Gene cloning, recombinant expression, and bioassay of an allatotropin in Spodoptera litura Fabricius (Lepidoptera: Noctuidae)

Background Allatotropin, a neuropeptide found in several invertebrates indirectly regulates vitellogenesis by stimulating juvenile hormone biosynthesis by the corpora allata. Here, we cloned and expressed the gene encoding allatotropin of Spodoptera litura (tobacco cutworm), a polyphagous pest in the Asian tropics. This study is aimed at studying the effect of recombinant protein on egg-laying in females of S. litura as it could be used as a method to control the pests from a reproductive perspective. Results The protein encodes a full-length open reading frame consisting of 173 amino acids and was rich in arginine (10%) and glutamic acid (9.3%). The theoretical pI of the protein was 5.47 and a hydrophobic signal peptide of 22 residues was predicted. The recombinant allatotropin was expressed in Escherichia coli BL21 (DE3) and purified by nickel exchange chromatography. The molecular weight of the recombinant protein was about 37 kDa and expression levels up to 5.3 mg/ml were achieved. Injection assay in vitro indicated that allatotropin induces egg-laying during the first scotophase after treatment in females of Spodoptera litura. Conclusion Allatotropin induces egg-laying in female moths and could be a potential molecule for the development of control strategies against Spodoptera litura. In this strategy, the protein if delivered to the females before mating may lead to accelerated egg deposition much before she encounters the male moths, thus the population being checked as the eggs deposited by the females are unfertilized. Thus, the present work could lead to the development of a protein based biopesticide resulting into a species-specific and an eco-friendly way of pest control.

Neuroendocrine Regulation of Reproductive Dormancy in the Fruit Fly Drosophila melanogaster: A Review of Juvenile Hormone-Dependent Regulation

Animals can adjust their physiology, helping them survive and reproduce under a wide range of environmental conditions. One of the strategies to endure unfavorable environmental conditions such as low temperature and limited food supplies is dormancy. In some insect species, this may manifest as reproductive dormancy, which causes their reproductive organs to be severely depleted under conditions unsuitable for reproduction. Reproductive dormancy in insects is induced by a reduction in juvenile hormones synthesized in the corpus allatum (pl. corpora allata; CA) in response to winter-specific environmental cues, such as low temperatures and short-day length. In recent years, significant progress has been made in the study of dormancy-inducing conditions dependent on CA control mechanisms in Drosophila melanogaster. This review summarizes dormancy control mechanisms in D. melanogaster and discusses the implications for future studies of insect dormancy, particularly focusing on juvenile hormone-dependent regulation.

Alternative Use of the Insecticide Diofenolan on Musca domestica (Diptera: Muscidae): A Morphological and Ultrastructural Investigation

Musca domestica Linnaeus (Diptera: Muscidae), a relevant synanthropic pest, is the most frequent dipteran species in farms and waste landfills. Insect Growth Regulators (IGR), insecticides with species-specific toxicity and low health and environmental impact, are known to act mostly on larval stages but may induce sterility in adults. We investigated the effects of diofenolan, an IGR analogue of juvenile hormone, on M. domestica, with special attention to female reproductive ability (egg-laying and hatching), and ovarian and ovariole morphology, using optical and transmission electron microscopy. We also tested the interactions between diofenolan and the activity of corpora allata, endocrine gland producing juvenile hormone required for ovarian development, by morphological and ultrastructural investigations. The results show that diofenolan negatively affects the reproductive ability of M. domestica, causing extensive morphological alterations in ovaries and ovarioles. In treated females, ovarioles showed nine different morphotypes that could be arranged into three “transformation paths”, and these alterations were able to reduce egg-laying. The effects of diofenolan on corpora allata, investigated at the optical and ultrastructural level in M. domestica, showed extensive alterations of the nuclei, cytoplasms, and mitochondria, strongly suggesting a rapid transition of the gland from inactivity to degeneration. The sterilizing effects of diofenolan in M. domestica are very interesting for integrated pest management programs within sustainable defence strategies against this relevant and annoying pest.

