Lepidopteran Larvae Research Articles

Morphology, phylogeny, and mitogenomics reveal a new entomopathogenic fungus, Blackwellomyces changningensis (Hypocreales, Clavicipitaceae), from southwestern China

ABSTRACT Two species of Blackwellomyces (Clavicipitaceae, Hypocreales, Ascomycota) were discovered during an investigation of the diversity of entomopathogenic fungi. A new fungus and one known fungal species that were gathered from Yunnan Province were described in this study. Blackwellomyces changningensis, sp. nov. was described using morphology and phylogenetic evidence from 14 mitochondrial protein-coding gene (PCG) data sets (atp6, atp8, atp9, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad5, nad6, and nad4L) and six nuclear genes (ITS [ITS4 and ITS5], 18S nuc rDNA [18S], 28S nuc rDNA [28S], tef1-α, rpb1, and rpb2). B. changningensis were found parasitic to Lepidoptera larvae and to produce filiform ascospores with septations. The asexual conidia were ovoid to ellipsoid in shape. Phylogenetic analysis and morphological observations concurred that the fungus belonged to a different species within the genus of Blackwellomyces. The foundation for further taxonomic, genetic, and evolutionary biological studies of the genus Blackwellomyces was laid by this work.

Lepidoptera larvae (Insecta) responsible for herbivory on Vanilla spp. (Orchidaceae) in the Federal District, Brazil

Given its culinary importance and high market value, the cultivation of Vanilla (Orchidaceae) has been expanding annually in Brazil. As observed in many crops, the increase in cultivated areas leads to a greater incidence of insects associated with species of this genus. This study reports the herbivory of larvae from Cremna thasus (Stoll, 1780) (Papilionoidea: Riodinidae: Riodininae), Hypercompe cunigunda (Stoll, 1781) (Noctuoidea: Erebidae: Arctiinae), and Spodoptera cosmioides Walker, 1858 (Noctuoidea: Noctuidae: Noctuinae) on Vanilla bahiana Hoehne and V. planifolia Andrews in the Federal District, Brazil. A list of 19 Lepidoptera species from six families associated with Vanilla spp. worldwide is provided based on a literature review. Most (n=15) of them exhibit polyphagous larvae. Given reports of exotic larvae spreading with orchids worldwide and the discussed larval polyphagy, we address precautions that producers of Vanilla and other orchids should take to reduce the incidence of insect pests in crops, especially in areas with intensive cropping systems involving annual commodities such as maize and soybean.

SIMPLE REARER AND YOUNG REVOLUTIONER

Insects are known as one of the most successful class of animals on earth, being a major player in the ecosystem. The economic importance of insects encompasses both their negative and positive importance. Often found on leaves, flowers and stems of crop plants, insects cause both direct (eat the leaves, fruits or vegetables of crop plants and many times too, do destroy the crops prior to fruiting) and indirect damage (transmit infections; viral, bacterial and fungal) to crops. Some insects are predaceous, they feed upon and destroy a large number of injurious insects. Stagomantis, a mantis is voracious, it feeds on flies, grasshoppers and caterpillars, some of which are injurious to crops. The larvae and adults of Chilomenes, a lady-bird beetle, feed on aphids which infect cotton plants. Novius, a lady-bird bettle, destroys scale worms which are pests of orange and lemon trees. Epicauta is a blister beetle, it deposits eggs where locusts occur, the larvae on hatching enter egg capsules of locusts and eat up masses of eggs. Calasoma, a ground beetle preys upon many kinds of lepidopterous larvae which destroy cereals and cotton. KEY WORDS: Honey bees, honey products, venom, royal jelly, pollen, propolis

The cryptonephridial/rectal complex: an evolutionary adaptation for water and ion conservation.

