BACKGROUND
diseases caused by mosquito-borne viruses are world-wide among the most challenging public health burdens in tropical countries.
current measures to control arthropod-borne viruses  and their mosquito vectors are insufficient due to the lack of sustainable vector control strategies and vaccines  <cit> .
control efforts to reduce the vectorial capacity of mosquitoes for arboviruses in the field rely mainly on insecticide applications, removal of mosquito breeding sites, use of repellents, door/window curtains, and bed nets  <cit> .
aedes aegypti is the primary vector for the currently most prevalent arboviruses, which include dengue virus , chikungunya virus , zika virus , and yellow fever virus .
an important component of a mosquito's vectorial capacity is its vector competence for a particular virus.
vector competence refers to the mosquito's intrinsic ability, based on its genetic make-up, to acquire, maintain, and transmit an arbovirus.
innate immunity and tissue barriers in the mosquito are important determinants of its vector competence  <cit> .
following ingestion of a viremic bloodmeal from a vertebrate host, the midgut is the first organ that becomes infected by the virus.
following replication in the midgut, the virus then disseminates to secondary tissues including nerve tissue, hemocytes, fatbody, and the salivary glands.
however, to establish a systemic and persistent infection, an arbovirus needs to overcome at least four tissue barriers in the mosquito.
these barriers include midgut infection and escape barriers and salivary gland infection and escape barriers  <cit> .
once the latter has been overcome by the virus, the mosquito can transmit the virus to another vertebrate host during probing.
recently, our research group began to investigate the nature of the midgut escape barrier for arboviruses in ae.
aegypti .
our observations so far suggest that a virus such as chikv disseminates from the midgut to secondary tissue during bloodmeal digestion by crossing the basal lamina  surrounding the midgut organ  <cit> .
in insects, the bl is a layer of extracellular matrix consisting of collagen iv and laminin and synthesized during early development by hemocytes and the fat body  <cit> .
earlier studies showed that the pore size exclusion limit of the mosquito bl is only 9– <dig> nm  <cit> .
the authors concluded that the bl has to be remodeled/degraded to allow arbovirus virions  to pass through.
indeed, our preliminary observations suggest that during bloodmeal digestion, the midgut bl is temporarily degraded as shown by a diminished abundance of collagen iv .
furthermore, bl degradation coincides with the time window during which chikv is disseminating from the midgut.
matrix metalloproteinases  have been regarded as the principal proteinases to be involved in bl degradation/remodeling processes .
in order to obtain a comprehensive expression profile of mosquito genes contributing to the midgut escape of arboviruses, we conducted an rna-seq experiment based on rna samples from midguts of mosquitoes, which had received a saline meal  or a protein meal  containing/not containing chikv.
recently, bovine serum albumin  has been reported as an alternative protein meal for ae.
aegypti and ae.
albopictus, which is taken up by the mosquito midgut and upon its digestion facilitates female vitellogenesis and egg production .
thus, we substituted blood for a pm  and a sm  to reduce potential "background noise" caused by genes responsive to blood digestion rather than to the process of viral dissemination from the midgut.
genetic interactions between ae.
aegypti and arboviruses have been extensively investigated in whole-genome based transcriptome/proteome analyses to reveal the mechanisms of arbovirus infection and transmission .
as a result, numerous candidate genes have been shown to be regulated by replicating viruses and some of these genes appeared to have antiviral properties in ae.
aegypti .
for example,  <dig> upregulated and  <dig> downregulated genes with similar expression profiles were identified at day  <dig> of infection in the rockefeller strain of ae.
aegypti, which had been challenged with three different flaviviruses, west nile virus , denv <dig>  and yfv  <cit> .
these genes exhibited diverse functions including ion binding, transport, metabolic processes, and peptidase activity.
a specific outcome of this study was the identification of a pupal cuticle protein , which was able to bind wnv envelope protein, leading to inhibition of infection in vitro and the prevention of lethal wnv encephalitis in mice.
bonizzoni and colleagues showed that denv <dig> infection affected the expression of  <dig> genes in ae.
aegypti ,  <dig> of which exhibited significant differential transcript accumulation levels in midgut samples in comparison to the non-infected control  <cit> .
specifically, two of these genes  showed progressively higher transcript accumulation levels in denv <dig> samples from  <dig> to  <dig> days post-infection .
however, in all these studies, the viruses had been administered to the mosquitoes either along with a bloodmeal or via intrathoracic injections .
moreover, most whole-transcriptome analyses in ae.
aegypti were performed in context with flavivirus infections, particularly denv .
chikv, an alphavirus  of the semliki forest serocomplex, has a particularly short extrinsic incubation period and high infection/dissemination rates in ae.
aegypti  <cit> .
already within  <dig> days post-oral acquisition, the virus can build up relatively high titers in midgut epithelial cells and, following its dissemination from the midgut, in infected secondary tissues as well.
in this study, we established an alternative, blood-free method of delivering chikv to ae.
aegypti by feeding a mixture of infectious cell culture, bsa  or pbs  solution, and adenosine triphosphate .
we conducted a genome-wide transcriptome analysis to reveal gene expression patterns in midguts of pm and sm  fed mosquitoes in comparison to a sugarfed control.
eventually, we analyzed in vitro the detailed expression patterns and catalytic activities of two selected candidate genes, ae.
aegypti late trypsin  and serine collagenase  <dig> precursor , in an attempt to reveal their functions.
