Genetic transformation of mosquitoes by microparticle bombardment.

Mosquitoes constitute the major living beings causing human deaths in the world. They are vectors of malaria, yellow fever, dengue, zika, filariases, chikungunya, among other diseases. New strategies to control/eradicate mosquito populations are based on newly developed genetic manipulation techniques. However, genetic transformation of mosquitoes is a major technical bottleneck due to low efficiency, the need of sophisticated equipment, and highly trained personnel. The present report shows the transgenerational genetic transformation of Aedes aegypti, using the particle inflow gun (PIG), by integrating the ecfp gene in the AAEL000582 mosquito gene with the CRISPR-Cas9 technique, achieving a mean efficiency of 44.5% of bombarded individuals (G0) that showed ECFP expression in their tissues, and a mean of 28.5% transformation efficiency measured on G1 individuals. The same transformation technique was used to integrate the egfp/scorpine genes cloned in the Minos transposon pMinHygeGFP into the Anopheles albimanus genome, achieving a mean efficiency of 43.25% of bombarded individuals (G0) that showed EGFP expression in their tissues. Once the technique was standardized, transformation of Ae. aegypti neonate larvae and An. albimanus eggs was achieved when exposed to gold microparticle bombardment. Integration of genes and heterologous protein expression were confirmed by PCR, sequencing, fluorescent microscopy, mass spectrometry, Western blot and dot blot analyses. Transgenerational inheritance of the transgenes was observed only on Ae. aegypti, as all transformed An. albimanus individuals died at the pupal stage of the G0 generation.

Current immunity markers in insect ecological immunology: assumed trade-offs and methodological issues.

The field of ecological immunology currently relies on using a number of immune effectors or markers. These markers are usually used to infer ecological trade-offs (via conflicts in resource allocation), though physiological nature of these markers remains elusive. Here, we review markers frequently used in insect evolutionary ecology research: cuticle darkening, haemocyte density, nodule/capsule formation, phagocytosis and encapsulation/melanization via use of nylon filaments and beads, phenoloxidase activity, nitric oxide production, lysozyme and antimicrobial peptide production. We also provide physiologically based information that may shed light on the probable trade-offs inferred when these markers are used. In addition, we provide a number of methodological suggestions to improve immune marker assessment.

Prostaglandin E2 modulates the expression of antimicrobial peptides in the fat body and midgut of Anopheles albimanus.

Prostaglandins (PGs) participate in the regulation of vertebrate and in at least six insect orders' immune responses. We identified PGE2 in midgut, fat body, Malpighian tubules, and ovarioles of Anopheles albimanus (Aa) mosquitoes. Our data indicate that PGE2 synthesis in cultured midguts responds to the presence of two bacterial species, Micrococcus luteus and Klebsiella pneumoniae. The production of mRNA coding for antimicrobial peptides Aa-Attacin, Aa-Cecropin, and Aa-Gambicin was observed in cultured fat bodies and midguts. The production of these messengers was reduced in the presence of dexamethasone, and this effect was reversed by arachidonic acid. Adding PGE2 to cultures resulted in increased Aa-cecropin mRNA and decreased Aa-attacin and Aa-gambicin mRNAs.

The expression of a sexually selected trait correlates with different immune defense components and survival in males of the American rubyspot.

Recent studies have suggested that courtship trait expression indicates immune strength. However, most studies have measured only one immune parameter, have not assessed individual differences in immune ability according to time and have not controlled for ecological differences among individuals after an immune challenge. In this work, we tested this hypothesis and controlled for these factors using males of the American rubyspot damselfly which bear a wing red spot whose size is evolutionarily maintained via male-male territorial competition. Our general hypothesis was that territorial, large-spotted males, had a better immune ability compared to nonterritorial, small-spotted males. We expected that the following variables were greater in territorial males compared to nonterritorial males: spot size, phenoloxidase (PO) and hydrolytic enzymatic (HE) activity in males challenged and nonchallenged with a nylon implant, PO and HE activity rate; PO activity after a Serratia marcescens challenge, and survival after a nylon challenge controlling for activity and feeding differences. We found that territorial males showed larger spot areas, greater PO and HE activity (independently of whether they were challenged or not), a higher rate of PO and HE activity (but only expressed at 8h), greater PO production after the bacterial challenge, and a higher survival after the challenge. These results corroborate that males with more pronounced sexual traits have a superior immune function.

Entamoeba histolytica: erythrophagocytosis, collagenolysis, and liver abscess production as virulence markers.