Arthropods have integrated digestive and renal systems, which function to acquire and maintain homeostatically the substances they require for survival. The cryptonephridial complex (CNC) is an evolutionary novelty in which the renal organs and gut have been dramatically reorganised. Parts of the renal or Malpighian tubules (MpTs) form a close association with the surface of the rectum, and are surrounded by a novel tissue, the perinephric membrane, which acts to insulate the system from the haemolymph and thus allows tight regulation of ions and water into and out of the CNC. The CNC can reclaim water and solutes from the rectal contents and recycle these back into the haemolymph. Fluid flow in the MpTs runs counter to flow within the rectum. It is this countercurrent arrangement that underpins its powerful recycling capabilities, and represents one of the most efficient water conservation mechanisms in nature. CNCs appear to have evolved multiple times, and are present in some of the largest and most evolutionarily successful insect groups including the larvae of most Lepidoptera and in a major beetle lineage (Cucujiformia + Bostrichoidea), suggesting that the CNC is an important adaptation. Here we review the knowledge of this remarkable organ system gained over the past 200 years. We first focus on the CNCs of tenebrionid beetles, for which we have an in-depth understanding from physiological, structural and ultrastructural studies (primarily in Tenebrio molitor), which are now being extended by studies in Tribolium castaneum enabled by advances in molecular and microscopy approaches established for this species. These recent studies are beginning to illuminate CNC development, physiology and endocrine control. We then take a broader view of arthropod CNCs, phylogenetically mapping their reported occurrence to assess their distribution and likely evolutionary origins. We explore CNCs from an ecological viewpoint, put forward evidence that CNCs may primarily be adaptations for facing the challenges of larval life, and argue that their loss in many aquatic species could point to a primary function in conserving water in terrestrial species. Finally, by considering the functions of renal and digestive epithelia in insects lacking CNCs, as well as the typical architecture of these organs in relation to one another, we propose that ancestral features of these organs predispose them for the evolution of CNCs.

Rapid evasive behaviors of diamondback moth larvae against ants

Abstract Larvae of the diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae) wriggle vigorously backward when physically stimulated. To explore whether this behavior is effective against ant attacks on their host plants with three-dimensional structures, we experimentally observed DBM larval movements and consequences thereof when attacked by Formica japonica (Hymenoptera: Formicidae) ants on the upper and lower surfaces of host plant leaves. On the upper surfaces of leaves, DBM larvae wriggled backward in response to ant attacks, and most larvae hung from leaf edges by spinning silk threads. On the lower surfaces of leaves, DBM larvae did not wriggle backward but immediately hung from the lower surface in response to attacks. No ants were able to pursue hanging larvae. We conclude that wriggling backward by DBM larvae belongs to a sequence of evasive movements against ant attacks on upper leaf surfaces. To our knowledge, this is the first report of combined wriggling and hanging behaviors by Lepidopteran larvae that are effective against ants in three-dimensional habitats.

Mode of action underlying toxicity of Camptothecin to Spodoptera frugiperda (Lepidoptera) larvae

Camptothecin (CPT) is derived from Camptotheca acuminate and its main action is the inhibition of the DNA topoisomerase I (Top I) complex. Therefore, CPT and its derivatives are always used as antitumor and antimicrobial agents and also have some insecticidal effects. However, the mode of action underlying the toxicity of CPT to the virulence of pests is still not fully understood. In this study, the effects of CPT on Spodoptera frugiperda (S. frugiperda) were determined by performing toxicity assessments, metabolomics, and mechanism analysis. The results showed that CPT increased the efficiency of inhibition of larval weight in a dose and time-dependent manner. In addition, the metabolomics of 5th-instar larval hemolymph was determined by UPLC-MS/MS and 1H NMR analysis. UPLC-MS/MS analysis showed that the energy metabolic pathway was more important than the amino acid pathway for the resistance of S. frugiperda to CPT. Furthermore, metabolites involved in the energy metabolic pathway were analyzed using 1H NMR, and fumarate and pyruvate were identified as interesting metabolites. Bioassays confirmed that they play a key role in enhancing the resistance of S. frugiperda larvae to CPT. Finally, the underlying mechanisms were found to be related to the restorative effects of the two metabolites on metabolic pathways associated with the TCA cycle. In a word, CPT is a potential biological insecticide that exhibits a strong toxicological effect against S. frugiperda by inhibiting the TCA cycle, which provides theoretical support for applying CPT in the control of pests.

