Early Instar Larvae Research Articles

Evaluation of Different IPM Modules against Green Semilooper (Thysanoplusia orichalcea Fab.) and Capitulum Borer (Helicoverpa armigera Hub.) of Sunflower

During Khariff-2017 seven different IPM modules were evaluated against green semilooper, Thysanoplusia orichalcea Fab. and capitulum borer, Helicoverpa armigera Hub. infesting sunflower. of which spinosad based module (M5) (Seed treatment with imidacloprid 70 WS (5 g/kg) + metalaxyl 35 SD (5 g/kg) + hand picking & destruction of gregarious early instar larvae of defoliators (Spodoptera litura Fab. and Spilarctia obliqua Walker) + 2 sprays of spinosad (50 and 70 DAS) found superior against major defoliator T. orichalcea Fab. at 3, 5 and 10 days after second spray and capitulum borer, H. armigera Hub. at 3, 5 and 10 days after first spray, followed by IPM module (UAS-B) (Seed treatment with imidacloprid 70 WS (5 g/kg) + two sprays of 5 % NSKE and HaNPV (50 and 70 DAS). The present findings also revealed that spinosad based module and NSKE based modules were potential candidates for the suppression of green semilooper and capitulum borer among the different IPM modules that were evaluated.

Isolation and Evaluation of Indigenous Isolates of Beauveria bassiana and Synergistic Control of Spodoptera frugiperda with the Parasitoid Microplitis prodeniae

Entomopathogenic fungi (EPFs) are capable of infecting a variety of insect pests and are widely used as biopesticides worldwide. This study intended to isolate indigenous EPFs from cadavers of Protaetia brevitarsis and investigate their effects on the fall armyworm Spodoptera frugiperda (FAW), a globally widespread invasive pest. Morphological and molecular analyses confirmed four native EPF strains all belong to Beauveria bassiana. Pathogenicity tests showed they were virulent toward FAW 1st instar larvae. The application of EPFs either by dipping or spraying significantly increased the larval mortalities compared to the control group, with corrected mortalities ranging from 92% to 73% after dipping in a fungal suspension of 108 conidia/mL, and those ranging from 76% to 35% after spraying with a fungal suspension of 107 conidia/mL. Our findings revealed the infectivity of four strains to FAW larvae significantly changed in a dose- and time-dependent manner. In addition, the combination use of the local B. bassiana strain and parasitoid Microplitis prodeniae resulted in a significantly enhanced S. frugiperda 3rd instar larval mortality compared to a single inoculation with one of them, suggesting an apparent synergistic effect for the joint application of these two biological control agents. The mortality inflicted by B. bassiana was probably promoted by the release of parasitoids, since the parasitoids’ movements and attacks could strengthen the fungal distribution and infection processes. This study underscores the potential of the combination use of EPFs and parasitoids against S. frugiperda early-instar larvae, and provides insights into the consequences of integrating these EPFs into integrated pest management systems.

A short-term impact of enriched CO2 [eCO2] on select growth performance of Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) and its host plant Gossypium barbadense L. (Malvaceae)

Natural interactions between herbivorous insect pests and their host plants are expected to be altered significantly as atmospheric CO2 concentrations (aCO2) continue to rise according to climate change scenario. The possible effect of enriched CO2 (eCO2) environments on these interactions is under attention. To better understand such effects on select insect growth parameters; early (3rd) and penultimate (6th) instar larvae of the Cotton Leaf Worm Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae), reared on the cotton plant leaves Gossypium barbadense L. (Malvacae) grown under either ambient (aCO2 = 350 ppm) or enriched (eCO2 = 700 ppm) atmospheres were investigated.

