Final Instar Larvae Research Articles

HIF signaling in the prothoracic gland regulates growth and development in hypoxia but not normoxia in Drosophila.

The developmental regulation of body size is a fundamental life-history characteristic that in most animals is tied to the transition from juvenile to adult form. In holometabolous insects, this transition is ostensibly initiated at the attainment of a critical weight in the final larval instar. It has been hypothesized that the size-sensing mechanism used to determine attainment of critical weight exploits oxygen limitation as a larvae grows beyond the oxygen-delivery capacity of its fixed tracheal system; that is, developmentally induced cellular hypoxia initiates the synthesis of the molting hormone ecdysone by the prothoracic gland. We tested this hypothesis in Drosophila by assaying cellular hypoxia throughout the third larval instar at 21 and 10 kPa O2, using the activity of the HIF (hypoxia inducible factor)-signaling pathway as a measure of hypoxia. While HIF signaling was elevated at low levels of environmental O2, it did not markedly increase during development at either oxygen level, and was only suppressed by hyperoxia after feeding had ceased. Further, changes in HIF signaling in the prothoracic gland alone did not alter body size or developmental time in a way that would be expected if cellular hypoxia in the prothoracic gland was part of the critical weight mechanism. Our data do show, however, that reduced HIF signaling in the prothoracic gland decreases survival and retards development at 10 kPa O2, suggesting that prothoracic HIF signaling is a necessary part of the beneficial plasticity mechanism that controls growth and development in response to low oxygen level.

Behavioural assay of a newly identified lepidopteran, Pexicopia tungabhadrai (Lepidoptera: Gelechiidae) along with a first report of larval diapause

Abstract This study examined the ovipositional and olfactory preferences of gravid pod moths in both choice and non-choice settings. In terms of olfactory preferences, pod moth neonates strongly preferred Abutilon indicum under choice conditions, showing nil interest in other hosts. Under non-choice conditions, both neonates and final-instar larvae thrived on A. indicum but refrained from feeding on other hosts or diets, leading to neonate mortality. Remarkably, neonates exhibited an absolute preference for A. indicum seeds, being unable to feed on Abutilon seed diets or green cotton bolls, resulting in starvation-induced mortality. A majority of the final-instar larvae successfully fed, entered pupation and completed their life cycle on A. indicum seeds but the majority entered diapause when exposed to other hosts or diets as compared to A. indicum at a temperature of 21°C, a relative humidity of 60% and a photoperiod of 14:10 hours (light:dark). Cage studies confirmed pod moth’s strong preference for A. indicum, allowing for successful larval feeding and adult emergence. However, pod moth failed to establish on cotton green bolls, underscoring their exclusive reliance on A. indicum as a host. These findings illuminate the intricate host preferences and behaviour of pod moth, underscoring the pivotal role of A. indicum in their life cycle, and is helpful for further research to exploit pod moth as an agent for biological control of the weed A. indicum.

Effects of maternal body mass, age and crowding on offspring diapause induction and life-history traits in Monochamus alternatus alternatus (Coleoptera: Cerambycidae)

ABSTRACT Monochamus alternatus alternatus is a vector of pine wilt disease and has facultative diapause at the final larval instar. To determine the maternal effect on diapause and life-history traits of offspring, two sorts of rearing experiments were implemented. Two experiments indicated that the body mass of females did not affect the induction of offspring diapause and the heritability of adult body mass did not differ from zero. In another experiment, adults that underwent diapause at three crowding levels of 1, 2 and 4 larvae/pine bolt were allowed to oviposit during their lifetime. Newly hatched larvae were inoculated singly on Pinus thunbergii bolts. Development time from larval inoculation to adult emergence and adult body mass of diapause-forgoing offspring were not affected by larval crowding level in the maternal generation, maternal age, bark surface area of the bolt (a proxy for amount of food available) or offspring sex. Body mass and head capsule width of larvae in diapause decreased with the maternal age but increased with the bark surface area. However, there was no effect of crowding level in the maternal generation. In conclusion, this study found no maternal effects on the induction of offspring diapause, although the influence of maternal photoperiodic and temperature conditions remains to be determined.

Development of the Pronotal Explanate Margin, a Novel Evolutionary Trait in Tortoise Beetles.

