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RNA interference (RNAi) is a promising next generation technology for the development of species-specific pest management. The key to successful RNAi based-plant protection is dependent in part on data-driven target gene selection, a challenging task due to the absence of laboratory strains and the seasonality of most pest species. In this study, we aimed to identify novel target genes by performing a knowledge-based approach in order to expand the spectrum of known potent RNAi targets. Recently, the protein-coding genes ncm, Rop, RPII-140, and dre4 have been identified as sensitive RNAi targets for pest control. Based on these potent RNAi targets, we constructed an interaction network and analyzed a selection of 30 genes in the model beetle Tribolium castaneum via injection of dsRNA synthesized by in vitro transcription. Nineteen of these targets induced significant mortality of over 70%, including six that caused 100% lethality. Orthologs of active T. castaneum RNAi targets were verified in the economically important coleopteran pests Diabrotica virgifera virgifera and Brassicogethes aeneus. Knockdown of D. v. virgifera genes coding for transcription factor Spt5, Spt6, and RNA polymerase II subunit RPII-33 caused over 90% mortality in larval feeding assays. Injection of dsRNA constructs targeting RPII-215 or the pre-mRNA-processing factor Prp19 into adult B. aeneus resulted in high lethality rates of 93 and 87%, respectively. In summary, the demonstrated knowledge-based approaches increased the probability of identifying novel lethal RNAi target genes from 2% (whole genome) to 36% (transcription- and splicing-related genes). In addition, performing RNAi pre-screening in a model insect increased also the probability of the identification essential genes in the difficult-to-work-with pest species D. v. virgifera and B. aeneus.

BACKGROUND γ-Amino butyric acid (GABA) antagonists are proven targets for control of lepidopteran and other pests. New heterocyclic compounds with high insecticidal activity were discovered using a competitive-intelligence-inspired scaffold-hopping approach to generate analogs of fipronil, a known GABA antagonist. These novel aryl heterocyclic amines (AHAs) displayed broad-spectrum activity on a number of chewing insect pests. RESULTS Through >370 modifications of the core AHA structure, a 7-pyrazolopyridine lead molecule was found to exhibit much improved activity on a number of insect pests. In field trial studies, its performance was 2–4 times lower than commercial standards and also appeared to be species dependent, with good activity seen for larvae of Spodoptera exigua, but inactivity on larvae of Trichoplusia ni. CONCLUSION An extensive investigational biology effort demonstrated that these AHA analogs appear to have multiple modes of action, including GABA receptor antagonism and mitopotential or uncoupler activity. The limited capability in larvae of T. ni to convert the lead molecule to its associated open form correlates with the low toxicity of the lead molecule in this species. This work has provided information that could aid investigations of novel GABA antagonists. © 2016 Society of Chemical Industry

Transgenic maize plants expressing dsRNA targeting western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) v-ATPase subunit C mRNA for RNAi provided significant root protection from WCR larval feeding damage in greenhouse assays compared to negative controls. Transcribed hairpin dsRNA in WCR-resistant maize plants was present as both intact hairpin-derived dsRNA and plant-processed siRNA. Therefore, the ability of dsRNA and siRNA targeting Dv v-ATPase CmRNA to cause an RNAi response was studied in both WCR larvae and adults. In 9-day diet-based feeding assays, dsRNA of at least 60 bp in length resulted in high levels of larval mortality. In contrast, 15-, 25- or 27-bp dsRNAs or pooled 21-bp siRNAs did not cause mortality of exposed larvae. When larvae were fed with diet overlaid with siRNAs, Dv v-ATPase C transcript levels did not change. Conversely, when WCR larvae were fed with diet overlaid with 184-bp dsRNA, the mRNA level was reduced by >20-fold relative to yfp dsRNA negative control. Similarly, 184-bp dsRNA caused 100% mortality of WCR adults, whereas the mortality of adults fed on diet treated with siRNAs was similar to the negative control. Feeding adults with siRNAs on diet did not affect the level of Dv v-ATPase CmRNA transcripts, whereas adults fed with the 184-bp dsRNA showed approximately 35-fold reduction in the target mRNA level. Similar results were obtained with the WCR adults injected with 184-bp dsRNA or 21-bp siRNA. These results suggest that only long dsRNA or hairpin-derived dsRNA is effective in causing lethal knock-down of Dv v-ATPase CmRNA. These results have implications for efficacious plant-delivered dsRNA for the protection of transgenic maize from WCR feeding damage and for the risk assessment of transgenic maize expressing insecticidal dsRNA.