Sulfakinins influence lipid composition and insulin-like peptides level in oenocytes of Zophobas atratus beetles

Insect sulfakinins are pleiotropic neuropeptides with the homology to vertebrate gastrin/cholecystokinin peptide family. They have been identified in many insect species and affect different metabolic processes. They have a strong influence on feeding and digestion as well as on carbohydrate and lipid processing. Our study reveals that sulfakinins influence fatty acids composition in Zophobas atratus oenocytes and regulate insulin-like peptides (ILPs) level in these cells. Oenocytes are cells responsible for maintenance of the body homeostasis and have an important role in the regulation of intermediary metabolism, especially of lipids. To analyze the lipid composition in oenocytes after sulfakinins injections we used gas chromatography combined with mass spectrometry and for ILPs level determination an immunoenzymatic test was used. Because sulfakinin peptides and their receptors are the main components of sulfakinin signaling, we also analyzed the presence of sulfakinin receptor transcript (SKR2) in insect tissues. We have identified for the first time the sulfakinin receptor transcript (SKR2) in insect oenocytes and found its distribution more widespread in the peripheral tissues (gut, fat body and haemolymph) as well as in the nervous and neuro-endocrine systems (brain, ventral nerve cord, corpora cardiaca/corpora allata CC/CA) of Z. atratus larvae. The presence of sulfakinin receptor transcript (SKR2) in oenocytes suggests that observed effects on oenocytes lipid and ILPs content may result from direction action of these peptides on oenocytes.

Identification and function of ETH receptor networks in the silkworm Bombyx mori

Insect ecdysis triggering hormones (ETHs) released from endocrine Inka cells act on specific neurons in the central nervous system (CNS) to activate the ecdysis sequence. These primary target neurons express distinct splicing variants of ETH receptor (ETHR-A or ETHR-B). Here, we characterized both ETHR subtypes in the moth Bombyx mori in vitro and mapped spatial and temporal distribution of their expression within the CNS and peripheral organs. In the CNS, we detected non-overlapping expression patterns of each receptor isoform which showed dramatic changes during metamorphosis. Most ETHR-A and a few ETHR-B neurons produce multiple neuropeptides which are downstream signals for the initiation or termination of various phases during the ecdysis sequence. We also described novel roles of different neuropeptides during these processes. Careful examination of peripheral organs revealed ETHRs expression in specific cells of the frontal ganglion (FG), corpora allata (CA), H-organ and Malpighian tubules prior to each ecdysis. These data indicate that PETH and ETH are multifunctional hormones that act via ETHR-A and ETHR-B to control various functions during the entire development—the ecdysis sequence and associated behaviors by the CNS and FG, JH synthesis by the CA, and possible activity of the H-organ and Malpighian tubules.

A population of neurons that produce hugin and express thediuretic hormone 44 receptorgene projects to the corpora allata inDrosophila melanogaster

The corpora allata (CA) are essential endocrine organs that biosynthesize and secrete the sesquiterpenoid hormone, namely juvenile hormone (JH), to regulate a wide variety of developmental and physiological events in insects. Previous studies had demonstrated that the CA are directly innervated with neurons in many insect species, implying the innervations to be important for regulating JH biosynthesis in response to internal physiology and external environments. While this is also true for the model organism,Drosophila melanogaster, which neurotransmitters are produced in the CA-projecting neurons are yet to be clarified. In this study onD. melanogaster, we aimed to demonstrate that a subset of neurons producing the neuropeptide hugin, the invertebrate counterpart of the vertebrate neuromedin U, directly projects to the adult CA. A synaptic vesicle marker in the hugin neurons was observed at their axon termini located on the CA, which were immunolabeled with a newly-generated antibody to the JH biosynthesis enzyme JH acidO-methyltransferase (JHAMT). We also found the CA-projecting hugin neurons to likely express a gene encoding the specific receptor for diuretic hormone 44 (Dh44). Moreover, our data suggested that the CA-projecting hugin neurons have synaptic connections with the upstream neurons producing Dh44. To the best of our knowledge, this is the first study to identify a specific neurotransmitter of the CA-projecting neurons inD. melanogaster, and to anatomically characterize a neuronal pathway of the CA-projecting neurons and their upstream neurons.