Most tortoise beetles, belonging to the subfamily Cassidinae (Coleoptera: Chrysomelidae), possess distinctive explanate margins, comprising elongations of the pronotum and elytra outer margins. These margins flatten against the ground, serving as a unique defensive mechanism against predators. To understand the developmental and evolutionary origins of explanate margins, we examined the development of the pronotal part of these structures in two tortoise beetle species: Thlaspida biramosa (Boheman) (tribe Cassidini) and Laccoptera nepalensis (Boheman) (tribe Aspidimorphini). Although final (fifth) instar larvae of both species exhibited no external prothoracic structures associated with explanate margins, pupae possessed a plate-shaped structure projecting anterolaterally on their pronotum. This plate-shaped structure was identified as the pupal primordium of the pronotal explanate margin, as the explanate margin emerged from inside the structure during eclosion. In prepupae of T. biramosa, the primordial tissue exhibited three region-specific folding and furrowing patterns, beneath larval cuticles. These epithelial structures expanded within minutes at the onset of pupation, resulting in pupal primordial formation. Thus, pronotal explanate margins originate from pronotal epithelia, with the furrowing and folding patterns of the epithelia shaping pupal and even adult pronotal explanate margins. The presence of morphologically similar pupal pronotal projections in various Cassidinae suggests that the development of pronotal explanate margins is likely common in these beetles.

Cross-instar larval identification leads divergent decision on pest management: An example from white grubs on peanuts

Accurate larval identification is a prerequisite for integrated pest management, but remains a long-term difficulty in insect taxonomy. Previous descriptions are mainly focused on the most disruptive final instar larvae. However, earlier instars especially cross-instar larval morphological comparisons were rarely delivered hitherto. Polyphylla laticollis and Maladera orientalis are pests that damage peanut crops at various developmental stages. The first instar larvae of P. laticollis and the mature larvae of M. orientalis attack peanuts simultaneously and cause different types of damage. In this study, we compared the life histories and larval morphologies through field investigations and scanning electron microscopy (SEM) observations. These cross-instar white grubs are similar in sizes, but exhibit morphological differences on their mouthparts, thoracic legs, anal slits, and rasters. This study presents a novel approach to larval identification, with a goal of generating new insights into integrated pest control practices on white grubs.

Helicoverpa zea Boddie (Lepidoptera: Noctuidae) pupal success and adult eclosion across variable soil type and moisture.

Helicoverpa zea Boddie (Lepidoptera: Noctuidae) is an important pest in many crops in the southern United States. Upon reaching the final larval instar, H. zea quests for a pupation site in the soil. Pupae are vulnerable to mortality since their movement is limited. Soil type and moisture can influence H. zea emergence, but the interaction of these factors has not been demonstrated. We compared sandy and clay soils in greenhouse and laboratory experiments. In the first experiment, we evaluated the preference of larvae to choose either sandy or clay soil for pupation. In a second experiment, we set the sandy soils at different moisture levels and observed prepupae pupation preference in a choice scenario. In a third experiment, we observed prepupae pupation in different moisture levels in a no-choice scenario. In a 4th experiment, we evaluated adult emergence following pupation when we increased moisture or kept it constant. In a final experiment, we evaluated pupation behavior in sandy or clay soils with a webcam and a glass arena. We found that larvae preferred to pupate in sandy soils over clay soils and that pupal success was highest at intermediate moisture levels. In addition, elevated soil moisture levels did not impact the emergence of H. zea between sandy or clay soil. Finally, H. zea did not take longer to burrow in either sandy or clay soil, but the tunnels of the pupal burrow were larger in sandy soil compared to clay soil. Our results clarify H. zea behavior across soil moisture and soil type.

Description of final stadium larva of Dysphaea gloriosa Fraser, 1938, (Odonata: Euphaeidae) with notes on its habitat and biology

The final instar larva of the genus Dysphaea Selys, 1853 is described and figured for the first time based on specimens of D. gloriosa Fraser, 1938 from Khao Yai National Park, Thailand. The larvae of D. gloriosa are very rarely encountered. The larvae are characterized by the relatively larger head, the swollen heavily spined genae extending well beyond the eyes, the swollen postocular lobes bearing a disordered row of dense strong marginal spines, the outer armature of the mandibles and the unusual labrum. Notes on the habitat and biology of the larvae are also provided.