The sulfoximines, as exemplified by sulfoxaflor ([ N -[methyloxido[1-[6-(trifluoromethyl)-3-pyridinyl]ethyl]- λ 4 -sulfanylidene] cyanamide] represent a new class of insecticides. Sulfoxaflor exhibits a high degree of efficacy against a wide range of sap-feeding insects, including those resistant to neonicotinoids and other insecticides. Sulfoxaflor is an agonist at insect nicotinic acetylcholine receptors (nAChRs) and functions in a manner distinct from other insecticides acting at nAChRs. The sulfoximines also exhibit structure activity relationships (SAR) that are different from other nAChR agonists such as the neonicotinoids. This review summarizes the sulfoximine SAR, mode of action and the biochemistry underlying the observed efficacy on resistant insect pests, with a particular focus on sulfoxaflor.

γ-Amino butyric acid (GABA) antagonists are proven targets for control of lepidopteran and other pests. New heterocyclic compounds with high insecticidal activity were discovered using a competitive-intelligence-inspired scaffold-hopping approach to generate analogs of fipronil, a known GABA antagonist. These novel aryl heterocyclic amines (AHAs) displayed broad-spectrum activity on a number of chewing insect pests.Through370 modifications of the core AHA structure, a 7-pyrazolopyridine lead molecule was found to exhibit much improved activity on a number of insect pests. In field trial studies, its performance was 2-4 times lower than commercial standards and also appeared to be species dependent, with good activity seen for larvae of Spodoptera exigua, but inactivity on larvae of Trichoplusia ni.An extensive investigational biology effort demonstrated that these AHA analogs appear to have multiple modes of action, including GABA receptor antagonism and mitopotential or uncoupler activity. The limited capability in larvae of T. ni to convert the lead molecule to its associated open form correlates with the low toxicity of the lead molecule in this species. This work has provided information that could aid investigations of novel GABA antagonists. © 2016 Society of Chemical Industry.

Several strains of Drosophila melanogaster possess mutant alleles in nicotinic acetylcholine receptor (nAChR) subunits, Dα1 and Dβ2 that confer resistance to neonicotinoids such as imidacloprid and nitenpyram, and Dα6, that confers resistance to spinosyns. These mutant strains were bioassayed with a selected set of nAChR active insecticides including neonicotinoids, spinosad, and sulfoxaflor, a new sulfoximine insecticide. All of the neonicotinoids examined, except dinotefuran showed reduced insecticidal efficacy on larvae of the Dα1 mutant, suggesting that this subunit may be important in the action of these insecticides. All of the neonicotinoids, including dinotefuran, showed reduced insecticidal efficacy on larvae possessing the Dβ2 mutation. A similar pattern of broad neonicotinoid resistance to that of Dβ2 alone was also observed for larvae with both the mutations (Dα1 + Dβ2). The Dβ2 mutation exhibited a lower level of cross-resistance to sulfoxaflor ( 13-fold). In contrast, there was no cross-resistance for any of the neonicotinoids or sulfoxaflor in adult flies with the Dα6 mutation, which confers high levels of resistance to spinosad. Thus in the D. melanogaster strains studied, target site resistance observed for the neonicotinoids and the spinosyns does not translate directly to resistance towards sulfoxaflor.

Strains of Drosophila melanogaster with resistance to the insecticides spinosyn A, spinosad, and spinetoram were produced by chemical mutagenesis. These spinosyn-resistant strains were not cross-resistant to other insecticides. The two strains that were initially characterized were subsequently found to have mutations in the gene encoding the nicotinic acetylcholine receptor (nAChR) subunit Dalpha6. Subsequently, additional spinosyn-resistant alleles were generated by chemical mutagenesis and were also found to have mutations in the gene encoding Dalpha6, providing convincing evidence that Dalpha6 is a target site for the spinosyns in D. melanogaster. Although a spinosyn-sensitive receptor could not be generated in Xenopus laevis oocytes simply by expressing Dalpha6 alone, co-expression of Dalpha6 with an additional nAChR subunit, Dalpha5, and the chaperone protein ric-3 resulted in an acetylcholine- and spinosyn-sensitive receptor with the pharmacological properties anticipated for a native nAChR.

