The metabolism and role of free fatty acids in key physiological processes in insects of medical, veterinary and forensic importance

Insects are the most widespread group of organisms and more than one million species have been described. These animals have significant ecological functions, for example they are pollinators of many types of plants. However, they also have direct influence on human life in different manners. They have high medical and veterinary significance, stemming from their role as vectors of disease and infection of wounds and necrotic tissue; they are also plant pests, parasitoids and predators whose activities can influence agriculture. In addition, their use in medical treatments, such as maggot therapy of gangrene and wounds, has grown considerably. They also have many uses in forensic science to determine the minimum post-mortem interval and provide valuable information about the movement of the body, cause of the death, drug use, or poisoning. It has also been proposed that they may be used as model organisms to replace mammal systems in research. The present review describes the role of free fatty acids (FFAs) in key physiological processes in insects. By focusing on insects of medical, veterinary significance, we have limited our description of the physiological processes to those most important from the point of view of insect control; the study examines their effects on insect reproduction and resistance to the adverse effects of abiotic (low temperature) and biotic (pathogens) factors.

An MD-2-related lipid-recognition protein is required for insect reproduction and integument development

The myeloid differentiation factor 2 (MD-2)-related lipid-recognition protein is involved in immune responses through recognizing bacteria lipopolysaccharide in mammals, arthropods and plants. However, the physiological roles of MD-2 in other biological processes are largely unknown. Here, we identified three homologue MD-2 genes ( NlML1 , NlML2 and NlML3 ) by searching the genome and transcriptome databases of the brown planthopper Nilaparvata lugens , a hemipteran insect species. Temporospatial analysis showed that the NlML1 gene was highly expressed in the fat body but much less so in the other tissues, while the NlML2 and NlML3 genes were highly expressed in the testis or digestive tract. RNA interference-mediated depletion of the NlML1 gene significantly downregulated the transcription of numerous integument protein genes. The NlML1 knockdown led to moulting failure and mortality at the nymph–adult transition phase, impaired egg laying and hatching, and reduced 20-hydroxyecdysone (20E) production in the nymphs. 20E could rescue the deficient moulting phenotypes derived from ds NlML1 RNAi. These novel findings indicate that NlML1 is required for nymphal moulting and female reproductive success as it plays an important role in regulating 20E synthesis, lipid and chitin metabolisms in N. lugens , thus contributing to our understanding of developmental and reproductive mechanisms in insects.

Wolbachia cifB induces cytoplasmic incompatibility in the malaria mosquito vector

Wolbachia, a maternally inherited intracellular bacterial species, can manipulate host insect reproduction by cytoplasmic incompatibility (CI), which results in embryo lethality in crosses between infected males and uninfected females. CI is encoded by two prophage genes, cifA and cifB. Wolbachia, coupled with the sterile insect technique, has been used in field trials to control populations of the dengue vector Aedes albopictus, but CI-inducing strains are not known to infect the malaria vector Anopheles gambiae. Here we show that cifA and cifB can induce conditional sterility in the malaria vector An. gambiae. We used transgenic expression of these Wolbachia-derived genes in the An. gambiae germline to show that cifB is sufficient to cause embryonic lethality and that cifB-induced sterility is rescued by cifA expression in females. When we co-expressed cifA and cifB in male mosquitoes, the CI phenotype was attenuated. In female mosquitoes, cifB impaired fertility, which was overcome by co-expression of cifA. Our findings pave the way towards using CI to control malaria mosquito vectors.

Juvenile hormone signaling promotes ovulation and maintains egg shape by inducing expression of extracellular matrix genes

It is well documented that the juvenile hormone (JH) can function as a gonadotropic hormone that stimulates vitellogenesis by activating the production and uptake of vitellogenin in insects. Here, we describe a phenotype associated with mutations in the Drosophila JH receptor genes, Met and Gce: the accumulation of mature eggs with reduced egg length in the ovary. JH signaling is mainly activated in ovarian muscle cells and induces laminin gene expression in these cells. Meanwhile, JH signaling induces collagen IV gene expression in the adult fat body, from which collagen IV is secreted and deposited onto the ovarian muscles. Laminin locally and collagen IV remotely contribute to the assembly of ovarian muscle extracellular matrix (ECM); moreover, the ECM components are indispensable for ovarian muscle contraction. Furthermore, ovarian muscle contraction externally generates a mechanical force to promote ovulation and maintain egg shape. This work reveals an important mechanism for JH-regulated insect reproduction.