MicroRNA miR-7-5p targets MARK2 to control metamorphosis in Galeruca daurica

The MARK2 gene, coding microtubule affinity-regulating kinase or serine/threonine protein kinase, is an important modulator in organism microtubule generation and cell polarity. However, its role in the metamorphosis of insects remains unknown. In this study, we found a conserved miRNA, miR-7-5p, which targets MARK2 to participate in the regulation of the larval-pupal metamorphosis in Galeruca daurica. The dual luciferase reporter assay showed that miR-7-5p interacted with the 3’ UTR of MARK2 and repressed its expression. The expression profiling of miR-7-5p and MARK2 displayed an opposite trend during the larval-adult development process. In in-vivo experiments, overexpression of miR-7-5p by injecting miR-7-5p agomir in the final instar larvae down-regulated MARK2 and up-regulated main ecdysone signaling pathway genes including E74, E75, ECR, FTZ-F1 and HR3, which was similar to the results from knockdown of MARK2 by RNAi. In contrast, repression of miR-7-5p by injecting miR-7-5p antagomir obtained opposite effects. Notably, both overexpression and repression of miR-7-5p in the final instar larvae caused abnormal molting and high mortality during the larval-pupal transition, and high mortality during the pupal-adult transition. The 20-hydroxyecdysone (20E) injection experiment showed that 20E up-regulated miR-7-5p whereas down-regulated MARK2. This study reveals that the accurate regulation of miRNAs and their target genes is indispensable for insect metamorphosis.

MicroRNA miR-285 modulates the metamorphosis in Galeruca daurica by targeting Br-C.

Galeruca daurica has become a new pest on the Inner Mongolia grasslands since an abrupt outbreak in 2009 caused serious damage. As a pupa indicator during insect metamorphosis, the early response gene of the ecdysone signaling pathway, Broad-Complex (Br-C), plays a vital role in the growth and development of insects. MicroRNAs (miRNAs) are small non-coding RNAs which mediate various biological activities, but it is unknown whether and how Br-C is regulated by miRNAs. Temporal expression profiles revealed that miR-285 and Br-C basically displayed an opposite trend during larval-adult development, and Br-C was sharply up-regulated on the last day of final-instar larvae while miR-285 was significantly down-regulated. Both dual-luciferase reporter assay and miRNA-mRNA interaction assay indicated that miR-285 interacts with the coding sequence of Br-C and represses its expression. Not only overexpression but also downexpression of miR-285 led to the failure of larval to pupal to adult metamorphosis. In addition, both overexpression of miR-285 and silence of Br-C inhibited the expression of Br-C and other ecdysone signaling pathway genes, including E74, E75, ECR, FTZ-F1, and HR3. On the contrary, suppressing miR-285 obtained opposite results. Further experiments showed that 20-hydroxyecdysone down-regulated miR-285 and up-regulated Br-C and above-mentioned genes, whereas juvenile hormone alalogue (JHA) resulted in opposite effects. Our results reveal that miR-285 is involved in mediating the metamorphosis in G. daurica by targeting Br-C in the ecdysone signaling pathway. miR-285 and its target Br-C could be as a potential target for G. daurica management. © 2024 Society of Chemical Industry.

Description of the Last Instar Larva of Platylestes platystylus (Rambur, 1842) from Kerala, India (Odonata: Lestidae).

The description of the final instar larva and the subsequent exuvia of Platylestes platystylus (Rambur, 1842) is given based on a single male larva collected from Kerala and reared in the laboratory. A brief account of the habitat of the damselfly and an updated key to the larvae of genera of Family Lestidae Calvert, 1901 are also provided.

Description of the final instar larva of Burmagomphus laidlawi Fraser, 1924, an endemic of the Western Ghats, India (Odonata: Gomphidae)

Description of the final instar larva of Burmagomphus laidlawi Fraser, 1924, an endemic of the Western Ghats, India (Odonata: Gomphidae)

Description of the final-instar larva of Psaironeura tenuissima (Odonata: Zygoptera: Coenagrionidae) from Amazonia

The final-instar larva of Psaironeura tenuissima is described based on reared specimens from Amazonas and Pará states in the Brazilian Amazon. Psaironeura larvae are grouped with Neoneura and Protoneura larvae by having nodated caudal lamellae, however, they can be differentiated by the number of labial palp setae. Psaironeura tenuissi­ma are morphologically compared with P. angeloi larvae as well as those of Neoneura kiautai and Protoneura aurantiaca. The P. tenuissima larva can be distinguished from that of P. ange­loi by the shape of the superior margin of prementum, projections on the prothorax, caudal lamellae apex shape, and the number of setae in the paraproct ventral margin setae.

