Distinct Roles of Hemocytes at Different Stages of Infection by Dengue and Zika Viruses in Aedes aegypti Mosquitoes.

Aedes aegypti mosquitoes are vectors for arboviruses of medical importance such as dengue (DENV) and Zika (ZIKV) viruses. Different innate immune pathways contribute to the control of arboviruses in the mosquito vector including RNA interference, Toll and Jak-STAT pathways. However, the role of cellular responses mediated by circulating macrophage-like cells known as hemocytes remains unclear. Here we show that hemocytes are recruited to the midgut of Ae. aegypti mosquitoes in response to DENV or ZIKV. Blockade of the phagocytic function of hemocytes using latex beads induced increased accumulation of hemocytes in the midgut and a reduction in virus infection levels in this organ. In contrast, inhibition of phagocytosis by hemocytes led to increased systemic dissemination and replication of DENV and ZIKV. Hence, our work reveals a dual role for hemocytes in Ae. aegypti mosquitoes, whereby phagocytosis is not required to control viral infection in the midgut but is essential to restrict systemic dissemination. Further understanding of the mechanism behind this duality could help the design of vector-based strategies to prevent transmission of arboviruses.

Specific Molecular Signatures for Type II Crustins in Penaeid Shrimp Uncovered by the Identification of Crustin-Like Antimicrobial Peptides in Litopenaeus vannamei.

Crustins form a large family of antimicrobial peptides (AMPs) in crustaceans composed of four sub-groups (Types I-IV). Type II crustins (Type IIa or "Crustins" and Type IIb or "Crustin-like") possess a typical hydrophobic N-terminal region and are by far the most representative sub-group found in penaeid shrimp. To gain insight into the molecular diversity of Type II crustins in penaeids, we identified and characterized a Type IIb crustin in Litopenaeus vannamei (Crustin-like Lv) and compared Type II crustins at both molecular and transcriptional levels. Although L. vannamei Type II crustins (Crustin Lv and Crustin-like Lv) are encoded by separate genes, they showed a similar tissue distribution (hemocytes and gills) and transcriptional response to the shrimp pathogens Vibrio harveyi and White spot syndrome virus (WSSV). As Crustin Lv, Crustin-like Lv transcripts were found to be present early in development, suggesting a maternal contribution to shrimp progeny. Altogether, our in silico and transcriptional data allowed to conclude that (1) each sub-type displays a specific amino acid signature at the C-terminal end holding both the cysteine-rich region and the whey acidic protein (WAP) domain, and that (2) shrimp Type II crustins evolved from a common ancestral gene that conserved a similar pattern of transcriptional regulation.

Antiviral Activity of Ctn[15-34], A Cathelicidin-Derived Eicosapeptide, Against Infectious Myonecrosis Virus in Litopenaeus vannamei Primary Hemocyte Cultures.

The shrimp farming has been converted into a mature aquaculture industry dealing with over millions of metric tonnes of processed commodities. Nevertheless, the global shrimp productions are constantly threatened by disease outbreaks, mainly triggered by rapidly disseminating viruses. Infectious myonecrosis virus (IMNV) is one of these epizootic agents affecting shrimp production in Brazil, of which no treatment exists. Herein, the antiviral activity against IMNV of an eicosapeptide, named Ctn[15-34], derived from a member of the cathelicidin family of antimicrobial peptides, was demonstrated. Cultures of hemocytes from Litopenaeus vannamei were established that support IMNV replication and infectivity titration. The cytotoxic effect of IMNV in culture and the in vitro anti-IMNV activity of Ctn[15-34] were assessed using a high-sensitive fluorescent-based method in combination with quantitative PCR. The Ctn[15-34] (<12.5 µM) neutralized the toxic effects of IMNV at loads sufficient to kill 50% of shrimp hemocytes. This study reported for the first time the replication of IMNV in vitro and the employment of a straightforward methodology to assess cell viability and viral/antiviral activities. In addition, it provided the basis for the development of the anti-infective multi-effector Ctn[15-34] eicosapeptide and analogs as components of antiviral formulations against shrimp viral diseases.

Two's a crowd: phenotypic adjustments and prophylaxis in Anticarsia gemmatalis larvae are triggered by the presence of conspecifics.

Defence from parasites and pathogens involves a cost. Thus, it is expected that organisms use this only at high population densities, where the risk of pathogen transmission may be high, as proposed by the "density-dependent prophylaxis" (DDP) hypothesis. These predictions have been tested in a wide range of insects, both in comparative and experimental studies. We think it pertinent to consider a continuum between solitarious and gregarious living insects, wherein: (1) solitarious insects are those that are constitutively solitary and do not express any phenotypic plasticity, (2) the middle of the continuum is represented by insects that are subject to fluctuations in local density and show a range of facultative and plastic changes; and (3) constitutively gregarious forms live gregariously and show the gregarious phenotype even in the absence of crowding stimuli. We aimed to chart some of the intermediary continuum with an insect that presents solitarious aspects, but that is subject to fluctuations in density. Thus, Anticarsia gemmatalis (Lepidoptera: Noctuidae) larvae reared at higher densities showed changes in coloration, a greater degree of encapsulation, had higher hemocyte densities and were more resistant to Baculovirus anticarsia, but not to Bacillus thuringiensis. Meanwhile, with increased rearing density there was reduced capsule melanization. Hemocyte density was the only variable that did not vary according to larval phenotype. The observed responses were not a continuous function of larval density, but an all-or-nothing response to the presence of a conspecific. As A. gemmatalis is not known for gregarious living, yet shows these density-dependent changes, it thus seems that this plastic phenotypic adjustment may be a broader phenomenon than previously thought.