RNA interference has been widely used to reduce the quantity of the proteins encoded by the targeted genes. A constitutively active, dominant allele of trp, TrpP365, causes massive degeneration of photoreceptors through a persistent and excessive Ca2+ influx. Here we show that a substantial reduction of the TRP channel protein by RNAi in TrpP365 heterozygotes completely rescues the neuronal degeneration and significantly improves the light-elicited responses of the eye. The reduction need not be complete, suggesting that rescue of degeneration may be possible with minimal side effects arising from overdepletion of the target protein.

A large number of mutants in the norpA gene, which encodes the phospholipase C (PLC) involved in Drosophila phototransduction, is available for the investigation of the effects of specific amino acid substitutions in PLC on biochemical and electrophysiological properties of these mutants. Of the 47 norpA mutants screened for PLC protein content, all but one (H43) displayed drastically decreased amounts of the protein suggesting that almost any mutational alteration has a deleterious effect on the integrity of the protein. Three new amino acids were identified in the catalytic domains X and Y that are important for PLC catalytic activity and the generation of photoreceptor responses (ERG). One of them was found substituted in H43, which showed a low specific PLC activity, a pronounced decrease in ERG sensitivity, and a wild-type-like response termination time. The response termination times obtained from three mutants was found to be approximately inversely proportional to the amount of PLC. In addition, we show that (i) the specific PLC activity is a key factor determining the photoreceptor sensitivity; (ii) the catalytic activity and response termination are separable functions of PLC; and (iii) a mutation in the putative G alpha-interacting C2 domain causes a preferentially strong defect in latency.

The trp gene encodes subunits of a highly Ca(2+)-permeable class of light-activated channels of Drosophila photoreceptors. The recently characterized mutation in this gene, Trp(P365), is semidominant and causes massive degeneration of photoreceptors by making the TRP channel constitutively active. We show that a single amino acid change, Phe-550 to Ile, near the beginning of the fifth transmembrane domain of TRP channel subunits is necessary to induce, and sufficient to closely mimic, the original mutant phenotypes of Trp(P365). Hypotheses are presented as to why the amino acid residues at position 550 and its immediate vicinity might be important in influencing the regulation of the TRP channel and why the substitution of Phe for Ile at this position, in particular, could result in constitutive activity of the channel.

The trp and trpl genes are thought to encode two classes of light-activated ion channels in Drosophila . A previous report indicated that a null trpl mutant does not display any mutant phenotype. This lack of detectable mutant phenotypes made it difficult to suggest functions for the transient receptor potential-like (TRPL) channel in photoreceptor responses. Here, the properties of trpl photoreceptor responses were studied by using electroretinogram (ERG) and intracellular recording techniques in combination with light stimuli of relatively long durations. Distinct mutant phenotypes were detectable under these conditions. These consisted of a reduced sustained component, oscillations superimposed on the response, a poststimulus hyperpolarization, and altered adaptation properties to dim background light. Comparison of photoreceptor responses obtained from wild type, trp , and trpl showed that the responses obtained from the trp and trpl null mutants did not sum up to that of the wild-type response. To explain the nonlinear summation at the peak of the response, [Reuss et al. (1997)][1] proposed that Ca2+ions entering through the TRP channel modulate TRP and TRPL channel activities differentially. However, nonlinear summation was present not only at the peak but throughout the duration of response. Two lines of evidence are presented to suggest that, in addition to the interaction proposed by [Reuss et al. (1997)][1], there are other forms of interactions between TRP and TRPL channels, probably involving the channel proteins themselves. [1]: #ref-18