Identification and Function of ETH Receptor Networks in The Silkworm Bombyx Mori

Insect ecdysis triggering hormones (ETHs) released from endocrine Inka cells act on specific neurons in the central nervous system (CNS) to activate the ecdysis sequence. These primary target neurons express distinct splicing variants of ETH receptor (ETHR-A or ETHR-B). Here, we characterized both ETHR subtypes in the moth Bombyx mori in vitro and mapped spatial and temporal distribution of their expression within the CNS and peripheral organs. In the CNS, we detected non-overlapping expression patterns of each receptor isoform which showed dramatic changes during metamorphosis. Most ETHR-A and a few ETHR-B neurons produce multiple neuropeptides which are downstream signals for the initiation or termination of various phases during the ecdysis sequence. We also described novel roles of different neuropeptides during these processes. Careful examination of peripheral organs revealed ETHRs expression in specific cells of the frontal ganglion (FG), corpora allata (CA), H-organ and Malpighian tubules prior to each ecdysis. These data indicate that PETH and ETH are multifunctional hormones that act via ETHR-A and ETHR-B to control various functions during the entire development - the ecdysis sequence and associated behaviors by the CNS and FG, JH synthesis by the CA, and possible activity of the H-organ and Malpighian tubules.

Crucial Role of Juvenile Hormone Receptor Components Methoprene-Tolerant and Taiman in Sexual Maturation of Adult Male Desert Locusts

Currently (2020), Africa and Asia are experiencing the worst desert locust (Schistocerca gregaria) plague in decades. Exceptionally high rainfall in different regions caused favorable environmental conditions for very successful reproduction and population growth. To better understand the molecular mechanisms responsible for this remarkable reproductive capacity, as well as to fill existing knowledge gaps regarding the regulation of male reproductive physiology, we investigated the role of methoprene-tolerant (Scg-Met) and Taiman (Scg-Tai), responsible for transducing the juvenile hormone (JH) signal, in adult male locusts. We demonstrated that knockdown of these components by RNA interference strongly inhibits male sexual maturation, severely disrupting reproduction. This was evidenced by the inability to show mating behavior, the absence of a yellow-colored cuticle, the reduction of relative testes weight, and the drastically reduced phenylacetonitrile (PAN) pheromone levels of the treated males. We also observed a reduced relative weight, as well as relative protein content, of the male accessory glands in Scg-Met knockdown locusts. Interestingly, in these animals the size of the corpora allata (CA), the endocrine glands where JH is synthesized, was significantly increased, as well as the transcript level of JH acid methyltransferase (JHAMT), a rate-limiting enzyme in the JH biosynthesis pathway. Moreover, other endocrine pathways appeared to be affected by the knockdown, as evidenced by changes in the expression levels of the insulin-related peptide and two neuroparsins in the fat body. Our results demonstrate that JH signaling pathway components play a crucial role in male reproductive physiology, illustrating their potential as molecular targets for pest control.

Approaches and Tools to Study the Roles of Juvenile Hormones in Controlling Insect Biology

The juvenile hormones (JHs) are a group of sesquiterpenoids synthesized by the corpora allata. They play critical roles during insect development and reproduction. To study processes that are controlled by JH, researchers need methods to identify and quantify endogenous JHs and tools that can be used to increase or decrease JH titers in vitro and in vivo. The lipophilic nature of JHs, coupled with the low endogenous titers, make handling and quantification challenging. JH titers in insects can easily be increased by the topical application of JH analogs, such as methoprene. On the other hand, experimentally reducing JH titers has been more difficult. New approaches to modulate JH homeostasis have been established based on advances in RNA interference and CRISPR/Cas9-based genome editing. This review will summarize current advances in: (1) the detection and quantification of JHs from insect samples; (2) approaches to manipulating JH titers; and (3) next-generation tools to modulate JH homeostasis.