Cytosolic manganese superoxide dismutase genes from the white shrimp Litopenaeus vannamei are differentially expressed in response to lipopolysaccharides, white spot virus and during ontogeny.

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme usually located in mitochondria. There are only a few examples of cytosolic MnSOD (cMnSOD). In the shrimp Litopenaeus vannamei, we have previously characterized three cMnSOD cDNAs and their differential tissue-specific expression. To obtain insights about their genomic organization, we characterized the three corresponding cMnSOD genes, named them cMnsod1, cMnsod2, and cMnsod3 and studied their specific expression during ontogeny, response to lipopolysaccharides (LPS) and white spot virus infection (WSSV) in hemocytes from shrimp. The first two genes contain five introns flanked by canonical 5'-GT-AG-3' intron splice-site junctions, while the third one is intron-less. We analyzed 995 nucleotides upstream cMnsod2, but no classical promoter sequences were found. The deduced products of the three cMnSOD genes differ in two amino acids and there are four silent changes. cMnsod3 expression is modulated by WSSV and cMnsod2 by LPS. cMnsod2 is expressed from eggs to post larval stage during ontogeny. This is the first report of crustacean cMnSOD multigenes that are differently induced during the defense response and ontogeny.

An Anticarsia gemmatalis multiple nucleopolyhedrovirus mutant, vApAg, induces hemocytes apoptosis in vivo and displays reduced infectivity in larvae of Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae).

An Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) mutant, vApAg, induces apoptosis in a cell culture derived from Anticarsia gemmatalis (UFL-AG-286), reducing viral progeny. We have investigated apoptosis induction in vivo by vApAg in A. gemmatalis larvae and its correlation to infectivity reduction. LC(50), LD(50), LT(50) and the mean time to death of larvae were determined for vApAg and AgMNPV. Apoptosis was accessed for hemocytes of infected larvae using light and transmission electron microscopy. All types of hemocytes can be infected by vApAg. After 12h post-infection (h p.i.), typical cellular modifications associated to nucleopolyhedrovirus infection were observed. Apoptosis becomes evident after 24h p.i., and massive after 72h p.i. Necrosis of infected cells was also observed. Despite cell death, hemocytes produced budded viruses and polyhedra. Pl and gh1-type hemocytes presented phagocytic activity. Agarose gel electrophoresis revealed fragmentation of hemocytes DNA at late times post-infection. The LC(50) and LD(50) were between five- and six-fold higher for vApAg. The LT(50) and the mean time to death were higher for vApAg in a same treatment or for a similar mortality induced by AgMNPV. These results show correlation of apoptosis and the reduced infectivity of vApAg in A. gemmatalis larvae.

In vivo apoptosis induction and reduction of infectivity by an Autographa californica multiple nucleopolyhedrovirus p35(-) recombinant in hemocytes from the velvet bean caterpillar Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae).

Baculoviruses have long been shown to regulate apoptosis in cultured insect cells. Recently, this phenomenon was also reported to occur in vivo, reinforcing the importance of apoptosis in insect immunity against viruses. The vP35del virus, an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) recombinant, was previously shown to induce apoptosis in Anticarsia gemmatalis cultured cells. In order to verify the AcMNPV interaction with hemocytes, apoptosis induction in vivo and its effects upon infectivity, we studied the course of intrahemocoelic infection of recombinant viruses (vHSGFP and vHSGFP/P35del) in A. gemmatalis larvae. Insect development and mortality were monitored and infection progress was followed by light, fluorescence and electron microscopy. For all doses tested, vHSGFP/P35del caused lower mortality than vHSGFP. Mortality of 95% occurred with a dose of 4x10(6) PFUs of vHSGFP, which was reduced to 60% for vHSGFP/P35del. GFP expression was first observed at 3 h p.i. for the two viruses, increasing for vHSGFP (40% at 120 h p.i.) and decreasing for vHSGFP/P35del (0% at 120 h p.i.). The virus vHSGFP/P35del induced apoptosis in hemocytes, with some budded virus being produced, and fragmented cells were observed between 24 and 72 h p.i. The recombinant vHSGFP induced typical wild-type cytopathic effects, with low production of occluded viruses until 120 h p.i. Plasmatocytes and granular hemocytes type 1 were the hemocytes most susceptible to both viruses. For these experimental conditions, we concluded that A. gemmatalis is a semi-permissive host to AcMNPV; moreover, apoptosis reduces AcMNPV infectivity and the p35 gene is essential for blocking apoptosis in this system.

Morphological characterization of Anticarsia gemmatalis M nucleopolyhedrovirus infection in haemocytes from its natural larval host, the velvet bean caterpillar Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae).

For a better understanding of virus x host interactions, transmission electron microscopy was used to characterize the intrahaemocoelic infection of Anticarsia gemmatalis larval haemocytes by A. gemmatalis M nucleopolyhedrovirus (AgMNPV). At 12 h post-infection (h p.i.), we observed nuclear hypertrophy, budded virus assembling, and protrusion towards the cytoplasm, virion envelopment, and accumulation of fibrillar aggregates in the cytoplasm. Around 24 h p.i., fibrillar aggregates also appeared inside nuclei of infected cells. By 48 h p.i., virogenic stroma and polyhedra were visualised in nuclei and at 72 h p.i., widespread infection in haemocytes was observed. Cell remnants and free polyhedra were phagocytosed by granular haemocyte 1 and plasmatocytes. Entire cells were phagocytosed only by plasmatocytes. Necrosis of infected cells was quite common, suggesting a putative cytotoxic response. Granular haemocyte 1 presented a more exuberant protrusion of budded viruses in comparison to other haemocytes. All types of haemocytes were shown to be infected, and the intense virus replication in some of these cells reveals the importance of haemolymph for AgMNPV spread in its natural host, a critical factor for permissiveness.