forests depends on survival of immature ash that were too small to be attacked during the initial outbreak. This study was designed to quantify impacts of established introduced parasitoids and native woodpeckers on the population growth of emerald ash borer infesting regenerating ash saplings in white ash forests. Most emerald ash borer larvae were killed by Tetrastichus planipennisi Yang and woodpeckers in the fourth instar. Life-table analyses revealed that the estimated net reproductive rate of emerald ash borer was R0 = 4.2 in eastern New York and R0 = 0.0 in western New York. Without mortality from parasitoids and woodpeckers, R0 values would have been 16.4 and 7.9 in eastern and western New York, respectively. We monitored the health of mature and sapling ash trees from 2012 to 2017 and found that large trees were significantly more likely to be infested with emerald ash borer and to die than smaller trees. Fifty-four percent to 81% of ash saplings contained no living emerald ash borer, and the density of emerald ash borer at all sites was very low (< 4/m2). This study adds to the growing body of evidence that emerald ash borer biocontrol is contributing to population control and can help promote survival of young ash trees as forests recover from emerald ash borer outbreaks.

Tyrosine hydroxylase involved in cuticle tanning and reproduction in the 28-spotted potato ladybeetle, Henosepilachna vigintioctopunctata.

Tyrosine hydroxylase (TH), a melanin synthesis pathway enzyme hydroxylating tyrosine into 3,4-dihydroxyphenylalanine, is involved in the pigmentation and sclerotization of insect cuticles. However, the role of TH in 28-spotted potato ladybeetle (Henosepilachna vigintioctopunctata), an emerging pest of the solanaceous crops has been explored to a limited extent. In this study, we integrated dietary RNA interference (RNAi) and hematoxylin and eosin (H&E) staining with various bioassays to analyze the role of tyrosine hydroxylase (HvTH) throughout the developmental processes of Henosepilachna vigintioctopunctata. The results revealed that ingestion of dsHvTH led to cuticle tanning impairment, arrested larval feeding in the first and second instars of Henosepilachna vigintioctopunctata, and subsequently resulted in 100% mortality. The H&E staining assays revealed that dsHvTH prevented new abdominal cuticle formation. A pharmacological study using 3-iodo-tyrosine (3-IT), a HvTH inhibitor, disrupted larval-larval-pupal cuticle tanning during the third-fourth instar larval development and eventually failed to pupate. Similarly, dsHvTH fed to fourth instars hindered larval-pupal-adult cuticle tanning, and the eclose adults were 100% malformed. Ingestion of dsHvTH or 3-IT significantly down-regulated HvTH, HvDDC, Hvebony, and Hvlaccase2 expression and reduced dopamine levels. Finally, HvTH silencing in adult females substantially reduced the offspring hatching rates. The collective results of the study suggested that HvTH plays conserved roles in larval-pupal-adult cuticle melanization and sclerotization while exhibiting a novel function in Henosepilachna vigintioctopunctata reproduction. © 2022 Society of Chemical Industry.

Effect of cannibalism phenomenon on some biological aspects and predation efficiency on Tetranychus urticae Koch of the ladybird beetle, Coccinella undecimpunctata Linnaeus

The coccinellid predator, Coccinella undecimpunctata (L.) is one of the most important insect predator, which plays an important role in regulation of too many insect pests in Egypt and worldwide. Cannibalism phenomenon is a well-known behavioral feature in this predator, as the larvae and adults prey upon each other. The consumption capacity of this predator on Tetranychus urticae Koch, as well the eggs, first and second larval instars of the this coccinellid was evaluated. The adults and 4th larval instar and adults of C. undecimpunctata devoured more prey compared to those devoured by 1st, 2 and 3rd larval instars. The fourth larval instar of the coccinellid fed upon an average of 96.7 ± 1.2 eggs and 12.0 ± 0.7 larvae of first larvae instar of the same species in Petri dish, in the absence of the spider mite (non – choice feeding). In the presence of the mite and eggs of C. undecimpunctata, the fourth larval instar fed upon 2.2 ± 0.8 eggs and consumed only 1.0 ± 0.5 larvae in 1st larval instar of C. undecimpunctata (choice feeding), and consumed and consumed 126.0 ± 1.0 individual of spider mite in the absence of other prey (non- choice feeding). The adult of the coccinellid fed upon 10.52 ± 0.9 eggs and 5.09 ± 0.5 1st larval instars, in the absence of the mite, but fed upon 1.08 ± 0.15 eggs in the presence of the spider mite and C. undecimpunctata eggs in the same dich. Also, the coccinellid adult fed upon 0.19 ± 0.21 first larval instars in the presence of C. undecimpunctata larval and mites found in the same Petri dish. The adults of C. undecimpunctata consumed 45.83 ± 1.17 individual of the spider mite in the absence of eggs and larvae of C. undecimpunctata. Data of cannibalism and predatory efficiency show that eggs and 1st larval instars of C. undecimpunctata are more devoured by C. undecimpunctata adults comparing to 4th larval inster and larvae of the coccinellid. The two-spotted spider mite is the preferred to all stage of the coccinellid. Results show that scarcity of the prey is one the main reason to cannibalism phenomenon, but it is a conspecific behavior of all coccinellids to keep all species.