High rates of erythrophagocytosis and collagenolysis in vitro have been regarded as indicative of virulence in vivo of Entamoeba histolytica trophozoites. In the present study, the erythrophagocytic index and the collagenolytic activity of 3 axenic lines of E. histolytica, strain HM1:IMSS, were measured. The 3 lines shared the same pathogenic zymodeme but showed clear-cut differences in the extent of liver damage induced in hamsters. A direct correlation between collagenolysis in vitro and the size of liver abscesses produced by each line of E. histolytica trophozoites was found. In contrast, the line with the highest erythrophagocytic index produced small amoebic abscesses in hamsters, whereas the line with a relatively low erythrophagocytic index produced the largest liver lesions. It is concluded that the extent of collagenolytic activity is a better marker of virulence of E. histolytica cultured under axenic conditions than is erythrophagocytosis.

Cost of immune priming within generations: trade-off between infection and reproduction.

Immune priming is a new paradigm in innate immunity. However, most studies have focused on the benefits of priming (enhanced survival and parasite clearance after a second challenge), while little attention has been paid to the costs. In this study, both factors were investigated in Anopheles albimanus primed against Plasmodium berghei. As previously observed in other invertebrates, compared to un-primed mosquitoes, those primed better controlled a challenge from the same parasite, and had a higher survival rate. Although there was no difference in the number of oviposited eggs between primed and control females, hatching rate was lower in primed than in control mosquitoes and it was more likely for control females to produce eggs than for primed females. Furthermore, a trade-off between parasite elimination and egg production was observed among primed mosquitoes, as primed females that successfully fought the infection were unable to produce eggs, but primed females that produced eggs were similarly infected as control un-primed ones. These results concord with recent mathematical models suggesting that reproduction affects immune priming outcomes, and may explain why in some species and under some conditions it seems that immune priming is not occurring.

Recombinant scorpine: a multifunctional antimicrobial peptide with activity against different pathogens.

Scorpine is an antimicrobial peptide whose structure resembles a hybrid between a defensin and a cecropin. It exhibits antibacterial activity and inhibits the sporogonic development of parasites responsible for murine malaria. In this communication we report the production of scorpine in a heterelogous system, using a specific vector containing its cloned gene. The recombinantly expressed scorpine (RScp) in (Anopheles gambie) cells showed antibacterial activity against (Bacillus subtilis) and (Klebsiella pneumoniae), at 5 and 10 microM, respectively. It also produced 98% mortality in sexual stages of (Plasmodium berghei) at 15 microM and 100% reduction in (Plasmodium falciparum) parasitemia at 5 microM. RScp also inhibited virus dengue-2 replication in C6/36 mosquito cells. In addition, we generated viable and fertile transgenic (Drosophila) that overexpresses and correctly secretes RScp into the insect hemolymph, suggesting that the generation of transgenic mosquitoes resistant to different pathogens may be viable.

Free radical generation during the activation of hemolymph prepared from the homopteran Dactylopius coccus.

Superoxide anion (O(-) (2)) and nitric oxide (NO) generation in Dactylopius coccus hemolymph obtained by perfusion and activated with zymosan was studied. Activated hemolymph reduced 3-[4,5 dimethylthiazolil-2]-2,5-diphenyl tetrazolium bromide. This reduction was prevented by superoxide dismutase (SOD) indicating O(-) (2) generation. This activity was dependent on temperature, and hemolymph incubated at 75 degrees C lost its activity. Chromatocytes incubated with zymosan released their content and produced O(-) (2). Activated hemolymph also produced NO and this activity was prevented in the presence of NG-nitro-L-arginine methyl ester, suggesting that nitric oxide synthase (NOS) might be present in D. coccus hemolymph. The probable source of O(-) (2) in the D. coccus hemolymph is the anthraquinone oxidation, since commercial carminic dye produced O(-) (2) during its oxidation by Agaricus bisporus tyrosinase. Gram+ Micrococcus luteus exposed to activated hemolymph were killed in vitro, and addition of NG-nitro-L-arginine methyl ester and D-Mannitol (a hydroxyl radical scavenger) prevented their killing. The cytotoxic effect produced by the activated hemolymph was not observed with the Gram- bacteria Serratia marcescens. These results suggest that D. coccus activated hemolymph generates reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) that may limit M. luteus growth.

Induction of DNA synthesis in Anopheles albimanus tissue cultures in response to a Saccharomyces cerevisiae challenge.

DNA synthesis was detected by the incorporation of 5-bromo-2' deoxy-uridine (BrdU) in adult Anopheles albimanus organs in culture in response to a challenge with Saccharomyces cerevisiae. Abdomens of mosquitoes inoculated with Roswell Park Memorial Institute medium (RPMI, control) or yeast were cultivated in RPMI plus ConA and BrdU for 5 days. DNA was obtained by phenolic extraction and the incorporated BrdU was quantified by ELISA using anti-BrdU peroxidase-labeled antibodies. Abdomen tissues of mosquitoes inoculated with yeast showed higher DNA synthesis than controls. Organs from untreated mosquitoes cultured in the presence of zymosan also synthesized DNA but at a lower level than tissues from yeast-inoculated mosquitoes. In similar experiments, DNA synthesis was inhibited by the addition of colchicine. DNA synthesis, evidenced by epifluorescence using an anti-BrdU fluorescein-labeled antibody, occurred in fat body, epithelial cells in pleural membranes, and the dorsal vessel. Pleural membranes showed the highest number of labeled cells. These tissues were also labeled with anti-PCNA (proliferating cell nuclear antigen) antibodies, two of which were able to produce polytene chromosomes under yeast stimulation. These results demonstrate that different An. albimanus tissues undergo DNA synthesis in response to foreign particles.