RESULTS
chikv orally acquired along with a pm or sm productively infected the midgut and disseminated to secondary tissues
ae.
aegypti  mosquitoes were fed with a  blood meal ,  40% bsa solution , or  2x pbs solution , each diluted 1: <dig> with cell culture supernatant collected from chikv infected  or uninfected vero cells.
all three meal types were deposited in the midgut lumen of the mosquitoes for digestion, whereas the sugarmeal  was deposited in the crop and therefore did not enter the midgut.
sm was digested at a faster rate  than pm or bm.
interestingly, sm feeding did not lead to peritrophic matrix formation in the midgut as observed in ultrastructural studies .
however, all three meal types allowed chikv to efficiently infect the midgut epithelium as early as  <dig> dpi  and to disseminate from the midgut within  <dig> dpi as shown by immunofluorescence assays and plaque assays .
chikv-containing sm and pm feeding led to the development of distinct viral infection foci in the mosquito midgut at  <dig> and  <dig> dpi, which by  <dig> dpi encompassed most of the midgut tissue .
plaque assays showed that overall titers in midguts were significantly lower for chikv acquired along with sm or pm in comparison to bm.
surprisingly, chikv titers in carcasses  were significantly higher for pm and sm acquired virus in comparison to bm acquired virus at  <dig> dpi  and significantly higher dissemination rates were observed for pm acquired chikv .
in summary, all three meal types supported the midgut infection with chikv and its dissemination to secondary tissues.fig.
<dig> comparison of chikv infection patterns and titers in midguts and carcasses of mosquitoes, which had received a bloodmeal , protein meal , or saline meal  mixed with chikv containing cell culture medium at  <dig>   <dig>   <dig> days post-infection .
a immunofluorescence assay detection of chikv-antigen in midguts at different time points post-infectious sm/pm  and non-infectious sm/pm  at  <dig> days post-feeding .
images  and  are higher magnification views of  and , respectively as outlined by the white boxes.
bars:   <dig> μm;   <dig> μm.
virus titers of individual midguts  and carcasses  as analyzed by plaque assays in vero cells.
each data point represents the chikv titer of a single midgut or carcass.
only infected mosquitoes were included in the statistical analysis based on the mann–whitney u-test to determine p values.
black bars indicate medians.
the tables below b and c show the numbers of chikv infected midguts or carcasses in relation to the total numbers of midgut and carcass samples tested.
d prevalence of chikv infection in carcasses of mosquitoes.
fisher’s exact test was used to determine p values
global changes in the midgut transcriptome in response to sugarfeeding, sm, pm, and chikv infection
a total of  <dig> rna-seq libraries from midguts of mosquitoes that had received pm or sm containing/not containing chikv at  <dig> and  <dig> dpi/days post-feeding  were generated and sequenced.
libraries from midguts of sugarfed mosquitoes were used as control.
since the bm associated midgut transcriptome of ae.
aegypti has been already reported  <cit> , we decided to not include bm in our study.
each experimental variant consisted of three biological replicates .
rna sequencing  generated on average  <dig>  million quality reads per rna-seq library of which ~ <dig> % mapped to the reference genome .
of  <dig>  protein encoding genes of ae.
aegypti, 86–89% of them were detected as transcripts in the different midgut rna-seq libraries .
around 50% of those genes showed a low read coverage  and less than 1% of them had read coverages exceeding  <dig>  fpkm.table  <dig> classification of genes based on transcript accumulation levels  in mosquito midguts
fpkm are average values from rna-seq libraries of three biological replicates.
sm, saline meal ; pm, protein meal
abundances of reads mapped to genes were quantified by featurecounts and the read counts of all genes across all samples were subjected to principal component analysis  and hierarchical clustering .
as a result of pca, the  <dig> libraries were divided into four large clusters: sm-day1/sugar, sm-day <dig>  pm-day <dig>  and pm-day <dig> .
of these four groups, pm-day <dig> and pm-day <dig> showed the steepest contrast from each other, whereas sm-day1/sugar and sm-day <dig> were more similar, with data values within the cluster sm-day1/sugar only weakly contributing to component variation.
hierarchical clustering revealed two major clusters, sm/sugar and pm, which were then subdivided according to the pca results .
based on the branch height  and the pca clustering, changes in gene expression levels were far less pronounced in midguts of sugarfed/sm fed females than in midguts of pm fed mosquitoes between  <dig> and  <dig> dpf/pi.
together, these results suggest that pm ingestion may have caused more dramatic transcriptional changes in the mosquito midgut over time than sm ingestion.fig.
<dig> principal component analysis   and hierarchical cluster analysis  of gene expression variation among  <dig> rna-seq libraries obtained from midguts of sugarfed, sm fed, and pm fed mosquitoes.
for pca, data visualization and plotting were performed using r. the euclidean distance measure was used to calculate distances and data fitting was performed by ward's method  <cit> .
sm, saline meal ; pm, protein meal
expression profiles of de genes in response to chikv infection of the mosquito midgut
similar to previous reports demonstrating that arboviruses only induced a relatively low number of differentially expressed  genes when acquired along with a bm, we observed only  <dig> de genes  at  <dig> dpi and  <dig> de genes  at  <dig> dpi in midguts of sm fed mosquitoes responding to oral challenge with chikv .
based on gene ontology  terms, the majority of these de genes matched the functional categories metabolic process, catalytic activity, response to stimulus, immune system process, cellular process, single-organism process, and binding .
an obvious change in the composition of the functional go categories was caused by the appearance of increasingly abundant transcripts belonging to the category immune system process in midguts of sm fed, chikv infected mosquitoes at  <dig> and  <dig> dpi  and in midguts of pm fed, chikv infected mosquitoes at  <dig> dpi .
