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8 pages, 6231 KiB

Open AccessCommunication

Chemical Treatments for Insect Cell Differentiation: The Effects of 20-Hydroxyecdysone and Veratridine on Cultured Spodoptera frugiperda (Sf21) Insect Cell Ultrastructure

by Lacey J. Jenson, James J. Becnel and Jeffrey R. Bloomquist

Insects 2022, 13(1), 32; https://doi.org/10.3390/insects13010032 - 28 Dec 2021

Viewed by 1559

Abstract

Previous studies have shown that insect cell cultures stop dividing, form clumps, and can be induced to grow processes reminiscent of axons, when the culture medium is supplemented with 20-hydroxyecdysone, insulin, or an agent that mimics their action, such as the ecdysone agonist, [...] Read more.

Previous studies have shown that insect cell cultures stop dividing, form clumps, and can be induced to grow processes reminiscent of axons, when the culture medium is supplemented with 20-hydroxyecdysone, insulin, or an agent that mimics their action, such as the ecdysone agonist, methoxyfenozide. Those cell growing processes resemble nerve cells, and the present study evaluates the ultrastructure of these cultures by transmission electron microscopy. Sf21 cells treated with 20-hydroxyecdysone (with or without veratridine amendment) and subjected to ultrastructural analysis had a similar somatic appearance to control cells, with slight changes in organelles and organization, such as a greater number of cytoplasmic vacuoles and mitochondrial granules. Finger-like projections were observed between control and treated cells. However, no structural markers of synaptic contacts (e.g., vesicles or synaptic thickenings) were observed in controls, 20-hydroxyecdysone, or 20-hydroxyecdysone + veratridine treated cells. It is concluded that additional agents would be required to induce functional synaptogenesis in Sf21 cells. Full article

(This article belongs to the Special Issue Advances in the Use of Insect Cell Culture and Biotechnology)

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17 pages, 5433 KiB

Open AccessArticle

Tick Cell Culture Analysis of Growth Dynamics and Cellular Tropism of Rickettsia buchneri, an Endosymbiont of the Blacklegged Tick, Ixodes scapularis

by Cody J. Thorpe, Xin-Ru Wang, Ulrike G. Munderloh and Timothy J. Kurtti

Insects 2021, 12(11), 968; https://doi.org/10.3390/insects12110968 - 27 Oct 2021

Cited by 2 | Viewed by 2994

Abstract

The blacklegged tick, Ixodes scapularis, a species of significant importance to human and animal health, harbors an endosymbiont Rickettsia buchneri sensu stricto. The symbiont is largely restricted to the ovaries, but all life stages can harbor various quantities or lack R. buchneri [...] Read more.

The blacklegged tick, Ixodes scapularis, a species of significant importance to human and animal health, harbors an endosymbiont Rickettsia buchneri sensu stricto. The symbiont is largely restricted to the ovaries, but all life stages can harbor various quantities or lack R. buchneri entirely. The endosymbiont is cultivable in cell lines isolated from embryos of Ixodes ticks. Rickettsia buchneri most readily grows and is maintained in the cell line IRE11 from the European tick, Ixodes ricinus. The line was characterized by light and electron microscopy and used to analyze the growth dynamics of wildtype and GFPuv-expressing R. buchneri. qPCR indicated that the genome copy doubling time in IRE11 was >7 days. Measurements of fluorescence using a plate reader indicated that the amount of green fluorescent protein doubled every 11 days. Two 23S rRNA probes were tested via RNA FISH on rickettsiae grown in vitro and adapted to evaluate the tissue tropism of R. buchneri in field-collected female I. scapularis. We observed strong positive signals of R. buchneri in the ovaries and surrounding the nucleus of the developing oocytes. Tissue tropism in I. scapularis and in vitro growth dynamics strengthen the contemporary understanding of R. buchneri as a transovarially transmitted, non-pathogenic endosymbiont. Full article

(This article belongs to the Special Issue Advances in the Use of Insect Cell Culture and Biotechnology)

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18 pages, 2566 KiB

Open AccessArticle

Isolation in Natural Host Cell Lines of Wolbachia Strains wPip from the Mosquito Culex pipiens and wPap from the Sand Fly Phlebotomus papatasi

by Lesley Bell-Sakyi, Alexandra Beliavskaia, Catherine S. Hartley, Laura Jones, Lisa Luu, Lee R. Haines, James G. C. Hamilton, Alistair C. Darby and Benjamin L. Makepeace

Insects 2021, 12(10), 871; https://doi.org/10.3390/insects12100871 - 26 Sep 2021

Cited by 9 | Viewed by 3344

Abstract

Endosymbiotic intracellular bacteria of the genus Wolbachia are harboured by many species of invertebrates. They display a wide range of developmental, metabolic and nutritional interactions with their hosts and may impact the transmission of arboviruses and protozoan parasites. Wolbachia have occasionally been isolated [...] Read more.