Life cycles and larval and pupal habitats of eleven Japanese Rhyacophila (Trichoptera) species at the mountain streams in central Honshu

The life cycles and microhabitats involved in aquatic stages of eleven Rhyacophila Stephens, 1836 species living in two neighboring Japanese mountain streams in Nara Prefecture, central Honshu, were investigated. Eight species had univoltine life cycles; seven species had spring to early summer emergence seasons. One species had an autumnal emergence. Four univoltine species had periods where no larvae or pupae were collected in aquatic stages, possibly a period of egg diapause. Three species had bivoltine life cycles, with early summer and autumn emergence seasons. Rhyacophila nipponica Iwata, 1927, R. manuleata AV Martynov, 1934, and R. shikotsuensis Iwata, 1927, in the nigrocephala species group Ross, 1956, have larvae which spend most of their larval stage in the hyporheos. The larvae of R. transquilla Tsuda, 1940, also inhabit the hyporheic zone, although the vertical microhabitat used by this species differed from the former three species. All four of these species have hyporheic larval stages, but in these species the first and final instar larvae reside on the surfaces of cobbles. The first instar larvae seem to live on stone surfaces immediately after hatching, and subsequently move to the interstices of sand. Final instar larvae live in the interstices of sandy bottom, and move to stone surfaces for pupation. Eleven species of the genus Rhyacophila had diverse life histories, microhabitat preferences, and experienced differing water temperature regimes during larval stages at the sites. Life history and habitat preference variation enhances the species richness of the genus and coexistence of species in upper stream of the Kinokawa River.

The immunostimulatory role of an Enterococcus-dominated gut microbiota in host protection against bacterial and fungal pathogens in Galleria mellonella larvae.

Understanding the intricate interplay between the gut microbiota and the immune response in insects is crucial, given its diverse impact on the pathogenesis of various microbial species. The microbiota's modulation of the host immune system is one such mechanism, although its complete impact on immune responses remains elusive. This study investigated the tripartite interaction between the gut microbiota, pathogens, and the host's response in Galleria mellonella larvae reared under axenic (sterile) and conventional (non-sterile) conditions. The influence of the microbiota on host fitness during infections was evaluated via two different routes: oral infection induced by Bacillus thuringiensis subsp. galleriae (Btg), and topical infection induced by Metarhizium robertsii (Mr). We observed that larvae without a microbiota can successfully fulfill their life cycle, albeit with more variation in their developmental time. We subsequently performed survival assays on final-instar larvae, using the median lethal dose (LD50) of Btg and Mr. Our findings indicated that axenic larvae were more vulnerable to an oral infection of Btg; specifically, a dose that was calculated to be half-lethal for the conventional group resulted in a 90%-100% mortality rate in the axenic group. Through a dual-analysis experimental design, we could identify the status of the gut microbiota using 16S rRNA sequencing and assess the level of immune-related gene expression in the same group of larvae at basal conditions and during infection. This analysis revealed that the microbiota of our conventionally reared population was dominated entirely by four Enterococcus species, and these species potentially stimulated the immune response in the gut, due to the increased basal expression of two antimicrobial peptides (AMPs)-gallerimycin and gloverin-in the conventional larvae compared with the axenic larvae. Furthermore, Enterococcus mundtii, isolated from the gut of conventional larvae, showed inhibition activity against Btg in vitro. Lastly, other immune effectors, namely, phenoloxidase activity in the hemolymph and total reactive oxygen/nitrogen species (ROS/RNS) in the gut, were tested to further investigate the extent of the stimulation of the microbiota on the immune response. These findings highlight the immune-modulatory role of the Enterococcus-dominated gut microbiota, an increasingly reported microbiota assemblage of laboratory populations of Lepidoptera, and its influence on the host's response to oral and topical infections.

The larva of Microptila orienthula Kjrandsen & Ito (Trichoptera, Hydroptilidae), with a description of its microhabitat in Japan.

The final instar larva, case, and microhabitat of the East Palearctic caddisfly species Microptila orienthula Kjrandsen & Ito 2009 (Trichoptera, Hydroptilidae) are described from Japan. The laterally compressed larva occurs in small plant tufts (primarily mosses and liverworts) in hygropetric habitats.

Phylogeny of Holocacista capensis (Lepidoptera: Heliozelidae) from Vineyards and Natural Forests in South Africa Inferred from Mitochondrial and Nuclear Genes

In South Africa, the family Heliozelidae in the order Lepidoptera is restricted to four known species. The grapevine leaf miner, Holocacista capensis, feeds between the epidermal layers of a grapevine leaf, predominantly along the leaf margin. A final instar larva will descend from the blotch mine/gallery to attach its cocoon casing (constructed from the epidermal layers of the mined gallery) to any object below the infested leaf. Five monophyletic clades and a polyphyletic group have been identified within the Heliozelidae, using a mitochondrially encoded gene cytochrome c oxidase I (COI) and a nuclear gene, histone 3 (H3). An exploratory study of the genetic diversity within H. capensis populations was conducted using these genes. The phylogenetic analyses of COI indicate that H. capensis that are currently being collected from South Africa fall within three clades/haplotypes, of which one is well supported and contains only one species from Gauteng, and one has three specimens from two different areas in the Western Cape province, while 80% belong to haplotype 1 (H1). The current study can be used as a starting point for future DNA-based studies aimed at gaining insight into possible patterns of diversity in H. capensis to confirm switching from native to commercial grapevine hosts. However, more samples need to be collected from different areas in South Africa.