Toxic Effects of Bt-(Cry1Ab+Vip3Aa) Maize (“DBN3601T’’ Event) on the Asian Corn Borer Ostrinia furnacalis (Guenée) in Southwestern China

Asian corn borer (ACB), Ostrinia furnacalis, is an important agricultural pest affecting maize production in southwestern China, but knowledge of the toxic effect of Bt maize on the pest has been insufficient until now. In this study, we determined the susceptibility of ACB to Cry1Ab, Vip3Aa, and their complex proteins and evaluated the efficacy of Chinese domestic Bt-(Cry1Ab+Vip3Aa) maize (“DBN3601T” event) against the pest in Yunnan Province of southwestern China. The susceptible bioassay indicated that the LC50 values of the Cry1Ab and Cry1Ab+Vip3Aa proteins expressed by the Bt maize varieties against ACB larvae were 51.42 and 46.85 ng/g, respectively; however, the ACB larva was insensitive to the Vip3Aa protein. The Cry1Ab+Vip3Aa protein contents in V6–V8 leaves, VT tassels, R1 silks, R2 kernels, R3 stalks and R3 cobs of the Bt-(Cry1Ab+Vip3Aa) maize were 114.20, 30.69, 3.77, 8.92, 11.09 and 10.99 μg/g, respectively. The larval feeding test indicated that the Bt-(Cry1Ab+Vip3Aa) maize was more toxic to the early instar larvae, and the survival time of larvae fed on the leaves was the shortest, while it survived the longest on stalks. The identification of maize resistance levels in the field showed that both larval density and plant damage score of Bt-(Cry1Ab+Vip3Aa) maize were significantly lower than those in conventional maize. It is concluded that the Bt-(Cry1Ab+Vip3Aa) maize can be used for control of the ACB in southwestern China.

Expanding the transgene expression toolbox of the malaria vector Anopheles stephensi.

Anopheles stephensi Liston, 1901 (Diptera: culicidae) is a competent vector of Plasmodium falciparum (Haemosporida: plasmodiidae) malaria, and its expansion in the African continent is of concern due to its viability in urban settings and resistance to insecticides. To enhance its genetic tractability, we determined the utility of a ~2 kb An. stephensi lipophorin (lp) promoter fragment in driving transgene expression. Lipophorin genes are involved in lipid transport in insects, and an orthologous promoter in An. gambiae (AGAP001826) was previously demonstrated to successfully express a transgene. In the present study, we qualitatively characterised the expression of a ZsYellow fluorescent marker protein, expressed by An. stephensi lp promoter fragment. Our study indicated that the lp promoter fragment was effective, generating a distinct expression pattern in comparison to the commonly utilised 3xP3 promoter. The lp:ZsYellow fluorescence was largely visible in early instar larvae and appeared more intense in later instar larvae, pupae and adults, becoming especially conspicuous in adult females after a blood meal. Different isolines showed some variation in expression pattern and intensity. Aside from general transgene expression, as the lp promoter produces a suitable fluorescent protein marker expression pattern, it may facilitate genotypic screening and aid the development of more complex genetic biocontrol systems, such as multi-component gene drives. This study represents an expansion of the An. stephensi genetic toolbox, an important endeavour to increase the speed of An. stephensi research and reach public health milestones in combating malaria.

Reproductive Biology and Rearing Improvements of Apanteles opuntiarum, Potential Biocontrol Agent of the Argentine Cactus Moth, Cactoblastis cactorum.

The cactus moth, Cactoblastis cactorum (Berg) (Lepidoptera: Pyralidae) is native to South America and has been used as a biocontrol agent of Opuntia (Cactaceae) in Australia and South Africa. Its invasion in North America has raised concerns for the native Opuntia in the USA and Mexico. We investigated the reproductive biology and rearing procedures of a host-specific potential biocontrol agent, Apanteles opuntiarum Martínez and Berta (Hymenoptera: Braconidae). Given the gregarious nature of the parasitoid larvae, we studied the morphology of the immature stages and evaluated evidence of polyembryony and superparasitism. We also investigated the effects of host exposure arena and host density on parasitism rates and wasp production. The morphological descriptions provide a basis for comparison with other species. Early larval instars of A. opuntiarum are similar to other microgastrine immature stages. However, the mature larva exhibits placoid sensilla in the epistomal region, a unique character not previously reported. We provide evidence that A. opuntiarum eggs are not polyembryonic; females frequently superparasitize and have an oviposition preference for larvae parasitized 1-2 d previously. Exposing larvae of C. cactorum to wasps while inside the cactus resulted in lower parasitism and fewer offspring from each host than exposing larvae in the arena without the cactus. Parasitism and mortality rates were higher at lower host densities, possibly due to reduced host group defensive behavior. These results suggest that preference for superparasitism, host defensive behavior, and interactions with the cactus may play an important role in per-host wasp production under laboratory conditions.