Regulation of the insect immune response: the effect of hemolin on cellular immune mechanisms.

Hemolin is a bacteria-inducible protein of the immunoglobulin superfamily identified in the silk moth Hyalophora cecropia. The role of this protein, in hemocyte aggregation and phagocytosis, was studied in vitro. Hemocyte aggregation, stimulated by phorbol myristate acetate or lipopolysaccharide (LPS), was prevented by hemolin in a dose-dependent fashion, but hemolin did not disrupt aggregates once they had been formed. Furthermore, hemolin was able to stimulate phagocytic activity in both hemocytes and hemocytic mbn-2 cells and this activity was enhanced by LPS. The enhanced phagocytosis produced by a combination of hemolin and LPS was prevented by the protein kinase C (PKC) inhibitors staurosporine and H-7, and PKC activity in hemocyte crude extracts was enhanced by hemolin and LPS, with the highest activity observed in the presence of both. Hemolin affected tyrosine phosphorylation of hemocyte proteins, enhancing the phosphorylation of two proteins of 20 and 30 kDa and preventing tyrosine phosphorylation of two proteins of 35 and 40 kDa. These results suggest that hemolin is involved in the regulation of the cellular immune responses via a pathway that includes PKC activation and protein tyrosine phosphorylation.

Cell adhesion properties of hemolin, an insect immune protein in the Ig superfamily.

The isolation of antibacterial peptides from the giant silkmoth Hyalophora cecropia has opened the area of animal antibiotics [Boman, H. G. (1991) Cell 65, 205-207] and the study of insect immune genes has revealed striking similarities to many immune response genes in mammals [Hultmark, D. (1994) Nature 267, 116-117]. However, the molecules and mechanisms behind primordial immune recognition are not understood. One candidate for one such recognition molecule is hemolin, a 48-kDa immunoglobulin-related protein first isolated from H. cecropia, where it is up-regulated upon infection and secreted into the hemolymph. Hemolin was shown to bind to bacteria and to hemocytes, giving rise to changes in hemocyte adhesiveness and intracellular phosphorylation patterns [Faye, I. & Kanost, M. (1997) in Molecular mechanisms of immune responses in insects (Brey, P. T. & Hultmark, D., eds) Chapman and Hall, London]. In the present publication, we give evidence for the presence of a 52-kDa membrane form of hemolin on hemocytes, based on flow-activated cell sorting and membrane protein extractions. In addition we reveal calcium-dependent homophilic binding properties of hemolin, using hemolin-coated microspheres. When biotinylated recombinant hemolin was allowed to bind to hemocyte membranes, higher molecular-mass complexes were formed. Furthermore, we used immunological methods and Northern-blot analysis to demonstrate the presence of hemolin in embryos and retinal discs, suggesting that hemolin is expressed in several tissues at different developmental stages. These results show novel cell adhesion features of hemolin, corroborating its multifunctional character with putative roles in cellular and humoral immunity and in development.

Ectonucleotide diphosphohydrolase activities in hemocytes of larval Manduca sexta.

In this work, we describe the ability of living hemocytes from an insect (Manduca sexta, Lepidoptera) to hydrolyze extracellular ATP. In these intact cells, there was a low level of ATP hydrolysis in the absence of any divalent metal (8.24 +/- 0.94 nmol of Pi/h x 10(6) cells). The ATP hydrolysis was stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 15.93 +/- 1.74 nmol of Pi/h x 10(6) cells. Both activities were linear with cell density and with time for at least 90 min. The addition of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.33 mM MgCl2. This stimulatory activity was not observed when Ca2+ replaced Mg2+. The apparent Km values for ATP-4 and Mg-ATP2- were 0.059 and 0.097 mM, respectively. The Mg2+-independent ATPase activity was unaffected by pH in the range between 6.6 and 7.4, in which the cells were viable. However, the Mg2+-dependent ATPase activity was enhanced by an increase of pH. These ecto-ATPase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, sodium fluoride, tartrate, and levamizole. To confirm the observed hydrolytic activities as those of an ecto-ATPase, we used an impermeant inhibitor, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), as well as suramin, an antagonist of P2-purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different extents. Interestingly, lipopolysaccharide, a component of cell walls of gram-negative bacteria that increase hemocyte aggregation and phagocytosis, increased the Mg2+-dependent ecto-ATPase activity in a dose-dependent manner but did not modify the Mg2+-independent ecto-ATPase activity.