Studies on influence of various stages of mulberry leaf in the growth and cocoonic parameters of silkworm Bombyx mori (L.)

The objective of this work was to determine the effects of different stages of mulberry leaves on growth performance of treated larvae of Bombyx mori L. (Lepidoptera, Bombycidae) and characters of the resulted cocoons were evaluated. Results showed that the food consumption rate and assimilation rate were greatest for larvae fed on tender and over matured mulberry leaves as compared to larvae fed on mature leaves. Assimilation and conversion efficiencies were very high in the larvae grown on matured mulberry leaves (0.189 and 1.99% respectively). Moreover, the larvae fed on matured mulberry leaves showed higher growth rate, cocoon weight and shell ratio. Among the three different growth stages of leaves, the matured leaves were found to be the best food for final instar larvae of Bombyx mori L. which promotes maximum larval growth and it gains to quality cocoon and raw silk production.

Morphology and biology of Homoplectra gracilis Nozaki, 2019 in a headwater stream in Yata Hills, Japan

Homoplectra Ross, 1938 (Hydropsychidae) comprises 24 species. Knowledge of the immature stages of species in this genus is quite poor. This study aimed to describe for the first time morphology of Homoplectra gracilis Nozaki, 2019 larvae, found in small headwater streams flowing throughout ‘Satoyama’, a unique and important ecosystem in Japan, and clarify its biology. Morphological observations showed that the larva of this species is unique among known Japanese species in having a small median triangular process on the anterior margin of the head. Final instar larvae were found throughout the year, whereas the second instar larvae were observed during July and November. The species retreated using various kinds of substrates; on/between leaves, plants roots, and under stones. Analysis of digestive tracts showed predominantly detritus in their diet along with remains of invertebrates. The larvae appeared to withstand drought which occur in the streams.

Description of the Larva of Oxyallagma dissidens (Selys, 1876) (Odonata: Coenagriidae, Ischnurinae)

<i>Oxyallagma dissidens</i> is a species of high elevations 2600-2700m in Ecuador and Peru. The larva of <i>Oxyallagma dissidens</i> was hitherto unknown. Based on exuviae of reared specimens, the final instar larva of <i>Oxyallagma dissidens</i> is described. Exuviae of <i>Oxyallagma dissidens</i> have two parallel rows of dark spots on the dorsum of the abdomen, the prementum has 5-6 setae and the labial palps 6 setae. Antenna 7-segmented, distal margin of labial palp with four crenulations and a large spine. Head brownish and between the eyes at the front with a light band with three loops to behind. Pterothorax with parallel stripes on the wing bags at the inner side. Metathoracic femur length 2.75 mm. All femora with four darker edges with fine spines. Abdominal dorsum with fine punctuations and with a darker brownish distal half. <i>Oxyallagma dissidens</i> has a comparable strong armation on the femurs. The gill lamellae have a medial broad dark brown vein, with several veins at all sides to the border of gills. In one case a description on <i>Oxyallagma dissidens </i>was not given, but a figure was published. <i>Oxyallagma dissidens </i>was mentioned from<i> </i>Peru (Quito, La Granadilla) and<i> from </i>Ecuador (Lago San Pablo, Campos of Guayaquil). <i>Oxyallagma dissidens </i>is the recent name in contrast to the alternative name <i>runtuni</i>. Bota-Sierra and Andrés described <i>Oxyallagma colombianum</i> as new species of the genus from Colombia.

Influence of hot water and UV radiation on host infectivity of entomopathogenic nematode Steinernema glaseri (Glaser, 1932)

Entomopathogenic nematodes act as good bio-control agents to manage lepidopteron and coleopteran pests. Steinernematidae and Heterorhabditae are considered important families in entomopathogenic nematodes. An in-vitro experiment was conducted to enhance the efficacy of Steinernema glaseri using hot water and UV radiation on final instar larvae of Corcyra Cephalonia. The results showed that the highest number of larvae were infected at 35 °C and 45 °C of hot water immersion. Among the UV-exposed larvae, the highest number of larvae infected was obtained by UV exposure for 10 minutes.