Molecular Characterization of Native Isolates of Metarhizium rileyi (Farlow) Samson (Ascomycetes: Hypocreales) from Major Cropping Ecosystems of Northeastern Karnataka, India

Aims: In India, commercial biopesticides have been recommended against different insect pests on different crops, but their application has shown only a reduced efficiency attributed to differences in susceptibility of target insect pests or non-adaptability to Indian agro-climatic conditions. Therefore, there is a need to isolate region-specific biopesticide strains to enhance the efficacy of such biopesticides for pest management. The current study aimed to explore the new isolates of the entomopathogenic fungus, Metarhizium rileyi (Farlow) Samson species across the major cropping ecosystems of kalyana Karnataka. M. rileyi is an important entomopathogenic fungus of more than 30 species of Lepidoptera larvae. The aim of this research was to characterize the isolate of M. rileyi from Northeastern Karnataka by using morphological and molecular approaches. Methodology: The fungus was isolated from the naturally infected cadavers collected from different cropping ecosystems and it was cultured on Sabouraud’s Maltose Yeast Extract Agar (SMAY) media for morphological characterization, SMY media used for molecular characterization. The vegetative hyphae of fungi were 2-3 µm in diameter and hyaline, septate. The conidia ellipsoidal, smooth, and pale green were observed under the optical microscope. The DNA was isolated using the CTAB method and an internal transcribed spacer (ITS1, ITS4) was used as the primers for the amplification. The amplified products of 650 bp were purified and sequenced at ClustaL W in both directions using the above primers. A consensus sequence was obtained by alignment of the forward and reverse sequences for this region and deposited in GenBank (UASRBC Mr-24, UASRBC Mr-3, UASRBC Mr-20, UASRBC Mr-29, UASRBC Mr-7, UASRBC Mr-18). Result: A BLAST search of the sequence in GenBank revealed a 50-100 percent identity with several strains of M. rileyi (Eg. JQ686668, MN907775 and KJ725726 etc.,). Conclusion: The phylogenetic relationship was derived from comparisons of the ITS gene sequences and a Dendrogram was constructed. The homology and variations based on amino acid sequences among the isolates showed 71 per cent (OK184897) and 51 per cent similarity with four isolates (OK184899, OK177835, OK178862 and OK184898).

Effectiveness of a formulation based on Indoxacarb 120 g/l + flonicamid 200 g/l (SC) in controlling sucking biting insects and carpophagous Lepidoptera larvae of cotton in Côte d'Ivoire

Effectiveness of a formulation based on Indoxacarb 120 g/l + flonicamid 200 g/l (SC) in controlling sucking biting insects and carpophagous Lepidoptera larvae of cotton in Côte d'Ivoire

Metarhiziumpuerense (Hypocreales, Clavicipitaceae): a new species from Yunnan, south-western China.

As a genus within the Clavicipitaceae, Metarhizium exhibits rich morphological and ecological diversity, with a wide distribution and a variety of hosts. Currently, sixty-eight species of Metarhizium have been described. A new species of Metarhizium, M.puerense (Hong Yu bis), was described in Pu'er City, Yunnan Province, south-western China. Based on morphological characteristics and multilocus phylogenetic analyses, Metarhiziumpuerense was confirmed to be phylogenetically related to M.album, but was clearly separated and formed a distinct branch. In contrast, the host of Metarhiziumalbum was plants and leafhoppers and that lepidopteran larvae were the host of M.puerense. The diagnostic features of M.puerense were solitary to multiple stromata and smooth-walled, cylindrical with rounded apices conidia.

Erratum for Fayad et al., "Complete genome sequences of two Bacillus thuringiensis serovar kurstaki strains isolated from Lebanon and Tunisia, highly toxic against lepidopteran larvae".

Erratum for Fayad et al., "Complete genome sequences of two Bacillus thuringiensis serovar kurstaki strains isolated from Lebanon and Tunisia, highly toxic against lepidopteran larvae".

Native Polistes wasps (Hymenoptera: Vespidae) hold potential as biocontrol agents for lepidopteran pests of Brassica.