other profound changes among the rna-seq libraries generated from the chikv infected midguts were observed for the categories transporter activity, localization, regulation of biological processes, biological regulation, and signaling.
the median logarithmic fold-change  value for upregulated genes in response to chikv infection was  <dig>  for  <dig> dpi and  <dig>  for  <dig> dpi, and that for downregulated genes was  <dig>  for  <dig> dpi and  <dig>  for  <dig> dpi .
in midguts of pm fed mosquitoes,  <dig> de genes  at  <dig> dpi and  <dig> de genes  at  <dig> dpi were responsive to the presence of chikv .
the majority of these de genes matched the same functional categories as observed for de genes in chikv infected midguts of sm fed mosquitoes .
the median logfc value for upregulated genes in the pm rna-seq libraries in response to chikv infection was  <dig>  for  <dig> dpi and  <dig>  for  <dig> dpi, and that for downregulated genes was  <dig>  for  <dig> dpi and  <dig>  for  <dig> dpi .
interestingly, only seven de genes were identical among the rna-seq libraries obtained from chikv infected midguts of sm and pm fed mosquitoes at day  <dig> and  <dig> .
this suggests that the presence of chikv induced specific gene expression patterns for each of the different treatments.fig.
<dig> overview of the functional categories of differentially expressed  genes in response to chikv infection  and sm/pm feeding .
de genes were determined based on statistical analysis by edgerrobust.
the total number of de genes for each comparison is shown in parentheses in each figure.
gene ontology analysis of de genes was performed using david bioinformatics resources  <dig> , and pie charts were generated using graphpad prism  <dig> .
up, upregulated de genes; down, downregulated de genes; sm, saline meal ; pm, protein meal
expression profiles of de genes related to immunity in response to chikv infection and their putative network interactions
according to a heatmap based on fpkm values, >50% and >80% of over  <dig> immunity related de genes were upregulated in midguts of pm fed mosquitoes at  <dig> and  <dig> dpf/pi, respectively, but not in those of sm fed mosquitoes .
surprisingly, the majority of these immunity related genes were significantly upregulated when chikv was absent , suggesting that ingestion of non-viral protein can induce an immune response in the mosquito midgut.
however, in midguts of sm fed mosquitoes at day  <dig>  only nine de genes related to immunity were significantly upregulated in response to chikv .
these included genes encoding five anti-microbial peptides , three fibrinogen-related proteins , and a peptidoglycan recognition protein .
at day  <dig> post-infection with chikv, seven immunity related genes were significantly upregulated encoding three amps, a thioester-containing protein , a c-type lysozyme, a gram-negative binding protein , and a frep .
in midguts of pm fed mosquitoes, only one de gene related to innate immunity, which encoded a gnbp was responsive to the presence of chikv at  <dig> dpi and none at  <dig> dpi.table  <dig> immunity related genes significantly upregulated in the presence of chikv
logfc logarithmic fold-change, cpm counts per million, fdr false discovery rate, pgrp peptidoglycan recognition protein, frep fibrinogen related protein, gnbp gram-negative binding protein, sm saline meal , pm protein meal , dpi days post-infection with chikv, dpf days post-feeding without chikv infection
the maximum relevance minimum redundancy network analysis showed interactions of these immunity related genes in response to chikv infection .
by implementing degree centrality statistics to predict key players within gene expression networks, we predicted a member of the frep family  as a key player in establishing cross-talks among the immunity gene networks in response to chikv infection of sm and pm fed mosquitoes.
freps are known to play an important role in anopheles as well as in aedes mosquitoes by triggering infection related immune responses  <cit> .
frep <dig> was predicted as the most central of all immunity genes in chikv infected midguts of sm and pm fed mosquitoes.
the variation of the centrality score for frep <dig> between midgut rna-seq libraries of  sm and pm fed mosquitoes indicates that the diet taken up by the midgut affected the gene's role in differentially modulating the cross-talk of immunity related genes.fig.
<dig> maximum relevance minimum redundancy  expression network of immunity related genes responding to chikv infection in midguts of sm and pm fed mosquitoes.
the arrows indicate that frep <dig> was predicted as the most central immunity gene in chikv infected midguts of sm and pm fed mosquitoes when compared to non-infected midguts demonstrating its importance in the innate immune response to chikv infection of the mosquito midgut.
the centrality score indicated is based on a degree centrality measure, showing how central the gene is to the given network.
differences in the centrality scores underline their role in differentially modulating the cross-talk of immunity genes depending on sm or pm ingestion.
genes used for the network analysis were: frep <dig> , frep <dig> , gnbp <dig> , pgrp <dig> , frep <dig> , ml26b , tep <dig> , cecg , tpx <dig> , att , lysc <dig> , defc , defd , frep <dig> , gam , cecn , and ceca
expression of rna interference and apoptotic pathway related genes
none of the rnai or apoptotic pathway related genes showed any significant de values in any of the libraries irrespective of the presence or absence of chikv .
based on fpkm values, uniform expression patterns were observed in all rna-seq libraries for  <dig> rnai pathway-related genes: with the exceptions of piwi <dig> , tsn , and ago <dig> , all rnai related genes were upregulated in chikv infected and non-infected midguts of pm fed mosquitoes at  <dig> dpf/dpi .
curiously,  <dig> day earlier, most of these genes were downregulated, whereas in the sm rna-seq libraries their expression levels did not change.
when analyzing  <dig> apoptosis-related genes, the overall expression pattern occurred to be reversed.
only casps <dig>  and an inhibitor of apoptosis-like protein encoding gene  showed upregulation in any of the pm rna-seq libraries whereas all other apoptosis-related genes were upregulated in the sm rna-seq libraries .