Biology and integrated management of tobacco caterpillar, Spodoptera litura Fab.: A systematic review

Tobacco caterpillar (Spodoptera litura) has damaged in great extent in different cultivated crops. Mainly it affects solanaceous crops. It feeds on the leaves of the crop and crops seems as it is fed by the animals. In later stage it damages whole plant causing rotting of plant. Commercial farmers are normally relying on chemical pesticides. Integrated Pest Management (IPM) strategy is suggested as a best option of pest management. Using respective trap crop of major crops has been found most effective method as trap has multiple advantages for the small-scale farmers. Handpicking can be effectively used for the fourth and fifth instar of the larva. This systematic review aims to collect the general biology of tobacco caterpillar and related species as well as various IPM measures of pest management. The details pest biology, ecology, host distribution and integrated pest management are reviewed systematically and presented in review section. Reviews were carried out citing different journal articles, books, conference proceedings, thesis, reports, books sections etc. This review can be useful for the researchers, academicians and extension workers to develop an IPM protocol for the tobacco caterpillar.

Feeding preference of Helicoverpa armigera larvae (Lepidoptera: Noctuidae) on vegetative and reproductive organs of soybean plants

Helicoverpa armigera is a pest insect with a large dispersal range, a high degree of polyphagy in the larval stage, and of difficult control. The pest's ability to survive due to the availability of food and persist in grain production systems present in several Brazilian regions allows it to infest a wide range of crops, including soybeans and invasive plants during the off-season. There is general knowledge that the larva prefers to feed on plant reproductive organs, but it is unknown if this is true for soybeans, a crop to which it is adapting following its recent recognition as a pest. Basic knowledge of herbivore feeding behavior is necessary to support control within the integrated pest management philosophy. The purpose of this study was to examine the preference behavior of H. armigera larvae in their first, fourth, and sixth instars for vegetative and reproductive organs of soybean plants. It is concluded that the larvae, regardless of instar, feed on all available vegetative and reproductive organs, but they have preferences based on their current developmental stage.

Reversion of CYP450 monooxygenase-mediated acetamiprid larval resistance in dengue fever mosquito, Aedes aegypti L.

Aedes-borne diseases are on the rampant rise despite continued application of chemical insecticide-based interventions. The appearance of high degree of insecticide resistance in Aedes species and noxious effects on environment and non-targets have raised further concerns. Among new chemical interventions, neonicotinoids are considered a safe and effective approach. The present study investigated the control potency of acetamiprid and development of resistance in Aedes aegypti larvae; and the involvement of CYP450 monooxygenases in inducing resistance. The early fourth instars of Ae. aegypti parent susceptible strain (PS) were selected with acetamiprid for 15 generations (ACSF strain) increasing the resistance to 19.74-fold in ACSF-10 and 36.71-fold in ACSF-15. The ACSF-10 larvae were assayed with acetamiprid combined with piperonyl butoxide (PBO) in three different ratios (1:1, 1:5 and 1:10) and selected for next five generations with 1:10 combination. Selection with synergized acetamiprid (APSF strains) reversed as well as reduced the rate of resistance development resulting in only 1.35-fold resistance in APSF-15. The APSF strains showed %monooxygenase dependency ranging from 86.71 to 96.72%. The estimation of the monooxygenases levels in parent and selected larvae showed increased monooxygenase level in the ACSF strains by 2.42-2.87-fold. The APSF-15 strains exhibited 57.95% lower enzyme production than ACSF-15 strain. The reduction and reversion of resistance by using PBO and the elevated levels of monooxygenases in ACSF and reduction in APSF strains recommend the involvement of CYP450-mediated mechanism in the development of acetamiprid resistance in Ae. aegypti. These studies could help in devising resistance management strategies in order to preserve the efficiency of pre-existing insecticides.