The cultivable gut bacteria Enterococcus mundtii promotes early-instar larval growth of Conogethes punctiferalis via enhancing digestive enzyme activity.

Gut bacteria are crucial in influencing insect development and even phenotypic plasticity. The yellow peach moth Conogethes punctiferalis, as a significant borer pest, has been the subject of limited reports regarding the structural and diversification changes in its gut microbiota during feeding, and their potential impacts on the growth and development of the host insects. This study, employing 16S rRNA sequencing, demonstrates distinct shifts in the larvae gut microbiome of C. punctiferalis between different feeding stages, highlighting a pronounced diversity in the early-instar with Enterococcus as a predominant genus in laboratory populations. Through in vitro cultivation and sequencing, three bacterial strains - Micrococcus sp., Brevibacterium sp. and Enterococcus mundtii - were isolated and characterized. Bioassays revealed that E. mundtii-infused corn significantly boosts early-instar larval growth, enhancing both body length and weight. Quantitative PCR and spectrophotometry confirmed a higher abundance of E. mundtii in younger larvae, correlating with increased digestive enzyme activity and total protein levels. This study reveals the heightened gut microbiota diversity in early instars of C. punctiferalis larvae, highlighting that Enterococcus represent a predominant bacteria in laboratory populations. In vitro cultivation and bioassays unequivocally demonstrate the significant role of the cultivable gut bacteria E. mundtii in promoting the growth of early-instar larva. These findings provide a solid theoretical foundation for advancing the comprehension of the intricate interactions between gut microbiota and insect hosts, as well as for the development of eco-friendly pest control technologies based on targeted manipulation of insect gut microbial communities. © 2024 Society of Chemical Industry.

Efficacy of sugar‐protein non‐membranous dietary system for diapause egg production in Aedes albopictus mosquitoes under short‐day conditions

AbstractThe Asian tiger mosquito, Aedes albopictus (Skuse) (Diptera: Culicidae), is a global vector of dengue, chikungunya and zika viruses. With extreme adaptability of survival as diapause eggs, this mosquito has invaded and established in temperate climatic zones. The diapause eggs are specially programmed to overcome the harsh winter conditions in temperate habitats. In the laboratory, diapausing females are reared from early larval instars under short‐day conditions (21°C and 16D:8 L photoperiods). Aedes albopictus is normally fed on Guinea pigs for the rearing of diapause mosquitoes which requires ethical approval from the animal use committee and costly arrangements. Therefore, the present study was conducted to find whether bovine serum albumin protein (10%) with sucrose (5%) solution (SAP‐10) offered through a membrane‐free feeding system would result in an alternative diet for the production of diapause eggs in comparison to blood‐feeding. This feeding system shows a considerable rate of engorgement of diapausing females under short‐day conditions and produced 30.8 diapause eggs/female in comparison to blood‐feeding on a guinea pig (40.9 eggs/female). The diapause rate of viable eggs for the SAP‐10 diet (95.28%) was similar to the blood‐feeding (96.32%). The findings suggest that the present diet feeding system is a promising tool for the rearing of diapause Ae. albopictus mosquitoes under short‐day conditions. Further studies are suggested to evaluate the physiological and developmental aspects of mosquitoes reared on the diet.

Pathogens of the oak processionary moth Thaumetopoea processionea: Developing a user-friendly bioassay system and metagenome analyses for microorganisms

The oak processionary moth (OPM) Thaumetopoea processionea is a pest of oak trees and poses health risks to humans due to the urticating setae of later instar larvae. For this reason, it is difficult to rear OPM under laboratory conditions, carry out bioassays or examine larvae for pathogens. Biological control targets the early larval instars and is based primarily on commercial preparations of Bacillus thuringiensis ssp. kurstaki (Btk). To test the entomopathogenic potential of other spore-forming bacteria, a user-friendly bioassay system was developed that (i) applies bacterial spore suspensions by oak bud dipping, (ii) targets first instar larvae through feeding exposure and (iii) takes into account their group-feeding behavior. A negligible mortality in the untreated control proved the functionality of the newly established bioassay system. Whereas the commercial Btk HD-1 strain was used as a bioassay standard and confirmed as being highly efficient, a Bacillus wiedmannii strain was ineffective in killing OPM larvae. Larvae, which died during the infection experiment, were further subjected to Nanopore sequencing for a metagenomic approach for entomopathogen detection. It further corroborated that B.wiedmannii was not able to infect and establish in OPM, but identified potential insect pathogenic species from the genera Serratia and Pseudomonas.