Distinct gene expression dynamics in germ line and somatic tissue during ovariole morphogenesis in Drosophila melanogaster

The survival and evolution of a species is a function of the number of offspring it can produce. In insects the number of eggs that an ovary can produce is a major determinant of reproductive capacity. Insect ovaries are made up of tubular egg-producing subunits called ovarioles, whose number largely determines the number of eggs that can be potentially laid. Ovariole number is directly determined by the number of cellular structures called terminal filaments, which are stacks of cells that assemble in the larval ovary. Elucidating the developmental and regulatory mechanisms of terminal filament formation is thus key to understanding the regulation of insect reproduction through ovariole number regulation. We systematically measured mRNA expression of all cells in the larval ovary at the beginning, middle and end of terminal filament formation. We also separated somatic and germ line cells during these stages and assessed their tissue-specific gene expression during larval ovary development. We found that the number of differentially expressed somatic genes is highest during late stages of terminal filament formation and includes many signaling pathways that govern ovary development. We also show that germ line tissue, in contrast, shows greater differential expression during early stages of terminal filament formation, and highly expressed germ line genes at these stages largely control cell division and DNA repair. We provide a tissue-specific and temporal transcriptomic dataset of gene expression in the developing larval ovary as a resource to study insect reproduction.

Feeding and Oviposition Behaviour of Trioza erytreae (Hemiptera: Triozidae) on Different Citrus Rootstock Material Available in Europe

Trioza erytreae (Del Guercio, 1918) (Hemiptera: Triozidae) is a vector of Candidatus Liberibacter spp., the causal agent of Huanglongbing disease (HLB). This study evaluates the preference of T. erytreae in different citrus seedlings. Thus, six different non-grafted citrus rootstocks were used for these experiments: (a) Carrizo citrange; (b) Citrus macrophylla; (c) ‘Cleopatra’ mandarin; (d) Forner-Alcaide No. 5; (e) Forner-Alcaide No. 517, and (f) Poncirus trifoliata (‘Flying Dragon’). The behaviour and survival of this psyllid was evaluated through the feeding preference of T. erytreae adults for different rootstocks (in a choice trial under greenhouse conditions) and oviposition and survival of T. erytreae adults on the different citrus material (in a no-choice trial under laboratory conditions). Trioza erytreae showed a clear preference for hosting and feeding on C. macrophylla, and Carrizo citrange was the most suitable rootstock for insect reproduction and survival followed by C. macrophylla. Conversely, Poncirus trifoliata was the least attractive rootstock to T. erytreae adults in the greenhouse trial and led to significantly lower T. erytreae survival. Our results suggest that conventional citrus rootstocks, such as Carrizo citrange and C. macrophylla, could increase T. erytreae populations.

Distinct gene expression dynamics in germ line and somatic tissue during ovariole morphogenesis in Drosophila melanogaster

The survival and evolution of a species is a function of the number of offspring it can produce. In insects the number of eggs that an ovary can produce is a major determinant of reproductive capacity. Insect ovaries are made up of tubular egg-producing subunits called ovarioles, whose number largely determines the number of eggs that can be potentially laid. Ovariole number is directly determined by the number of cellular structures called terminal filaments, which are stacks of cells that assemble in the larval ovary. Elucidating the developmental and regulatory mechanisms of terminal filament formation is thus key to understanding the regulation of insect reproduction through ovariole number regulation. We systematically measured mRNA expression of all cells in the larval ovary at the beginning, middle and end of terminal filament formation. We also separated somatic and germ line cells during these stages and assessed their tissue-specific gene expression during larval ovary development. We found that the number of differentially expressed somatic genes is highest during late stages of terminal filament formation and includes many signaling pathways that govern ovary development. We also show that germ line tissue, in contrast, shows greater differential expression during early stages of terminal filament formation, and highly expressed germ line genes at these stages largely control cell division and DNA repair. We provide a tissue-specific and temporal transcriptomic dataset of gene expression in the developing larval ovary as a resource to study insect reproduction.