casps <dig> was described as a putative sensor or enhancer for casps <dig> as the former is lacking its own catalytic activity  <cit> .
however, casps <dig> was not upregulated in the pm rna-seq libraries.
of all caspases, casps <dig> exhibited the strongest overall gene expression levels  followed by casps <dig>
thus, in presence of sm there was a tendency for an apoptotic response whereas pm ingestion had the tendency to trigger rnai and other immune responses.
de genes in midguts of sm and pm fed mosquitoes in comparison to midguts of sugarfed mosquitoes
sm feeding induced in midguts  <dig> de genes  at day  <dig> and  <dig> de genes  at day  <dig> when compared to midguts of sugarfed mosquitoes .
the median logfc value for upregulated genes in response to sm feeding was  <dig>  for day  <dig> and  <dig>  for day  <dig>  whereas that for downregulated genes was  <dig>  for day  <dig> and  <dig>  for day  <dig> .
however, pm ingestion induced a considerably higher number of de genes,  <dig> genes  at day  <dig> and  <dig> genes  at day  <dig> when compared to midguts of sugarfed mosquitoes .
the median logfc value for upregulated genes in response to pm feeding was  <dig>  for day  <dig> and  <dig>  for day  <dig>  whereas that for downregulated genes was  <dig>  for day  <dig> and  <dig>  for day  <dig> .fig.
<dig> differential expressed  genes induced by sm and pm at  <dig> and  <dig> days post-feeding  and functional annotation of de genes at  <dig> dpf.
a venn diagram representing shared de genes between sugarfed controls and sm/pm samples at  <dig> and  <dig> dpf.
b histogram presentation of functional categories  of upregulated  and downregulated  de genes shared between sm and pm samples at  <dig> dpf.
gene ontology was analyzed using biomart at vectorbase.
the x-axis denotes the number of genes in a functional category
similar to the functional categories revealed for de genes in response to chikv infection, de genes in response to sm or pm feeding belonged to the six largest categories unknown > catalytic activity > cellular process > binding > metabolic > single organism process .
profound changes in transcript abundance between sm and pm fed mosquitoes and between  <dig> and  <dig> dpf occurred for the go categories transporter activity, response and stimulus, regulation of biological processes, localization, signaling, and single organism process.
at day  <dig>  sm and pm rna-seq libraries shared  <dig> upregulated de genes and  <dig> downregulated genes .
one day later, the number of commonly upregulated de genes decreased by about half to  <dig> genes and that of commonly downregulated genes to just  <dig>  the sm libraries of day  <dig> and  <dig> had nine upregulated and  <dig> downregulated de genes in common.
pm had by far the strongest effect on de gene upregulation in the midgut.
of those upregulated de genes,  <dig> were commonly expressed at  <dig> and  <dig> dpf and both libraries shared  <dig> downregulated de genes.
the profound increase in the number of upregulated genes  in the pm libraries when compared to sm libraries suggests that most of these genes were involved in protein  digestion.
common de genes among the sm and pm rna-seq libraries obtained from midguts of non-infected mosquitoes at  <dig> dpf belonged to the categories catalytic activity, hydrolase activity, serine-type endopeptidase activity, metalloproteinase activity, protein binding, and oxidoreductase activity, three of which  may contain proteinase genes involved in bl degradation and remodeling .
comparing expression levels of genes encoding trypsins, metalloproteinases, and serine-type endopeptidases
in view of their possible involvement in bl degradation and remodeling, we focused on three groups of proteinase genes: trypsins, metalloproteinases, and serine-type endopeptidases whose relative expression levels are presented in heatmaps based on fpkm values .
sm or pm ingestion caused increased expression of >50% of the trypsin, metalloproteinase and serine-type endopeptidase encoding genes, including genes for two mmps, aemmp <dig>  and aemmp <dig> , two adams/adamts , and two predicted serine collagenases .
as a general trend, most of the trypsin, metalloproteinase, and serine-type endopeptidase genes showed upregulation in the pm rna-seq libraries in comparison to the sugarfed library.
ten, seven, and four de genes encoding trypsins, serine-type endopeptidases, and metalloproteinases, respectively, were significantly upregulated in midguts of sm or pm fed mosquitoes at  <dig> dpf .
in addition, two serine collagenase-like genes and one glutamate carboxypeptidase encoding gene were identified.
most of these genes were highly expressed in midguts and their expression levels were increased by logfc >  <dig> in sm and logfc >  <dig> in pm.fig.
<dig> median-normalized expression levels  of three groups of proteinase genes .
data show a comparison between sugarfed controls and sm/pm samples at  <dig> and  <dig> days post-feeding  and are presented as heatmaps for each transcript  at each time point , with yellow and blue indicating high and low levels of expression, respectively
logfc logarithmic fold-change, cpm counts per million, fdr false discovery rate, sm saline meal , pm protein meal , dpf days post-feeding without chikv infection
mmps, adams/adamts, and collagenases are known to be involved in extracellular matrix remodeling and tissue repairing .
it is possible that trypsins, other metalloproteinases, and serine-type endopeptidases are also involved in midgut bl degradation and/or remodeling either directly by taking part in proteinase activation cascades or indirectly by participating in relevant signaling processes  <cit> .
assessing the catalytic activities of aesp <dig> and aelt in vitro
being a major component of the midgut bl, we wanted to investigate whether collagen iv abundance was changed in mosquito midguts following sm ingestion.
when we conducted a time series study of collagen iv degradation in midguts of sm fed mosquitoes, it became evident that midgut collagen iv was less abundant at  <dig> and  <dig> h post-feeding in comparison to other time points .
concurrent with midgut collagen iv degradation, generic mmp activity was significantly increased in mosquito midguts at  <dig> day post-sm  in comparison to the sugarfed control.
no significant difference was observed between midguts of sm and sugarfed mosquitoes at  <dig> dpf while bm and pm acquisition resulted in a significantly higher mmp activity .
these results support the conclusion that bm, sm, and pm feeding can stimulate or support bl remodeling by affecting the abundance of collagen iv via an increased mmp activity.
the results also suggest that genes encoding proteinases involved in collagen iv degradation/remodeling were expressed in midguts of sm fed mosquitoes.
the potential involvement of aemmps in midgut bl remodeling has been investigated in a recent study  <cit> .fig.