Social paper wasps regularly prey upon lepidopteran larvae, some of which are economically impactful agricultural pest species. We examined the potential of native North American Polistes metricus Say (Hymenoptera: Vespidae) and Polistes fuscatus (Fabricius) for biocontrol of Brassica L. pests in an experimental setting. First, we translocated P. metricus to one-half of a divided screened hoop house and placed a mix of 4th-5th instar lepidopteran larvae (Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), Pieris spp. Schrank (Lepidoptera: Pieridae), and Plutella xylostella (L.) (Lepidoptera: Plutellidae)) on half of the broccoli plants on both sides of the hoop house. We recorded and replaced missing caterpillars daily, and assessed leaf damage after 1 wk. P. metricus was 100% effective at removing all caterpillars from the wasp side of the hoop house, and plant damage by caterpillars was significantly reduced in the presence of wasps. We then replicated the study using a different combination of Polistes (fuscatus) and Brassica crop (kale) with a 2 × 2 experimental design in which plants had either T. ni larvae added or absent (factor 1), and were either covered with insect mesh or left exposed (factor 2). By the second day of exposure to these pest species, wasps removed over 80% of the larvae within 3 h of placing them on the plants. We discuss implications of this study for the potential use of native Polistes wasps as an integrated pest management strategy.

Detoxification and neurotransmitter clearance drive the recovery of Arma chinensis from β-cypermethrin-triggered knockdown

Natural enemies of arthropods contribute considerably to agriculture by suppressing pests, particularly when combined with chemical control. Studies show that insect recovery after insecticide application is rare. Here, we discovered the recovery of the predatory bug Arma chinensis from knockdown following the application of β-cypermethrin but not five other insecticides. A. chinensis individuals were more tolerant to β-cypermethrin than lepidopteran and coleopteran larvae, which did not recover from knockdown. We assessed A. chinensis recovery by monitoring their respiration and tracking locomotion through the entire process. We identified and verified the trans-regulation of detoxifying genes, including those encoding cytochrome P450s and α/β-hydrolase, which confer recovery from β-cypermethrin exposure in A. chinensis, by mitogen-activated protein kinase (MAPK) and cAMP response element binding protein (CREB). Furthermore, we discovered a novel mechanism, the neurotransmitter clearance, in vivo during the recovery process, by which the insect initiated the removal of excessive dopamine with a degrading enzyme ebony. Overall, these results provide mechanistic insights into the detoxification and neurotransmitter clearance that jointly drive insect recovery from insecticide exposure.

Specialist fig-consuming lepidopterans can inflict costs to plant reproductive success that are mitigated by ant bodyguards

Insect herbivores can inflict substantial costs on plant reproductive success. Seed herbivory impacts directly by reducing the number of seeds and therefore the dispersal and reproductive potential of the plant. Fig trees, Ficus, provide keystone resources for tropical forests. The pollinating fig wasps develop inside figs, so consumption of unripe figs results in trees not only losing seeds but also their pollen dispersers. Selection to defend figs should therefore be strong. Seed herbivory is understudied in tropical forests and most data has been collected from fallen fruits. Here we use canopy sampling to identify fig-consuming larvae in central Panama and quantify both their consequences for the fig trees’ reproductive success and the defensive value that ants provide against these larvae. Field surveys of 46 crops from nine fig species revealed that larvae could destroy up to 80% of figs on a tree. From seven Ficus species we barcoded (using COI) 51 individual fig consuming larvae (mainly Lepidoptera) that grouped into seven molecular operational taxonomic units. Lepidopteran larvae formed two feeding strategies, either stationary within a fig or tunneling between figs. Within the context of our study, stationary larvae were specialists whereas tunneling larvae were either specialists or generalists found on different Ficus species. Trees with ants had significantly fewer figs consumed by larvae (9% ± 17% (mean ± SD) for trees with azteca ants and 16% ± 24% for trees with other ants) than did trees without ants (51% ± 27%). Our results corroborate earlier findings that hosting ants can be an effective defensive mechanism for trees against seed herbivores or other antagonistic insects. Our study contributes to a wider body of research around the networks of insects associated with figs that highlights the importance of a multitrophic approach for understanding mutualism stability and persistence in the face of antagonism.