Molecular characterization and enzyme inhibition studies of NADP+- farnesol dehydrogenase from diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)

Juvenile hormone III (JH III) plays an important role in insect reproduction, development, and behavior. The second branch of JH III production includes oxidation of farnesol to farnesal by farnesol dehydrogenase. This study reported the identification and characterization of Plutella xylostella farnesol dehydrogenase (PxFoLDH). Our results showed that PxFoLDH belongs to the short-chain dehydrogenase/reductase superfamily, consisting of a single domain with a structurally conserved Rossman fold, an NAD(P) (H)—binding region and a structurally diverse C- terminal region. The purified enzyme displayed maximum activity at 55 °C with pH 9.5 and was stable in the temperature below 70 °C. PxFoLDH was determined to be a monomer with a relative molecular weight of 27 kDa and highly specific for trans, trans-farnesol and NADP+. Among analog inhibitors tested, farnesyl acetate was the most effective inhibitor with the lowest Ki value of 0.02 µM. Our findings showed this purified enzyme may represent as NADP+-farnesol dehydrogenase.

Neutral Ceramidase Is Required for the Reproduction of Brown Planthopper, Nilaparvata lugens (Stål)

Ceramides are bioactive sphingolipids that have been implicated in insect development; however, their role in insect reproduction remains poorly understood. Here, we report the pivotal role of neutral ceramidase (NCER) in the female reproduction of the brown planthopper (BPH), Nilaparvata lugens (Stål), a significant pest in rice cultivation in Asia. LC-MS/MS demonstrated that, among different developmental stages of BPH, the levels of ceramides were highest in 1st instar nymphs and lowest in adults. The transcription of NCER was negatively correlated with the levels of ceramides at different developmental stages of BPH, in that the transcript levels of NCER were the highest, whereas ceramides levels were the lowest in BPH adults. Knocking down NCER through RNA interference (RNAi) increased the levels of ceramides in BPH females and ovaries, which resulted in a delay in oocyte maturation, a reduction in oviposition and egg hatching rate, as well as the production of vulnerable offspring. Transmission electron microscopy (TEM) analysis and TdT-mediated dUTP Nick-End Labeling (TUNEL) assays showed mitochondrial deficiency and apoptosis in NCER-deficient oocytes. Taken together, these results suggest that NCER plays a crucial role in female reproduction in BPH, likely by regulating the levels of ceramides.

Proteolytic activity of the proteasome is required for female insect reproduction

Non-ATPase regulatory subunits (Rpns) are components of the 26S proteasome involved in polyubiquitinated substrate recognition and deubiquitination in eukaryotes. Here, we identified 15 homologues sequences of Rpn and associated genes by searching the genome and transcriptome databases of the brown planthopper, Nilaparvata lugens , a hemipteran rice pest. Temporospatial analysis showed that NlRpn genes were significantly highly expressed in eggs and ovaries but were less-highly expressed in males. RNA interference-mediated depletion of NlRpn genes decreased the proteolytic activity of proteasome and impeded the transcription of lipase and vitellogenin genes in the fat bodies and ovaries in adult females, and reduced the triglyceride content in the ovaries. Decrease of the proteolytic activity of the proteasome via knockdown of NlRpn s also inhibited the transcription of halloween genes, including NlCYP307A2 , NlCYP306A2 and NlCYP314A1 , in the 20-hydroxyecdysone (20E) biosynthetic pathway in the ovaries, reduced 20E production in adult females, and impaired ovarian development and oocyte maturation, resulting in reduced fecundity. These novel findings indicate that the proteolytic activity of the proteasome is required for female reproductive processes in N. lugens , thus furthering our understanding of the reproductive and developmental strategies in insects.