Potential of the mirid bug, Engytatus nicotianae, for the biological control of the tomato-potato psyllid in greenhouses

The tomato-potato psyllid (TPP), Bactericera cockerelli, is a serious pest of solanaceous crops. As an alternative to the use of insecticides, we tested the potential of the mirid bug Engytatus nicotianae (adults) as a biocontrol agent of TPP on greenhouse tomato plants. The experiment comprised: a) a laboratory choice feeding assay to investigate the preferences of E. nicotianae when offered TPP eggs and nymphs and b) a greenhouse experiment to assess the potential of E. nicotianae as a biocontrol agent under close to commercial conditions. The laboratory choice experiment highlighted a preference by E. nicotianae for the first two TPP instars, followed by the third instar and then the eggs. Consumption of fourth instars occurred only when all other life-stages had been consumed. In the cage experiment, the E. nicotianae-only treatment produced the same numbers of fruits and flowers as the control (no insects), even though TPP feeds on tomato leaves and stems. In contrast, the TPP-only treatment resulted in fewer fruits and flowers. The simultaneous introduction of E. nicotianae and TPP markedly reduced TPP build up compared to the TPP-only treatment and prevented the pest from establishing at all in four of the seven replicates of the experiment. These findings suggest that E. nicotianae should be considered as a potential biocontrol tool against TPP and further research is needed to determine an optimum release configuration.

Warming Causes Atypical Phenology in a Univoltine Moth With Differentially Sensitive Larval Stages

Climate change profoundly alters the phenology of insects, yet the mechanisms at play remain particularly elusive for univoltine species. Those species typically have to deal with contrasting thermal conditions across their development and life stages occurring at different seasons may have different thermal sensitivity. A modeling framework taking into account stage-specific thermal biology is lacking to predict the effect of climate change on the phenology of such species. Insect development rate scales non-linearly with temperature. This can be described with a thermal performance curve within each developmental stage, enabling higher accuracy near developmental thresholds than linear degree-day models. This approach, however, requires ample data to be correctly estimated. We developed a phenological model based on stage-specific performance curves to predict the phenology of a univoltine species undergoing uninterrupted larval development from summer to next spring, the pine processionary moth (Thaumetopoea pityocampa). This gregarious species is an important pine defoliator and is known to readily respond to climate change with a consistent and sustained range expansion/shift since the 1990s, as winter warming facilitates its survival in previously unsuitable areas. First, we determined the thermal performance curve of development rate for each stage from the egg to the fourth larval instar by monitoring molting in larval colonies exposed to fluctuating thermal treatments in controlled conditions. Second, we developed a phenology model to simulate the cumulated development rate across successive life stages, using observation data of adult flights and daily mean temperatures as input variables. A good fit was found between predictions and observations. Finally, the model was used to explore phenological consequences of hypothetical climate variations. With a simulated increase of temperature by 3°C, the model successfully predicted atypical ends of larval development before winter, which are being observed in nature in some regions or during years with autumnal heatwaves. With a simulated heatwave, carry-over effect on life stages development were predicted. On this winter-active species, we illustrate how variations in development rate caused by climate variations in early development can feedback into subsequent stages typically developing slowly in the cold season.

Passability of Chironomid Larvae in Granular Activated Carbon.

We conducted an experiment to confirm the passability of chironomid larvae (Glyptotendipes tokunagai) in granular activated carbon (GAC) used in water treatment plants. After injecting larvae at different growth stages (first through fourth instars) into circular columns filled with GAC, the number of individuals and their locations within the GAC columns were recorded after 168 h. We found that more than 80% of the injected larvae in the first instar and 3.3% in the second instar passed, whereas none from the third and fourth instars had passed through the column. The second instar larvae were evenly distributed within the column, whereas the third and fourth instar larvae were mostly distributed within 10 cm of the upper layer of the GAC. Our results demonstrate the passability of chironomid larvae in GAC and can be used as basic information for water quality management in water treatment plants.