TheDrosophila trpgene encodes a light-activated Ca2+channel subunit, which is a prototypical member of a novel class of channel proteins. Previously identifiedtrpmutants are all recessive, loss-of-function mutants characterized by a transient receptor potential and the total or near-total loss of functional TRP protein. Although retinal degeneration does occur in these mutants, it is relatively mild and slow in onset. We report herein a new mutant,TrpP365, that does not display the transient receptor potential phenotype and is characterized by a substantial level of the TRP protein and rapid, semi-dominant degeneration of photoreceptors. We show that, in spite of its unusual phenotypes,TrpP365is atrpallele because aTrpP365transgene induces the mutant phenotype in a wild-type background, and a wild-typetrptransgene in aTrpP365background suppresses the mutant phenotype. Moreover, amino acid alterations that could cause theTrpP365phenotype are found in the transmembrane segment region of the mutant channel protein. Whole-cell recordings clarified the mechanism underlying the retinal degeneration by showing that the TRP channels ofTrpP365are constitutively active. Although several genes, when mutated, have been shown to cause retinal degeneration inDrosophila, the underlying mechanism has not been identified for any of them. The present studies provide evidence for a specific mechanism for massive degeneration of photoreceptors inDrosophila. Insofar as some human homologs of TRP are highly expressed in the brain, a similar mechanism could be a major contributor to degenerative disorders of the brain.

During sporulation, Bacillus thuringiensis produces crystalline inclusions comprised of a mixture of δ-endotoxins. Following ingestion by insect larvae, these inclusion proteins are solubilized, and the protoxins are converted to toxins. These bind specifically to receptors on the surfaces of midgut apical cells and are then incorporated into the membrane to form ion channels. The steps required for toxin insertion into the membrane and possible oligomerization to form a channel have been examined. When bound to vesicles from the midguts of Manduca sexta larvae, the Cry1Ac toxin was largely resistant to digestion with protease K. Only about 60 amino acids were removed from the Cry1Ac amino terminus, which included primarily helix α1. Following incubation of the Cry1Ab or Cry1Ac toxins with vesicles, the preparations were solubilized by relatively mild conditions, and the toxin antigens were analyzed by immunoblotting. In both cases, most of the toxin formed a large, antigenic aggregate of ca. 200 kDa. These toxin aggregates did not include the toxin receptor aminopeptidase N, but interactions with other vesicle components were not excluded. No oligomerization occurred when inactive toxins with mutations in amphipathic helices (α5) and known to insert into the membrane were tested. Active toxins with other mutations in this helix did form oligomers. There was one exception; a very active helix α5 mutant toxin bound very well to membranes, but no oligomers were detected. Toxins with mutations in the loop connecting helices α2 and α3, which affected the irreversible binding to vesicles, also did not oligomerize. There was a greater extent of oligomerization of the Cry1Ac toxin with vesicles from the Heliothis virescens midgut than with those from the M. sexta midgut, which correlated with observed differences in toxicity. Tight binding of virtually the entire toxin molecule to the membrane and the subsequent oligomerization are both important steps in toxicity.

The spinosyns are insecticidal natural products originated from Saccharopolyspora spinosa. Spinosad, the first commercial product, is a mixture of two naturally occurring spinosyns (A & D). A second product, spinetoram, is a mixture composed of two synthetically modified spinosyns that, compared to spinosad, provides improved insecticidal potency and a broader pest insect spectrum. The spinosyns act on a subgroup of insect nicotinic acetylcholine receptors (nAChR), which are distinct from those targeted by the neonicotinoid insecticides. Selection of Drosophila for resistance to spinosad helped pinpoint the Dα6 as the nAChR subunit involved in the insecticidal action of the spinosyns. Cases of both target-site- and metabolism-based spinosyn resistance in insect pests in the field have been reported, and programmes have been developed to manage spinosyn resistance. This review highlights the discovery of the spinosyns, elucidation of the spinosyn target site, spinosyn–receptor interaction, and progress in spinosyn resistance management.