<dig> effects of sm feeding on midgut collagen iv abundance and generic mmp activity.
a western blot showing abundance of midgut collagen iv at different time points post-sm feeding using polyclonal antibodies generated against human collagen iv.
β-actin was detected as a loading control.
molecular masses in kda are indicated.
b generic mmp activity of midgut lysates prepared from mosquitoes that had received a bm, pm, or sm at  <dig> and  <dig> dpf .
mean values with standard deviations  from three independent experiments are shown.
different letters indicate significant differences based on one-way analysis of variance  followed by tukey's multiple comparisons test .
bm, blood meal ; sm, saline meal ; pm, protein meal
here, we focus on the potential involvement of two putative collagenase genes, aelt and aesp <dig> in midgut bl degradation/remodeling.
as revealed by qrt-pcr, both genes were significantly upregulated in midguts at  <dig> dpf/pi in conjunction with bm or pm ingestion; however, only aelt was significantly upregulated in sm fed midguts at  <dig> dpf/pi .
as shown by western blot analysis, expression of aelt and aesp <dig> in drosophila s <dig> cells resulted in recombinant proteins with sizes of  <dig> kda and ~ <dig> kda for aelt and aesp <dig>  respectively, when using an anti-his-tag specific monoclonal antibody for detection .
following their activation via incubation with tpck-treated bovine trypsin, both proteins produced band signals that were reduced in size by ~ <dig> kda indicating that pro-domain cleavage had occurred .
in vitro, activated recombinant aelt showed strong generic mmp activity using fs- <dig> as substrate  whereas raesp <dig> did not .
further, addition of the recombinant aedes aegypti tissue inhibitor of metalloproteinases  did not inhibit raelt activity indicating that the catalytic domain of the proteinase is structurally different from that of mmps or adams both of which can be inhibited by timps  <cit> .fig.
<dig>
drosophila s <dig> cell expression of two putative serine collagenase genes and catalytic activities of the recombinant proteins.
a western blot detection of recombinant aelt and aesp <dig> proteins using an his tag-specific monoclonal antibody.
blank, s <dig> cells were transfected with non-insert containing plasmid vector; raelt and raesp <dig>  s <dig> cells were transfected with plasmid vectors expressing aelt or aesp <dig>  molecular masses in kda are indicated.
b sds-page showing purified raelt and raesp <dig> with or without additional trypsin activation .
molecular masses in kda are indicated.
kinetics of raelt  and raesp <dig>  in vitro activities and their inhibition by raetimp.
twenty ng of purified raelt or raesp <dig> were preincubated with/without tpck-treated trypsin for  <dig> min at rt, then incubated with  <dig> ng of raetimp, or reaction buffer  at rt for  <dig> h, followed by addition of fs- <dig> substrate.
fluorescence intensity was measured every  <dig> min.
raelt-pro and raesp1-pro, pro-protein of raelt and raesp1; raelt and raesp <dig>  activated raelt and raesp1
.
discussion
bloodmeal uptake by the mosquito midgut leads to a global upregulation of endogenous genes, many of which encode proteins specifically involved in bloodmeal digestion  <cit> .
thus, offering mosquitoes minimal meals lacking any substantial nutritional value seemed to be a promising strategy to limit gene expression to those candidate genes responding to structural changes in the midgut due to tissue expansion rather than to genes involved in digestion.
ingestion of pm, which consisted of bsa and this way was of nutritional value, caused upregulation of several thousand genes making this meal type physiologically look similar to a bloodmeal.
however, in contrast to blood, bsa is a highly purified protein and free of proteinases, enzymes and growth factors, which all are highly abundant in blood sources.
thus, the midgut transcriptome of pm fed mosquitoes should predominantly reflect any effects associated with feeding, protein digestion, and changes in tissue structure.
replacing protein with a substance without nutritional value such as salt  in sm, should strongly reduce the expression of genes involved in digestion.
in agreement with this hypothesis, we only recorded  <dig> de genes that were upregulated by sm at  <dig> dpf, compared to  <dig> genes which were upregulated in presence of pm at  <dig> dpf or  <dig> genes upregulated at  <dig> h post-bm   <cit> .
the presence of chikv in sm induced expression of  <dig> de genes in total at  <dig> and  <dig> dpi, which was similar to the number of genes observed to be upregulated in the chikv containing pm libraries .
to compare with another study, denv <dig> induced expression of  <dig>   <dig>  and  <dig> de genes in midguts of ae.
aegypti at  <dig>   <dig> and  <dig> dpi via bm, respectively  <cit> .
most of the de genes responding to chikv challenge along with sm feeding had been previously reported to be responsive to arboviruses such as denv <dig>  wnv and yfv  <cit> .
interestingly, ingestion of chikv along with sm or pm significantly reduced the midgut infection intensity of the virus but increased its carcass infection intensity in comparison to bm acquired chikv.