An illustrated identification key to early instar larvae of forensically important Muscidae (Diptera) of the western Palaearctic region

There is a significant gap in the availability of comprehensive identification keys for the early larval stages of forensically important fly species. While well-documented identification keys exist for the third instar larvae, particularly for the Calliphoridae, Muscidae and Sarcophagidae families, there is a notable scarcity of keys for the first, except Calliphoridae, and the second instar larvae, with no such resources available for muscid species. The second instar larvae suffer the most from the lack of morphological descriptions and available identification keys. The Muscidae is one of the most frequently reported dipteran families of forensic importance colonising animal cadavers and human corpses. Nevertheless, descriptions of the morphology of their early instars remain scarce and limited to only a few species, thus their larval identification is challenging or impossible. Considering the numerous challenges associated with studying small-sized entomological material, we tested whether it is feasible to identify muscid flies to the species or at least genus level based predominantly on the details of the cephaloskeleton. To overcome the obstacle of observing details of small sclerites, especially their shapes and interconnections, we effectively employed confocal laser scanning microscopy (CLSM) as a supplementary method for light microscopy (LM). This study provides an identification key for first and second instar larvae of forensically important muscid species from the western Palaearctic (Europe, North Africa, Middle East). The proposed key primarily utilises details of the cephaloskeleton with only addition of external morphology.

Larval mosquito predation: evaluation of the effectiveness of five aquatic arthropod species against larvae of Culex pipiens Linnaeus, 1758 (Diptera: Culicidae)

ABSTRACT Mosquitoes are insects of medical interest. They contribute to the transmission and spread of many viral and parasitic diseases. Various methods have been used to control mosquitoes such as conventional insecticides, which are widely used throughout the world and have a negative impact on the environment and targeted species. Predatory insects are significant for the biocontrol of various pests, including the important potential against the larval instars of mosquitoes. In this study, we tested the efficacy of five species Piona uncata Koenike, 1888; Notonecta glauca Linnaeus, 1758; Corixa punctata Illiger, 1807, Ilyocoris cimicoide Linnaeus, 1758; and Sympetrum striolatum Charpentier, 1840 against the fourth instars larvae of Culex pipiens Linnaeus, 1758, according to two exposure phases (Light phase, Twilight phase). The predation assays show a significant effect of tested species against mosquito larvae with the following decreasing order of efficacy: S. striolatum> N. gluca> P. uncata> I. cimicoides> C. pinctata. The early larval instars are the most favourable trophic choice of the five predatory species. On the other hand, we noticed a difference in the predation rate for each species during the two experimental periods. The light phase exhibits the highest predatory efficacy than the twilight phase.

Metarhizium dianzhongense sp. nov. and New Record of M. bibionidarum (Clavicipitaceae, Hyocreales) Attacking Insects from China

The genus Metarhizium is one of the most significant entomopathogenic fungi with diverse morphological characteristics and host species. Species of Metarhizium have been widely used for pest control as an environmentally safe alternative to chemical pesticides. This study reports a new species of Metarhizium and a new record of M. bibionidarum from China. The taxonomic positions of the two species within Metarhizium were assessed by morphological and multi-gene phylogenetic data. This assessment confirmed that the new species M. dianzhongense on white grubs (Coleoptera) is a sister to M. ellipsoideum on adult leafhoppers (Hemiptera) and represents a distinctive fungus according to the morphological and phylogenetic evidence. The two species, M. dianzhongense and M. bibionidarum, were described and illustrated. Pathogenicity tests by M. bibionidarum and M. dianzhongense were performed on early instar larvae of the significant agricultural pest Spodoptera frugipera (Lepidoptera). The results demonstrated that both M. bibionidarum and M. dianzhongense exhibit significant insecticidal activity against larvae of S. frugipera, providing new fungal resources for the development of an eco-friendly biocontrol agent against this pest.

Geographic variation in larval cold tolerance and exposure across the invasion front of a widely established forest insect.