The corpora allata (CA) of insects are innervated by axons of non-neurosecretory cerebral neurons, and of the various known neurotransmitters in the brain of the tobacco hornworm, Manduca sexta, only dopamine is detected in the CA by electrochemical detection HPLC. This neurotransmitter stimulates the biosynthetic activity of the CA in vitro for the first 2 days of the last larval stadium, but inhibits CA from day 3 through day 6, the beginning of the prepupal period. Stimulation of JH synthesis has previously been linked with an increase in the production of cyclic AMP (cAMP) in the CA, and dopamine stimulates the adenylyl cyclase system of CA from larvae early in the fifth stadium, while on day 6, its effect is inhibitory. These results suggest: (1) the existence in the CA of both D1- and D2-like dopamine receptors, which in vertebrates stimulate and inhibit, respectively, adenylyl cyclase; and (2) the developmental control of their expression. A potent D1 agonist, (+/-)-SKF 82958-HBr, did not stimulate JH biosynthesis by day 0 CA as expected, but appeared to inhibit it at a concentration of 10(-5)M. Thus the apparent D1-like receptor in Manduca CA may be pharmacologically distinct from vertebrate D1 receptors. The existence of D2-like receptors is supported by the finding that a vertebrate D2 receptor agonist, (+/-) PPHT-HCl, and an antagonist, eticlopride, have the predicted effects on JH acid biosynthesis and cAMP production by day 6 Manduca CA. However, the D1 agonist also significantly reduces JH acid biosynthesis and cAMP production, indicating that while the Manduca D2-like receptor is pharmacologically similar to the vertebrate D2, it shares some characteristics with D1 receptors. The developmental regulation of these receptors by ecdysteroids is suggested by the fact that when day 0 larvae are treated in vivo with exogenous ecdysone:20-hydroxyecdysone, the biosynthetic activity of the CA in vitro 24 h later is no longer stimulated by dopamine.

Whole body extracts of the two-spotted spider mite (Tetranychus urticae Koch) were analyzed using a 16-channel electrochemical array high performance liquid chromatography (HPLC)-based detection system that allows the simultaneous isolation and identification of a variety of biogenic amines. The spider mite extracts were found to contain the biogenic amines octopamine, dopamine, and 5-hydroxytryptamine (5-HT), as well as several precursors and metabolites including tyrosine, tyramine, tryptophan, and N-acetyl octopamine. Differences in the levels of biogenic amines were observed between eggs and the adult stages and between males and females. This is the first direct determination of biogenic amines in the Tetranychidae and the first demonstration of 5-HT in any mite species. © 1994 Wiley-Liss, Inc.

1. The extracts of brains (cerebral ganglion) of the tobacco hornworm larvae contain octopamine, dopamine and 5-hydroxytryptamine, but not norepinephrine via analysis using a 16-channel electrochemical array HPLC system. 2. A comprehensive daily analysis reveals the patterns of biogenic amine levels in the brain during larval—pupal metamorphosis. 3. Monoamine oxidase-based amine metabolites are not present at detectable levels. N -β-Alanyldopamine is the predominant metabolite of dopamine present in the brain during the postwandering phase of the last larval stadium.

A method was developed to analyze biogenic amines in extracts of the central nervous system of the tobacco hornworm, Manduca sexta by high-performance liquid chromatography with 16-sensor electrochemical detection (n-EC-HPLC). The amines, precursors, and metabolites were separated in two dimensions. The first dimension involves separation based upon retention time by reversed-phase HPLC, while the second dimension involves separation based upon the characteristic oxidation potentials achieved by n-EC. Biogenic amine identification was based upon maximum oxidation potential and peak height ratios in addition to retention time. The improved resolving power of this method allows for a simplified sample preparation procedure and simultaneous determination of a wide range of compounds, including phenylethylamine, catecholamines, indoleamines, and some of their precursors and metabolites.

We discuss in this chapter the role of Ca2+ homeostasis in maintaining the structural integrity of photoreceptor cells in Drosophila. Both insufficient and excessive amounts of Ca2+ in photoreceptor cells appear to lead to cell degeneration. Because one of the two classes of light-sensitive channels in Drosophila photoreceptors is highly Ca2+-permeable, how well this class of channels functions can profoundly affect Ca2+ homeostasis. We will begin by reviewing Drosophila phototransduction, emphasizing what is known about the mechanism of activation of light-sensitive channels. We will then describe Ca2+ entry through light-sensitive channels and the presumed mechanisms by which too little and too much Ca2+ entry can both cause photoreceptor degeneration. We will conclude the chapter with discussions of two examples of mutations known to cause unregulated Ca2+ entry through light-sensitive channels, leading to massive photoreceptor degeneration.

We discuss in this chapter the role of Ca2+homeostasis in maintaining the structural integrity of photoreceptor cells inDrosophila.Both insufficient and excessive amounts of Ca2+in photoreceptor cells appear to lead to cell degeneration. Because one of the two classes of light-sensitive channels inDrosophilaphotoreceptors is highly Ca2+-permeable, how well this class of channels functions can profoundly affect Ca2+homeostasis. We will begin by reviewingDrosophilaphototransduction, emphasizing what is known about the mechanism of activation of light-sensitive channels. We will then describe Ca2+entry through light-sensitive channels and the presumed mechanisms by which too Iittle and too much Ca2+entry can both cause photoreceptor degeneration. We will conclude the chapter with discussions of two examples of mutations known to cause unregulated Ca2+entry through light-sensitive channels, leading to massive photoreceptor degeneration.