these observations support the conclusion that blood may contain additional components that are advantageous for viral cell entry.
our data show that chikv induced expression of numerous genes related to immunity including those encoding amp, frep and pdgr.
we identified additional genes  that were significantly upregulated in the presence of chikv and had previously been reported to be involved in arbovirus infection of mosquitoes.
these genes encoded three synaptic vesicle  proteins , unc93a , cysteine-rich venom protein , and two c-type lectins  .
in mammals, unc93a and sv proteins are involved in intracellular transport  <cit>  and their transcripts were enriched more than 40-fold during sindbis virus  or denv <dig> infection of ae.
aegypti  <cit> .
unc93a and sv seem to play a role in virus assembly and/or budding from the infected cell as revealed by transient gene silencing of sv <dig> or unc93a in mosquitoes infected with sinv  <cit> .
crvp proteins are known to contain a trypsin inhibitor-like  domain, and therefore could be functioning as serine protease inhibitors  <cit> .
one of the ae.
aegypti crvp genes, crvp <dig> , has recently been reported to be significantly upregulated in mosquitoes infected with the flaviviruses yfv or wnv.
further, crvp <dig> expression proved to be essential during infection of ae.
aegypti mosquitoes or aag <dig> cells with denv <dig>  <cit> .
ctls are a family of proteins possessing carbohydrate-binding activity.
several mosquito ctls have been reported to be interacting with arboviruses as ligands to facilitate mosquito infection  <cit> .
ctls play a role in maintaining gut microbiome homeostasis by manipulating the bactericidal capacity of amps  <cit> .
in ae.
aegypti, the galactose specific c-type lectin- <dig>  increased the attachment efficiency of wnv to the cell membrane via binding to a membrane-bound protein   <cit> .
furthermore, mosgctl- <dig> enhanced denv <dig> infection via interacting with the e protein of the virus  <cit> .
besides those genes whose possible functions during arbovirus infections have been analyzed by others, we also observed numerous genes to be significantly upregulated in presence of chikv, whose functions do not appear to be directly related to virus infection.
these genes, for example, include a cytochrome p <dig> gene , two lethalessential for life efl) genes , a juvenile hormone-inducible protein gene , a heat shock protein gene , and a microtubule binding protein gene .
in a recent study in which we compared the vector competence of two ae.
aegypti strains  for chikv, we suggested that the virus exits the midgut tissue by crossing the bl surrounding the organ instead of using tracheal cells or the cardia as a conduit for dissemination  <cit> .
in agreement with this finding, we observed here that the abundance of collagen iv, a major component of the bl, was diminished between 18– <dig> h post-sm, which coincided with an increased generic mmp activity.
in eukaryotes, bl degradation/remodeling involves the activities of numerous metalloproteinases and inhibitors  <cit>  and an aim of this study was to identify potential candidate genes involved in mosquito bl degradation/remodeling.
in insects, several mmps such as drosophila mmp <dig> and mmp <dig> have been shown to cleave collagen in vitro  <cit> .
here, we also observed transcriptional upregulation of two mmps, aemmp1and aemmp <dig>  <cit>  and two adams/adamts in response to sm and/or pm ingestion.
in addition, two putative serine collagenase genes were significantly upregulated in the midgut rna-seq libraries of sm and pm fed mosquitoes and their expression patterns were further confirmed by qrt-pcr.
in insects, a serine collagenase was for the first time identified in the heel fly hypoderma lineatum  <cit>  with the purified enzyme being able to degrade native collagen but unable to degrade synthetic peptide substrates specific for trypsin or chymotrypsin  <cit> .
despite its designation as a trypsin, bacterially expressed recombinant aelt did not affect cleavage of the trypsin specific substrate dl-bapna, whereas transient silencing of aelt had no effect on the midgut infection efficiency of denv <dig> suggesting that aelt may actually lack any trypsin activity .
based on structural similarities, aelt does not resemble a canonical trypsin but a serine collagenase instead  <cit> .
we expressed the full-length aelt in drosophila s <dig> cells and the resulting raelt showed a high level of generic mmp activity.
therefore, our results strongly support the conclusion that aelt, indeed, is a serine collagenase.
in contrast, recombinant aesp <dig> lacked any mmp activity, which is not surprising because in aesp <dig>  his <dig> and ser <dig> in the archetypal his57-asp102-ser <dig> catalytic triad motif are changed to gln <dig> and val <dig>  <cit> .
CONCLUSIONS
substituting bm for pm or sm did not affect the persistent infection of ae.
aegypti with chikv, although virus titers in midguts were significantly lower when the virus was acquired along with sm instead of a bloodmeal.
on the other hand, pm and sm did not negatively affect the dissemination rates of the virus, indicating that the protein content of the ingested meal was not a major determinant of chikv midgut escape from the midgut.
instead, stretching of the midgut tissue due to meal ingestion may be the critical mechanism or trigger underlying viral midgut escape.
gene expression patterns in pm fed midguts largely resembled those previously observed for bloodfed midguts.
the presence of chikv in any of the ingested meals had relatively minor effects on the overall gene expression profiles in midguts, as only  <dig> genes  were being differentially expressed,  <dig>  of which were related to immunity.
in comparison to pm feeding resulting in the upregulation of  <dig> genes at day  <dig> and  <dig>  sm feeding substantially reduced the gene expression profile in the mosquito midgut with only  <dig>  genes being significantly upregulated, many of which belonged to the functional category catalytic activity.
these genes included seven serine-type endopeptidases, ten trypsins, two serine collagenases, four m <dig> zinc metalloproteases, and a glutamate carboxypeptidase, which could be involved in the midgut escape of chikv when traversing the midgut bl.
the recombinant serine collagenase, aelt, showed strong generic mmp activity in vitro.