Under global climate change, high and low temperature extremes can drive shifts in species distributions. Across the range of a species, thermal tolerance is based on acclimatization, plasticity, and may undergo selection, shaping resilience to temperature stress. In this study, we measured variation in cold temperature tolerance of early instar larvae of an invasive forest insect, Lymantria dispar dispar L. (Lepidoptera: Erebidae), using populations sourced from a range of climates within the current introduced range in the Eastern United States. We tested for population differences in chill coma recovery (CCR) by measuring recovery time following a period of exposure to a nonlethal cold temperature in 2 cold exposure experiments. A 3rd experiment quantified growth responses after CCR to evaluate sublethal effects. Our results indicate that cold tolerance is linked to regional climate, with individuals from populations sourced from colder climates recovering faster from chill coma. While this geographic gradient is seen in many species, detecting this pattern is notable for an introduced species founded from a single point-source introduction. We demonstrate that the cold temperatures used in our experiments occur in nature during cold spells after spring egg hatch, but impacts to growth and survival appear low. We expect that population differences in cold temperature performance manifest more from differences in temperature-dependent growth than acute exposure. Evaluating intraspecific variation in cold tolerance increases our understanding of the role of climatic gradients on the physiology of an invasive species, and contributes to tools for predicting further expansion.

A genetic toolkit underlying the queen phenotype in termites with totipotent workers

Social insect castes (e.g., queens, workers) are prime examples of phenotypic plasticity (i.e., different phenotypes arising from the same genotype). Yet, the mechanisms that give rise to highly fertile, long-lived queens versus non-reproducing, short-lived workers are not well understood. Recently, a module of co-expressed genes has been identified that characterizes queens compared to workers of the termite Cryptotermes secundus (Kalotermitidae): the Queen Central Module (QCM). We tested whether the QCM is shared in termite species, in which queens gradually develop via early larval and late larval instars, the latter functioning as totipotent workers (linear development). Similar as in C. secundus, gene expression profiles revealed an enrichment of QCM genes in Zootermopsis angusticollis queens, a species from another termite family (Archotermopsidae). The expression of these QCM genes became gradually enriched during development from early larval instars via workers to queens. Thus, our results support the hypothesis of a conserved genetic toolkit that characterizes termite queens with gradual linear development. Our data also imply a strong caste-specific tissue specificity with the QCM signal being restricted to head-prothorax tissues in termite queens. This tissue-specific expression of key aging-related genes might have facilitated the evolution of a long lifespan in termite queens.

Ecdysteroid UDP-Glucosyltransferase Expression in Beauveria bassiana Increases Its Pathogenicity against Early Instar Silkworm Larvae.

Beauveria bassiana (B. bassiana) is a broad-spectrum entomopathogenic fungus that can control pests in agriculture and forestry. In this study, encoding ecdysteroid uridine diphosphate glucosyltransferase gene (egt) was successfully screened in B. bassiana on the medium containing 500μg/mL G418 sulfate solution through the protoplast transformation method. This enzyme has the function of 20E (20-hydroxyecdysone) inactivation, thus increasing the mortality of the early instar larvae infected with B. bassiana. In this study, we transformed B. bassiana with the egt gene, which deactivates 20-hydroxyecdysone, a key hormone in insect development. The results showed that transgenic B. bassiana killed more silkworms of the 2nd instar larvae than the wild-type with a shorter LT50 time, which was reduced by approximately 20% (day 1 of the 2nd instar silkworm infection of B. bassiana) and 26.4% (day 2 of the 2nd instar silkworm infection of B. bassiana) compared to the wild-type, and also showed a higher mortality number before molting. The transgenic B. bassiana had a higher coverage of the body surface of silkworms compared to the wild type on the 3rd instar. In summary, improving entomopathogenic fungi using biological methods such as genetic engineering is feasible.