No evidence for the melanin desiccation hypothesis in a larval Lepidopteran

Water regulation is an important physiological challenge for insects due to their small body sizes and large surface area to volume ratios. Adaptations for decreasing cuticular water loss, the largest avenue of loss, are especially important. The melanin desiccation hypothesis states that melanin molecules in the cuticle may help prevent water loss, thus offering protection from desiccation. This hypothesis has much empirical support in Drosophila species, but remains mostly untested in other taxa, including Lepidoptera. Because melanin has many other important functions in insects, its potential role in desiccation prevention is not always clear. In this study we investigated the role of melanin in desiccation prevention in the white-lined Sphinx moth, Hyles lineata (Lepidoptera, Sphingidae), which shows high plasticity in the degree of melanin pigmentation during the late larval instars. We took advantage of this plasticity and used density treatments to induce a wide range of cuticular melanization; solitary conditions induced low melanin pigmentation while crowded conditions induced high melanin pigmentation. We tested whether more melanic larvae from the crowded treatment were better protected from desiccation in three relevant responses: i) total water loss over a desiccation period, ii) change in hemolymph osmolality over a desiccation period, and iii) evaporation rate of water through the cuticle. We did not find support for the melanin desiccation hypothesis in this species. Although treatment influenced total water loss, this effect did not occur via degree of melanization. Interestingly, this implies that crowding, which was used to induce high melanin phenotypes, may have other physiological effects that influence water regulation. There were no differences between treatments in cuticular evaporative water loss or change in hemolymph osmolality. However, we conclude that osmolality may not sufficiently reflect water loss in this case. This study emphasizes the context dependency of melanin’s role in desiccation prevention and the importance of considering how it may vary across taxa. In lepidopteran larvae that are constantly feeding phytophagous insects with soft cuticles, melanin may not be necessary for preventing cuticular water loss.

Bioactive Bioxanthracene and Cyclodepsipeptides from the Entomopathogenic Fungus Blackwellomyces roseostromatus BCC56290.

In the course of our ongoing research targeting the identification of potential biocontrol agents from entomopathogenic fungi (EPF), we explored a solid-state rice fungal extract of Blackwellomyces roseostromatus BCC56290 derived from infected lepidopteran larvae. Chemical and biological prospections afforded four unprecedentedly reported natural products differentiated into a dimeric naphthopyran bioxanthracene ES-242 derivative (1) and three cyclodepsipeptides (2-4) in addition to two known cyclodepsipeptides, cardinalisamides B (5) and C (6). Chemical structures of the isolated compounds were elucidated through comprehensive 1D/2D NMR and HR-ESI-MS data together with comparisons to the reported literature. The absolute configuration of the isolated cyclodepsipeptides was determined using Marfey's method. All isolated compounds were assessed for their antimicrobial, cytotoxic, and nematicidal activities with some compounds revealing significant activities.

Knock-Out of ACY-1 Like Gene in Spodoptera litura Supports the Notion that FACs Improve Nitrogen Metabolism.

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine were originally identified in the regurgitant of Spodoptera exigua larvae. These fatty acid amino acid conjugates (FACs) are known to be elicitors that induce plants to release volatile compounds which in turn attract natural enemies of the larvae such as parasitic wasps. FAC concentrations are regulated by enzymatic biosynthesis and hydrolysis in the intestine of Lepidoptera larvae. It has been proposed that FAC metabolism activates glutamine synthetase and plays an important role in nitrogen metabolism in larvae. In this study, we identified candidate genes encoding a FACs hydrolase in Spodoptera litura using genomic information of various related lepidopteran species in which FACs hydrolases have been reported. We analyzed the importance of FAC hydrolysis on caterpillar performance with CRISPR/Cas9 knock outs. Larvae of strains with an inactive FACs hydrolase excreted FACs in their feces. They absorbed 30% less nitrogen from the diet compared to WT caterpillars resulting in a reduction of their body weight of up to 40% compared to wild type caterpillars. These results suggest that the hydrolysis of FACs is an important metabolism for insects and that FACs are important for larval growth.