 diseases caused by mosquito-borne viruses are world-wide among the most challenging public health burdens in tropical countries. current measures to control arthropod-borne viruses and their mosquito vectors are insufficient due to the lack of sustainable vector control strategies and vaccines. control efforts to reduce the vectorial capacity of mosquitoes for arboviruses in the field rely mainly on insecticide applications, removal of mosquito breeding sites, use of repellents, door/window curtains, and bed nets. aedes aegypti is the primary vector for the currently most prevalent arboviruses, which include dengue virus, chikungunya virus, zika virus, and yellow fever virus. an important component of a mosquito's vectorial capacity is its vector competence for a particular virus. vector competence refers to the mosquito's intrinsic ability, based on its genetic make-up, to acquire, maintain, and transmit an arbovirus. innate immunity and tissue barriers in the mosquito are important determinants of its vector competence. following ingestion of a viremic bloodmeal from a vertebrate host, the midgut is the first organ that becomes infected by the virus. following replication in the midgut, the virus then disseminates to secondary tissues including nerve tissue, hemocytes, fatbody, and the salivary glands. however, to establish a systemic and persistent infection, an arbovirus needs to overcome at least four tissue barriers in the mosquito. these barriers include midgut infection and escape barriers and salivary gland infection and escape barriers. once the latter has been overcome by the virus, the mosquito can transmit the virus to another vertebrate host during probing. recently, our research group began to investigate the nature of the midgut escape barrier for arboviruses in ae. aegypti. our observations so far suggest that a virus such as chikv disseminates from the midgut to secondary tissue during bloodmeal digestion by crossing the basal lamina surrounding the midgut organ. in insects, the bl is a layer of extracellular matrix consisting of collagen iv and laminin and synthesized during early development by hemocytes and the fat body. earlier studies showed that the pore size exclusion limit of the mosquito bl is only 9– nm. the authors concluded that the bl has to be remodeled/degraded to allow arbovirus virions to pass through. indeed, our preliminary observations suggest that during bloodmeal digestion, the midgut bl is temporarily degraded as shown by a diminished abundance of collagen iv. furthermore, bl degradation coincides with the time window during which chikv is disseminating from the midgut. matrix metalloproteinases have been regarded as the principal proteinases to be involved in bl degradation/remodeling processes. in order to obtain a comprehensive expression profile of mosquito genes contributing to the midgut escape of arboviruses, we conducted an rna-seq experiment based on rna samples from midguts of mosquitoes, which had received a saline meal or a protein meal containing/not containing chikv. recently, bovine serum albumin has been reported as an alternative protein meal for ae. aegypti and ae. albopictus, which is taken up by the mosquito midgut and upon its digestion facilitates female vitellogenesis and egg production. thus, we substituted blood for a pm and a sm to reduce potential "background noise" caused by genes responsive to blood digestion rather than to the process of viral dissemination from the midgut. genetic interactions between ae. aegypti and arboviruses have been extensively investigated in whole-genome based transcriptome/proteome analyses to reveal the mechanisms of arbovirus infection and transmission. as a result, numerous candidate genes have been shown to be regulated by replicating viruses and some of these genes appeared to have antiviral properties in ae. aegypti. for example, upregulated and downregulated genes with similar expression profiles were identified at day of infection in the rockefeller strain of ae. aegypti, which had been challenged with three different flaviviruses, west nile virus, denv and yfv. these genes exhibited diverse functions including ion binding, transport, metabolic processes, and peptidase activity. a specific outcome of this study was the identification of a pupal cuticle protein, which was able to bind wnv envelope protein, leading to inhibition of infection in vitro and the prevention of lethal wnv encephalitis in mice. bonizzoni and colleagues showed that denv infection affected the expression of genes in ae. aegypti, of which exhibited significant differential transcript accumulation levels in midgut samples in comparison to the non-infected control. specifically, two of these genes showed progressively higher transcript accumulation levels in denv samples from to days post-infection. however, in all these studies, the viruses had been administered to the mosquitoes either along with a bloodmeal or via intrathoracic injections. moreover, most whole-transcriptome analyses in ae. aegypti were performed in context with flavivirus infections, particularly denv. chikv, an alphavirus of the semliki forest serocomplex, has a particularly short extrinsic incubation period and high infection/dissemination rates in ae. aegypti. already within days post-oral acquisition, the virus can build up relatively high titers in midgut epithelial cells and, following its dissemination from the midgut, in infected secondary tissues as well. in this study, we established an alternative, blood-free method of delivering chikv to ae. aegypti by feeding a mixture of infectious cell culture, bsa or pbs solution, and adenosine triphosphate. we conducted a genome-wide transcriptome analysis to reveal gene expression patterns in midguts of pm and sm fed mosquitoes in comparison to a sugarfed control. eventually, we analyzed in vitro the detailed expression patterns and catalytic activities of two selected candidate genes, ae. aegypti late trypsin and serine collagenase precursor, in an attempt to reveal their functions.  