Endosymbiotic intracellular bacteria of the genus Wolbachia are harboured by many species of invertebrates. They display a wide range of developmental, metabolic and nutritional interactions with their hosts and may impact the transmission of arboviruses and protozoan parasites. Wolbachia have occasionally been isolated during insect cell line generation. Here, we report the isolation of two strains of Wolbachia, wPip and wPap, during cell line generation from their respective hosts, the mosquito Culex pipiens and the sand fly Phlebotomus papatasi. wPip was pathogenic for both new C. pipiens cell lines, CPE/LULS50 and CLP/LULS56, requiring tetracycline treatment to rescue the lines. In contrast, wPap was tolerated by the P. papatasi cell line PPL/LULS49, although tetracycline treatment was applied to generate a Wolbachia-free subline. Both Wolbachia strains were infective for a panel of heterologous insect and tick cell lines, including two novel lines generated from the sand fly Lutzomyia longipalpis, LLE/LULS45 and LLL/LULS52. In all cases, wPip was more pathogenic for the host cells than wPap. These newly isolated Wolbachia strains, and the novel mosquito and sand fly cell lines reported here, will add to the resources available for research on host–endosymbiont relationships, as well as on C. pipiens, P. papatasi, L. longipalpis and the pathogens that they transmit. Full article

(This article belongs to the Special Issue Advances in the Use of Insect Cell Culture and Biotechnology)

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18 pages, 3082 KiB

Open AccessArticle

Evaluation of Total Female and Male Aedes aegypti Proteomes Reveals Significant Predictive Protein–Protein Interactions, Functional Ontologies, and Differentially Abundant Proteins

by Abubakar Shettima, Shaleni Joseph, Intan H. Ishak, Syahirah Hanisah Abdul Raiz, Hadura Abu Hasan and Nurulhasanah Othman

Insects 2021, 12(8), 752; https://doi.org/10.3390/insects12080752 - 20 Aug 2021

Cited by 2 | Viewed by 2187

Abstract

Aedes aegypti is a significant vector for many tropical and subtropical flavivirus diseases. Only the female mosquito transmits pathogens, while the male plays a vital role in mating and species continuity. This study explored the total proteomes of females and males based on [...] Read more.

Aedes aegypti is a significant vector for many tropical and subtropical flavivirus diseases. Only the female mosquito transmits pathogens, while the male plays a vital role in mating and species continuity. This study explored the total proteomes of females and males based on the physiological and genetic differences of female and male mosquitoes. Protein extracts from mosquitoes were analysed using LC–ESI–MS/MS for protein identification, protein interaction network analysis, functional ontology enrichment, and differential protein abundance analyses. Protein identification revealed 422 and 682 proteins exclusive to males and females, respectively, with 608 common proteins found in both sexes. The most significant PPIs (<1.0 × 10⁻¹⁶) were for common proteins, followed by proteins exclusive to females (<1.0 × 10⁻¹⁶) and males (1.58 × 10⁻¹²). Significant functional enrichments were observed in the biological process, molecular function, and cellular component for the male and female proteins. The abundance of the proteins differed, with one protein showing an increase (elongation factor 1 α, EF1α) and two showing reductions (actin family) in females versus males. Overall, the study verified the total proteomes differences between male and female Ae. aegypti based on protein identification and interactions, functional ontologies, and differentially abundant proteins. Some of the identified proteins merit further investigation to elucidate their roles in blocking viral transmission. Full article

(This article belongs to the Special Issue Advances in the Use of Insect Cell Culture and Biotechnology)

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22 pages, 4926 KiB

Open AccessArticle

Three-Dimensional Culture of Rhipicephalus (Boophilus) microplus BmVIII-SCC Cells on Multiple Synthetic Scaffold Systems and in Rotating Bioreactors

by Michael T. Suderman, Kevin B. Temeyer, Kristie G. Schlechte and Adalberto A. Pérez de León

Insects 2021, 12(8), 747; https://doi.org/10.3390/insects12080747 - 19 Aug 2021

Cited by 1 | Viewed by 2627

Abstract

Tick cell culture facilitates research on the biology of ticks and their role as vectors of pathogens that affect humans, domestic animals, and wildlife. Because two-dimensional cell culture doesn’t promote the development of multicellular tissue-like composites, we hypothesized that culturing tick cells in [...] Read more.