The trp gene of Drosophila encodes a subunit of a class of Ca 2+ -selective light-activated channels that carry the bulk of the phototransduction current. Transient receptor potential (TRP) homologs have been identified throughout animal phylogeny. In vertebrates, TRP-related channels have been suggested to mediate “store-operated Ca 2+ entry,” which is important in Ca 2+ homeostasis in a wide variety of cell types. However, the mechanisms of activation and regulation of the TRP channel are not known. Here, we report on the Drosophila inaF gene, which encodes a highly eye-enriched protein, INAF, that appears to be required for TRP channel function. A null mutation in this gene significantly reduces the amount of the TRP protein and, in addition, specifically affects the TRP channel function so as to nearly shut down its activity. The inaF mutation also dramatically suppresses the severe degeneration caused by a constitutively active mutation in the trp gene. Although the reduction in the amount of the TRP protein may contribute to these phenotypes, several lines of evidence support the view that inaF mutations also more directly affect the TRP channel function, suggesting that the INAF protein may have a regulatory role in the channel function.

The Drosophila receptor oscillation A (rosA) mutations, which cause electroretinogram (ERG) defects, including oscillations, were localized to the 24F4-25A2 region of chromosome 2L. Genomic fragments from this region, isolated from bacteriophage P1 clones, included those that detect transcriptional defects in rosA mutants in RNA blot experiments. One of these genomic fragments was used to screen a head cDNA library. The largest cDNA clone (3.6 kb) isolated was shown to rescue a rosA mutant in P element-germline transformation experiments. The ROSA protein deduced from the open reading frame in the 3.6 kb rosA cDNA is 943 amino acids long and is 36-41% identical to members of the superfamily of Na+/Cl(-)-dependent neurotransmitter transporters, with no indication of higher sequence identity to any one subgroup within the superfamily. RNA blot experiments revealed multiple transcripts in various developmental stages, the most abundant one being a 3.7 kb transcript, particularly in the adult head. Tissue in situ experiments identified the rosA transcript to be localized to many tissues, with higher levels of hybridization in the nervous system and digestive tract. The results demonstrate that the rosA gene encodes a novel Na+/Cl(-)-dependent transporter important for normal response properties of the photoreceptor.

Summary Little is known of the participation of insect hemolymph proteins in wound healing and clot formation. We describe the assembly of purified hemolymph protein from the tobacco hornworm into an extended fibrous coagulum in the absence of hemocytes. This coagulum resembles the clot formed from bovine fibrinogen and thrombin. Structural components of the coagulum are present in hemolymph, however, spontaneous assembly occurs only in hemolymph collected through a wound. The fibrous coagulum assembles from purified structural protein(s) following addition of a non-protein factor from hemolymph, which is also present in Grace's insect cell culture medium.

Both hemocytes and fat body from larvae of Manduca sexta, which have been injected with inducers of antibacterial protein synthesis, contain immunoreactive lysozyme. However, fat body is a richer source and has been demonstrated to synthesize and release lysozyme and cecropin-like peptides (bactericidins) in vitro. Fat body secretion of lysozyme and bactericidins is stimulated by addition of soluble peptidoglycan fragments to culture medium. The rate of lysozyme secretion by fat body varies as a function of peptidoglycan inducer concentration. These data are consistent with the hypothesis that, in vivo, bacteria must be phagocytized and partially degraded (processed) by hemocytes to generate a signal (peptidoglycan) that subsequently induces antibacterial protein synthesis by fat body.

Injection of bacteria and bacterial cell walls into larvae of the tobacco hornworm elicits a rapid, specific depletion of plasmatocytes from circulation. Plasmatocyte depletion in response to injection of bacteria is dose dependent with a threshhold for response between 10 3 –10 4 bacteria per insect and a maximal depletion at greater than 10 7 bacteria per insect. Injection of saline, latex beads that are phagocytized, or fragments of peptidoglycan that elicit antibacterial protein synthesis do not affect plasmatocyte abundance.