Laboratory scale evaluation of the feasibility of locally found bladderworts as biological agents to control dengue vector, Aedes aegypti in Sri Lanka

BackgroundThe carnivorous genus Utricularia also includes aquatic species that have the potential to trap a wide range of prey, leading its death due to anoxia. However, the effectiveness of such an approach with carnivorous plants for vector control has not been evaluated in Sri Lanka.MethodsEarly instar (i & ii) and late instar (iii & iv) larvae of Aedes aegypti were exposed to locally found bladderwort (U. aurea Lour and Utricularia sp.). The experimental design was set with 10 larvae (both early and late instars separately) in 250 mL of water with bladderworts containing approximately 100 bladders in plant segments of both species, separately. Each treatment and control were repeated 50 times. The survival status of larvae was recorded daily until death or adult emergence. The larvae found whole or partially inside the bladders were attributed to direct predation. The Cox-regression model and Mantel-Cox log rank test were carried out to assess the survival probabilities of larvae in the presence of two bladderworts separately.ResultsThe highest predation was observed when using early instar larvae in both U. aurea (97.8%) and Utricularia sp. (83.8%). The mortality caused due to predation by U. aurea was observed to be significantly higher according to the Mantel-Cox log-rank test (HR = 60.71, CI; 5.69–999.25, P = 0.004). The mortality rates of late instar stages of Ae. aegypti were observed to be lower in both U. aurea (82.6%) and Utricularia sp. (74.8%). Overall, the highest predation efficacy was detected from U. aurea (HR = 45.02; CI: 5.96–850.51, P = 0.017) even in late instar stages. The results suggested the cumulative predation in both plants on Ae. aegypti larvae was > 72%.ConclusionsUtricularia aurea is a competent predator of Ae. aegypti larvae. Further, it is recommended to evaluate the feasibility of this plant to be used in the field as a control intervention in integrated vector management programmes.

Larvicidal property and active compound profiling of Annona squamosa leaf extracts against two species of diptera, Aedes aegypti and Anopheles stephensi.

Vector control measures are important in lowering the spread of infections spread by mosquito. Synthetic pesticides used to suppress vector populations during the larval stage have had adverse impacts on people and the environment. The early III instar larvae of Aedes aegypti and Anopheles stephensi were the targets of the current experiment, which assessed the larvicidal ability of petroleum ether, chloroform, methanol, and aqueous extracts of Annona squamosa leaves. Using the standard World Health Organization (WHO) larval bioassay test, leaf extracts were evaluated for their activity against Ae. aegypti and An. stephensi to determine lethal doses. Phytochemical analysis and gas chromatography-mass spectrometry (GC-MS) were carried out to identify larvicidal components in the extract. Further analysis using a scanning electron microscope (SEM) was done to check the extracts toxicity for both mosquito larvae. The larvicidal active components were identified by GC-MS as tetradecanoic acid, cis-vaccenic acid, and 2,4-di-tert-butylphenol etc. Methanol leaf extracts of A. squamosa (ASME) exhibited strong larvicidal activity against the early 3rd instar larvae of Ae. aegypti and An. stephensi with Lethal concentration (LC50) values of 51.450 ppm and 107.121 ppm. Cell damages to the larva post exposure to ASME were examined. This finding showed that the ASME has better larvicidal activity and its components that may be used to kill larvae as larvicides. The extracts toxicity towards damage of midgut of larva further suggests that this plant methanol leaf extracts could be effective in larval growth control approaches.

Cydalima perspectalis (Walker) (Lepidoptera: Crambidae) üzerinde potansiyel biyolojik mücadele etmeni Bracon hebetor (Say) (Hymenoptera: Braconidae)’un etkinliği

Cydalima perspectalis (Walker, 1859) (Lepidoptera: Crambidae) is the main pest of boxwood fields in many countries. It has caused significant damage especially since 6-7 years in Türkiye. Chemical control is definitely not recommended against this pest because boxwoods are found in forest areas, parks and gardens. In this study, the potential use of the idibiont larval parasitoid Bracon hebetor (Say, 1836) (Hymenoptera: Braconidae) against the box tree moth was evaluated within the scope of biological control. Laboratory experiments were carried out with early and late instar larvae of C. perspectalis at 25±1°C, 60-70% R.H. and 16:8 (light: dark) conditions. The number of paralyzed and parasitized larvae, parasitism rate, number of eggs laid per female, rate of larvae and pupae were determined. Bracon hebetor, which has the ability to paralyze the host larvae, showed an insecticidal effect against the pest C. perspectalis. It was found that the number of paralyzed larvae (10.33±1.15) in early stage (3rd instar) larvae was higher than in late stage (5th instar) larvae (4.67±0.57). On the other hand, it was determined that the number of parasitized larvae (14.68±1.50) and eggs laid (7.50±0.32) was higher in late stage (5th instar) larvae. Parasitism rates were found to be 33% and 73% in early and late instar larvae, respectively. This detailed biological data has been obtained to for the first time. Bracon hebetor venom showed a high toxic effect on Cydalima larvae. The results of the study showed that the parasitoid may be recommended as an alternative to chemical control against the box tree moth.