Tick cell culture facilitates research on the biology of ticks and their role as vectors of pathogens that affect humans, domestic animals, and wildlife. Because two-dimensional cell culture doesn’t promote the development of multicellular tissue-like composites, we hypothesized that culturing tick cells in a three-dimensional (3-D) configuration would form spheroids or tissue-like organoids. In this study, the cell line BmVIII-SCC obtained from the cattle fever tick, Rhipicephalus (Boophilus) microplus (Canestrini, 1888), was cultured in different synthetic scaffold systems. Growth of the tick cells on macrogelatinous beads in rotating continuous culture system bioreactors enabled cellular attachment, organization, and development into spheroid-like aggregates, with evidence of tight cellular junctions between adjacent cells and secretion of an extracellular matrix. At least three cell morphologies were identified within the aggregates: fibroblast-like cells, small endothelial-like cells, and larger cells exhibiting multiple cytoplasmic endosomes and granular vesicles. These observations suggest that BmVIII-SCC cells adapted to 3-D culture retain pluripotency. Additional studies involving genomic analyses are needed to determine if BmVIII-SCC cells in 3-D culture mimic tick organs. Applications of 3-D culture to cattle fever tick research are discussed. Full article

(This article belongs to the Special Issue Advances in the Use of Insect Cell Culture and Biotechnology)

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11 pages, 2034 KiB

Open AccessArticle

Bombyx mori Nucleopolyhedrovirus p26 Is Associated with Viral Late Stage Replication

by Jun-Qing Ge, Zhu-Hong Wang, Xi Chen, Hua Chen and Jian Huang

Insects 2021, 12(8), 707; https://doi.org/10.3390/insects12080707 - 06 Aug 2021

Cited by 2 | Viewed by 1645

Abstract

Bombyx mori nucleopolyhedrovirus (BmNPV) p26 is conserved among all Lepidoptera baculoviruses that have been completely sequenced thus far, and some baculoviruses even have two copies of p26, which suggested that p26 may play an important role in the virus infection cycle. This [...] Read more.

Bombyx mori nucleopolyhedrovirus (BmNPV) p26 is conserved among all Lepidoptera baculoviruses that have been completely sequenced thus far, and some baculoviruses even have two copies of p26, which suggested that p26 may play an important role in the virus infection cycle. This study aimed to characterize BmNPV p26. We found that BmNPV p26 transcripts were detectable as early as 3 h post-infection (hpi), and the transcript levels rapidly increased starting from 12 hpi. Western blot analysis using an anti-p26 polyclonal antibody demonstrated that the corresponding protein was also detectable from 6 hpi in BmNPV-infected cell lysates. Immunofluorescence analysis demonstrated that p26 was mainly dispersed in the infected cell cytoplasm, whereas the over-expressed fusion protein EGFP-p26 also accumulated in the nucleus. These results indicated that p26 is an early BmNPV gene and has functions both in the cytoplasm and the nucleus. RNAi-based knockdown of p26 could produce infectious virus and normal-appearing virions but decreased budded virus (BV) production in BmNPV-infected cells at 72 hpi. Moreover, the results of further quantitative PCR (Q-PCR) analysis indicated that the gp64 and p74 transcripts levels decreased significantly. These results indicated that BmNPV p26 may be associated with BmNPV replication during the late infection stage. Full article

(This article belongs to the Special Issue Advances in the Use of Insect Cell Culture and Biotechnology)

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30 pages, 2130 KiB

Open AccessEditor’s ChoiceArticle

Investigating Virus–Host Interactions in Cultured Primary Honey Bee Cells

by Alexander J. McMenamin, Fenali Parekh, Verena Lawrence and Michelle L. Flenniken

Insects 2021, 12(7), 653; https://doi.org/10.3390/insects12070653 - 17 Jul 2021

Cited by 5 | Viewed by 5277

Abstract

Honey bee (Apis mellifera) health is impacted by viral infections at the colony, individual bee, and cellular levels. To investigate honey bee antiviral defense mechanisms at the cellular level we further developed the use of cultured primary cells, derived from either [...] Read more.