Lack of impact of Wolbachia on foraging behavior and morphological characteristics of the parasitoid wasp, Habrobracon hebetor (Braconidae)

Wolbachia are the most widespread intracellular alphaproteobacteria in insects with a variety of phenoptypic effects on the fitness and reproduction of their host, but much less is known about how these bacteria affect host behavior. In this study, we asked whether Wolbachia affects the foraging behavior of the parasitoid wasp Habrobracon hebetor (Hym.: Braconidae), an important biological control agent of many lepidopteran larvae. To test this, we analyzed the functional and numerical responses of Wolbachia-infected and uninfected (tetracycline cured) wasps, as well as morphological parameters. Functional response analysis showed Holling type II responses in both the Wolbachia-infected and uninfected females. The handling time and searching efficiency of Wolbachia-infected and uninfected females were similar, although the estimated maximum parasitism rate was shorter in infected females. Regardless of Wolbachia infection status, there was a negative non-linear relationship between the number of larvae parasitized by a female and an increase in the host density, reflecting a decrease in the total number of eggs laid. Our results also showed that Wolbachia had no effect on morphological traits of the parasitoid wasp. Together, these results suggest that Wolbachia which are prevalent in H. hebetor have limited impact on foraging behavior and morphology.

Establishing a fluorescence-based technique for ABC transporters functional analysis in metabolism of insecticides in a Lepidopteron

The insecticide resistance development poses challenges to the pest control effectiveness. As phase III detoxification enzyme, ATP-binding cassette (ABC) transporters play a crucial role in conferring resistance by facilitating the elimination of insecticides. However, the precise involvement of ABCs in insecticides metabolism remains unclear. In this study, a competitive assay utilizing microinjection of the ABC substrate Texas Red (TR) was established in Lepidoptera for the first time to investigate the contribution of ABCs to the detoxification process of Cydia pomonella towards lambda-cyhalothrin (LCT) and abamectin (ABM). Results revealed that the elimination of TR was hindered by ABC inhibitor verapamil, LCT, or a combination of both, implying a shared subsets of ABCs involved in the transportation of TR and LCT. Moreover, the application of ABM alone led to marked inhibition of TR elimination, with verapamil not further exacerbating this inhibitory effect, indicating that ABM exhibited a highly competitive ABC substrate characteristic and has a strong interaction with them. Furthermore, silencing key resistance associated ABC genes through RNAi resulted in increased mortality of larvae under LCT or ABM exposure, affecting TR elimination and ATP hydrolysis. These findings imply that ABC genes are critical for the active transport function of ABCs energized by ATP, ultimately reducing larvae tolerance to insecticides. In conclusion, our findings not only validated the feasibility of a competitive bioassay using fluorescent ABC substrate in lepidopteran larvae but also highlighted that ABCs in enhancing the efflux capacity of C. pomonella larvae, facilitating the binding and transportation of insecticides and their metabolites.

Two Prominent Entomopathogenic Fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metchnikoff) Sorokin as Natural Enemies of Lepidopteran Larvae from Rayalaseema Region of Andhra Pradesh

Two Prominent Entomopathogenic Fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metchnikoff) Sorokin as Natural Enemies of Lepidopteran Larvae from Rayalaseema Region of Andhra Pradesh

Discovering allatostatin type-C receptor specific agonists

Currently, there is no pesticide available for the selective control of the pine processionary moth (Thaumetopoea pityocampa-specific), and conventional methods typically rely on mechanical techniques such as pheromone traps or broad-spectrum larvicidal chemicals. As climate change increases the range and dispersion capacity of crop and forest pests, outbreaks of the pine processionary occur with greater frequency and significantly impact forestry and public health. Our study is carried out to provide a T. pityocampa-specific pesticide targeting the Allatostatin Type-C Receptor (AlstR-C). We use a combination of computational biology methods, a cell-based screening assay, and in vivo toxicity and side effect assays to identify, for the first time, a series of AlstR-C ligands suitable for use as T. pityocampa-specific insecticides. We further demonstrate that the novel AlstR-C targeted agonists are specific to lepidopteran larvae, with no harmful effects on coleopteran larvae or adults. Overall, our study represents an important initial advance toward an insect GPCR-targeted next-generation pesticide design. Our approach may apply to other invertebrate GPCRs involved in vital metabolic pathways.