The life history of Euborellia annulipes (Lucas) (Dermaptera: Anisolabididae) fed onlarvae and pupae of Plutella xylostella (L.) (Lepidoptera: Plutellidae)

Euborellia annulipes (Lucas) (Dermaptera: Anisolabididae) is reported as a potential predator of Plutella xylostella (L.) (Lepidoptera: Plutellidae). In this study, we aimed to investigate whether fourth instar larvae, 1 or 3-day-old pupae of P. xylostella are suitable food for the development and reproduction of E. annulipes. We evaluated the characteristics of the predation, development of each life stage, and reproduction of the predator, by comparing them with an artificial diet. The nymphs consumed larvae and pupae equally in the first instars, but, from the third to fifth, they consumed more larvae over pupae. Both males and females consumed more larvae over pupae as well. The shortest developmental period of nymphs was observed on larvae at the fourth instar. Males and females presented lower longevity fed on 3-day-old pupae. The highest longevity and survival rate of adults were observed on an artificial diet. Pupae of P. xylostella at any age provided the highest weight for nymphs (fourth and fifth instar) and adults. One-day-old pupae provided the highest fecundity (480 eggs/female); however, the egg hatching rate was higher when the predator fed on fourth instar larvae. The net reproductive rate was higher on 1-day-old pupae (292.8), but the intrinsic rate of increase (0.03 day$^{-1}$) and the finite rate of increase (1.034 day$^{-1}$) were lower, and both mean generation time (143.4 days) and doubling time (20.6 days) were higher on artificial diet. This study shows that fourth instar larvae, 1- and 3-day-old pupae of P. xylostella can be suitable food for the development and reproduction of E. annulipes.

Atividade inseticida de um inibidor de peptidase das sementes de Anadenanthera macrocarpa contra Anagasta kuehniella

ABSTRACT Protease inhibitors (PIs) are a part of the plant defense system and reduce the proteolytic activity of the digestive enzymes of insect pests. The current study aimed to isolate and characterize an inhibitor of trypsin (AmTI) within the seed of Anadenanthera macrocarpa (Benth) (Leguminosae-Mimosoideae). Moreover, we tried to assess the defense mechanism of the larvae of Anagasta kuehniella against this inhibitor. Protein seed extracts were purified using Sephadex G-50 and trypsin-Sepharose columns. Electrophoresis revealed the molecular weight of the inhibitor to be 25 kDa. The stability evaluation demonstrated that the inhibitor was not denatured at temperatures of up to 60 °C, pH 2-10, and concentrations of up to 100 mM dithiothreitol for one hour. The inhibitor reacted in a 1:1 ratio with bovine trypsin with an inhibition constant [Ki] = 2.517 ´ 10-8. Incorporating the inhibitor in a 1 mg per 100 mg proportion of artificial diet offered to A. kuehniella larvae led to a significant difference in the weight and survival of larvae of the fourth instar compared to the control. AmTI acted on the enzymatic activities of trypsin and chymotrypsin, not allowing until the fourth larval instar, A. kuehniella, to create adaptations against the inhibitor, as it had a simultaneous effect on larval weight and mortality. Therefore, a new trypsin inhibitor showing inhibitory activity against the digestive enzymes trypsin and chymotrypsin from A. kuehniella was isolated, indicating that these activities are correlated with the deleterious effects of this insect.

The study of the biological characters of three entomopathogenic fungi and their pathogenicity against the olive psyllid (Euphyllura olivina) under laboratory conditions

The effect of three different growth media, temperature, and pH on the mycelium growth of three isolates of entomopathogenic fungi, Fusarium sp., Verticillium lecanii and Isaria fumosorosea was investigated. PDA and SDA are the most suitable growth media. The three isolates of entomopathogenic fungi that can grow on pH varied between 4.5 and 8.5. Both temperatures, 20 and 25°C, were more suitable for the mycelia growth of these isolates. The tested isolates were sporulated on standard culture media. In this study, we evaluated the comparative entomopathogenicity of three important fungi: Fusarium sp., Verticillium lecanii and Isaria fumosorosea in infecting the fourth and fifth larval instars of E. olivina with different concentrations under laboratory conditions. The obtained results showed that these isolates caused the highest mortality in days and had the potential to limit the life stage of olive psylla.