Honey bee (Apis mellifera) health is impacted by viral infections at the colony, individual bee, and cellular levels. To investigate honey bee antiviral defense mechanisms at the cellular level we further developed the use of cultured primary cells, derived from either larvae or pupae, and demonstrated that these cells could be infected with a panel of viruses, including common honey bee infecting viruses (i.e., sacbrood virus (SBV) and deformed wing virus (DWV)) and an insect model virus, Flock House virus (FHV). Virus abundances were quantified over the course of infection. The production of infectious virions in cultured honey bee pupal cells was demonstrated by determining that naïve cells became infected after the transfer of deformed wing virus or Flock House virus from infected cell cultures. Initial characterization of the honey bee antiviral immune responses at the cellular level indicated that there were virus-specific responses, which included increased expression of bee antiviral protein-1 (GenBank: MF116383) in SBV-infected pupal cells and increased expression of argonaute-2 and dicer-like in FHV-infected hemocytes and pupal cells. Additional studies are required to further elucidate virus-specific honey bee antiviral defense mechanisms. The continued use of cultured primary honey bee cells for studies that involve multiple viruses will address this knowledge gap. Full article

(This article belongs to the Special Issue Advances in the Use of Insect Cell Culture and Biotechnology)

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Review

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19 pages, 692 KiB

Open AccessReview

Cell Line Platforms Support Research into Arthropod Immunity

by Cynthia L. Goodman, David S. Kang and David Stanley

Insects 2021, 12(8), 738; https://doi.org/10.3390/insects12080738 - 17 Aug 2021

Cited by 11 | Viewed by 2868

Abstract

Innate immune responses are essential to maintaining insect and tick health and are the primary defense against pathogenic viruses, bacteria, and fungi. Cell line research is a powerful method for understanding how invertebrates mount defenses against pathogenic organisms and testing hypotheses on how [...] Read more.

Innate immune responses are essential to maintaining insect and tick health and are the primary defense against pathogenic viruses, bacteria, and fungi. Cell line research is a powerful method for understanding how invertebrates mount defenses against pathogenic organisms and testing hypotheses on how these responses occur. In particular, immortal arthropod cell lines are valuable tools, providing a tractable, high-throughput, cost-effective, and consistent platform to investigate the mechanisms underpinning insect and tick immune responses. The research results inform the controls of medically and agriculturally important insects and ticks. This review presents several examples of how cell lines have facilitated research into multiple aspects of the invertebrate immune response to pathogens and other foreign agents, as well as comments on possible future research directions in these robust systems. Full article

(This article belongs to the Special Issue Advances in the Use of Insect Cell Culture and Biotechnology)

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18 pages, 926 KiB

Open AccessReview

Growth and Maintenance of Wolbachia in Insect Cell Lines

by Ann M. Fallon

Insects 2021, 12(8), 706; https://doi.org/10.3390/insects12080706 - 06 Aug 2021

Cited by 14 | Viewed by 3440

Abstract

The obligate intracellular microbe, Wolbachia pipientis (Rickettsiales; Anaplasmataceae), is a Gram-negative member of the alpha proteobacteria that infects arthropods and filarial worms. Although closely related to the genera Anaplasma and Ehrlichia, which include pathogens of humans, Wolbachia is uniquely associated with invertebrate [...] Read more.

The obligate intracellular microbe, Wolbachia pipientis (Rickettsiales; Anaplasmataceae), is a Gram-negative member of the alpha proteobacteria that infects arthropods and filarial worms. Although closely related to the genera Anaplasma and Ehrlichia, which include pathogens of humans, Wolbachia is uniquely associated with invertebrate hosts in the clade Ecdysozoa. Originally described in Culex pipiens mosquitoes, Wolbachia is currently represented by 17 supergroups and is believed to occur in half of all insect species. In mosquitoes, Wolbachia acts as a gene drive agent, with the potential to modify vector populations; in filarial worms, Wolbachia functions as a symbiont, and is a target for drug therapy. A small number of Wolbachia strains from supergroups A, B, and F have been maintained in insect cell lines, which are thought to provide a more permissive environment than the natural host. When transferred back to an insect host, Wolbachia produced in cultured cells are infectious and retain reproductive phenotypes. Here, I review applications of insect cell lines in Wolbachia research and describe conditions that facilitate Wolbachia infection and replication in naive host cells. Progress in manipulation of Wolbachia in vitro will enable genetic and biochemical advances that will facilitate eventual genetic engineering of this important biological control agent. Full article

(This article belongs to the Special Issue Advances in the Use of Insect Cell Culture and Biotechnology)

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