Toxicity of Elytraria Acaulis (L. F.) Lindau (Acanthaceae) to the Larvae of Vector Mosquitoes

Synthetic pesticides, which are non-biodegradable and have detrimental effects on the environment, non-targeted organisms, and human health, are often used to control mosquitoes. This situation fostered and prompted the creation of substitutes utilizing natural products like phytoextracts and phytochemicals. The current study was set out to determine the toxicity of leaf extracts from Elytraria acaulis on the early third instar larvae of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus at doses of 31.5, 62.5, 125, 250, 500 and 1000mg/L at 24 and 48 hours of exposure. All extracts, with the exception of aqueous, demonstrated potent larvicidal effectiveness with 100% larval death in all the three studied vector mosquitoes after 48 hours. The ethanol extract showed the maximum larvicidal activity and 100% larval mortality in Aedes aegypti after 24 hours, and its respective LC50 values against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus were 31.98, 560.29, 603.8108mg/L and 20.43, 46.13 and 60.08mg/L after 24 and 48 hours. The treated larvae exhibited extremely restless behaviour, including wiggling, sinking, floating, slowness, paralysis, sinking to the bottom of the glass beaker, and ultimately death. Qualitative phytochemical study of Elytraria acaulis leaves revealed the presence of alkaloids, flavonoids, glycosides, phenolics, saponins, steroids, tannins, terpenes, and terpenoids. The ethanolic extract GC-MS examination identified main phytocompounds, including imidazole, imidazolidinone, phytol, phytol acetate, octacosane, thymol 1-thiocarbonylimidazolide and methoxyacetic acid to determine the larvicidal mechanism of action and the cause of larval death. It is quite exciting to note, based on the results of the current investigation, that Elytraria acaulis leaf extracts, particularly ethanol extract, demonstrated good larvicidal efficacy. The present study documents the first report on the effectiveness of Elytraria acaulis ethanolic leaf extract against the larvae of Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus.

Dot-ELISA based on recombinant Hypodermin C of Przhevalskiana silenus for field diagnosis of goat warble fly infestation.

Goat warble fly infestation (GWFI) is an economically important myiasis caused by larvae of Przhevalskiana silenus (Diptera, Oestridae), prevalent in countries of the Mediterranean Basin and Indian subcontinent. GWFI is characterized by the presence of subcutaneous warbles at the lumbar and sacral region of dorsum in the infested animal. The early larval instars (L1 and L2) remain inaccessible to physical detection due to their small size and subcutaneous presence thus causing hindrance in the diagnosis. The objective of present study was to develop a field applicable early diagnostic intervention for GWFI monitoring and prophylactic management for effective control of the disease. Recombinant Hypodermin C (rHyC) antigen of P. silenus was expressed in Escherichia coli. The purified protein was used for optimizing dot-ELISA in a checkerboard titration using goat warble fly infested serum as known positive. The optimized assay was further tested for lower temperature (18°C) and incubation time (30 min). The optimized assay was assessed for inter-rater reliability and field samples. The optimized conditions require 188 ng of protein/dot, 1:800 dilution of serum sample, 1:4000 dilution of anti-goat IgG conjugate and 5% skim milk powder in phosphate buffer saline as blocking buffer. The assay was found to have a diagnostic sensitivity and specificity of 97.3% and 95.8%, respectively. The inter-rater reliability of dot ELISA with rHyC indirect ELISA was found to be almost perfect with a Cohen's kappa index of 0.973. Further testing at ambient temperature (18°C) and shorter incubation steps (30 min) supported suitability of the assay for field diagnosis of GWFI. The present study provides the first report of a sensitive and specific dot-ELISA for early diagnosis of GWFI which is rapid and cost effective. The test may provide an effective field applicable tool for sustainable control of GWFI.