chikv orally acquired along with a pm or sm productively infected the midgut and disseminated to secondary tissues ae. aegypti mosquitoes were fed with a blood meal, 40% bsa solution, or 2x pbs solution, each diluted 1: with cell culture supernatant collected from chikv infected or uninfected vero cells. all three meal types were deposited in the midgut lumen of the mosquitoes for digestion, whereas the sugarmeal was deposited in the crop and therefore did not enter the midgut. sm was digested at a faster rate than pm or bm. interestingly, sm feeding did not lead to peritrophic matrix formation in the midgut as observed in ultrastructural studies. however, all three meal types allowed chikv to efficiently infect the midgut epithelium as early as dpi and to disseminate from the midgut within dpi as shown by immunofluorescence assays and plaque assays. chikv-containing sm and pm feeding led to the development of distinct viral infection foci in the mosquito midgut at and dpi, which by dpi encompassed most of the midgut tissue. plaque assays showed that overall titers in midguts were significantly lower for chikv acquired along with sm or pm in comparison to bm. surprisingly, chikv titers in carcasses were significantly higher for pm and sm acquired virus in comparison to bm acquired virus at dpi and significantly higher dissemination rates were observed for pm acquired chikv. in summary, all three meal types supported the midgut infection with chikv and its dissemination to secondary tissues.fig. comparison of chikv infection patterns and titers in midguts and carcasses of mosquitoes, which had received a bloodmeal, protein meal, or saline meal mixed with chikv containing cell culture medium at days post-infection. a immunofluorescence assay detection of chikv-antigen in midguts at different time points post-infectious sm/pm and non-infectious sm/pm at days post-feeding. images and are higher magnification views of and, respectively as outlined by the white boxes. bars: μm; μm. virus titers of individual midguts and carcasses as analyzed by plaque assays in vero cells. each data point represents the chikv titer of a single midgut or carcass. only infected mosquitoes were included in the statistical analysis based on the mann–whitney u-test to determine p values. black bars indicate medians. the tables below b and c show the numbers of chikv infected midguts or carcasses in relation to the total numbers of midgut and carcass samples tested. d prevalence of chikv infection in carcasses of mosquitoes. fisher’s exact test was used to determine p values global changes in the midgut transcriptome in response to sugarfeeding, sm, pm, and chikv infection a total of rna-seq libraries from midguts of mosquitoes that had received pm or sm containing/not containing chikv at and dpi/days post-feeding were generated and sequenced. libraries from midguts of sugarfed mosquitoes were used as control. since the bm associated midgut transcriptome of ae. aegypti has been already reported, we decided to not include bm in our study. each experimental variant consisted of three biological replicates. rna sequencing generated on average million quality reads per rna-seq library of which ~ % mapped to the reference genome. of protein encoding genes of ae. aegypti, 86–89% of them were detected as transcripts in the different midgut rna-seq libraries. around 50% of those genes showed a low read coverage and less than 1% of them had read coverages exceeding fpkm.table classification of genes based on transcript accumulation levels in mosquito midguts fpkm are average values from rna-seq libraries of three biological replicates. sm, saline meal; pm, protein meal abundances of reads mapped to genes were quantified by featurecounts and the read counts of all genes across all samples were subjected to principal component analysis and hierarchical clustering. as a result of pca, the libraries were divided into four large clusters: sm-day1/sugar, sm-day pm-day and pm-day. of these four groups, pm-day and pm-day showed the steepest contrast from each other, whereas sm-day1/sugar and sm-day were more similar, with data values within the cluster sm-day1/sugar only weakly contributing to component variation. hierarchical clustering revealed two major clusters, sm/sugar and pm, which were then subdivided according to the pca results. based on the branch height and the pca clustering, changes in gene expression levels were far less pronounced in midguts of sugarfed/sm fed females than in midguts of pm fed mosquitoes between and dpf/pi. together, these results suggest that pm ingestion may have caused more dramatic transcriptional changes in the mosquito midgut over time than sm ingestion.fig. principal component analysis and hierarchical cluster analysis of gene expression variation among rna-seq libraries obtained from midguts of sugarfed, sm fed, and pm fed mosquitoes. for pca, data visualization and plotting were performed using r. the euclidean distance measure was used to calculate distances and data fitting was performed by ward's method. sm, saline meal; pm, protein meal expression profiles of de genes in response to chikv infection of the mosquito midgut similar to previous reports demonstrating that arboviruses only induced a relatively low number of differentially expressed genes when acquired along with a bm, we observed only de genes at dpi and de genes at dpi in midguts of sm fed mosquitoes responding to oral challenge with chikv. based on gene ontology terms, the majority of these de genes matched the functional categories metabolic process, catalytic activity, response to stimulus, immune system process, cellular process, single-organism process, and binding. an obvious change in the composition of the functional go categories was caused by the appearance of increasingly abundant transcripts belonging to the category immune system process in midguts of sm fed, chikv infected mosquitoes at and dpi and in midguts of pm fed, chikv infected mosquitoes at dpi. other profound changes among the rna-seq libraries generated from the chikv infected midguts were observed for the categories transporter activity, localization, regulation of biological processes, biological regulation, and signaling. the median logarithmic fold-change value for upregulated genes in response to chikv infection was for dpi and for dpi, and that for downregulated genes was for dpi and for dpi. in midguts of pm fed mosquitoes, de genes at dpi and de genes at dpi were responsive to the presence of chikv. the majority of these de genes matched the same functional categories as observed for de genes in chikv infected midguts of sm fed mosquitoes. the median logfc value for upregulated genes in the pm rna-seq libraries in response to chikv infection was for dpi and for dpi, and that for downregulated genes was for dpi and for dpi. interestingly, only seven de genes were identical among the rna-seq libraries obtained from chikv infected midguts of sm and pm fed mosquitoes at day and. this suggests that the presence of chikv induced specific gene expression patterns for each of the different treatments.fig. overview of the functional categories of differentially expressed genes in response to chikv infection and sm/pm feeding. de genes were determined based on statistical analysis by edgerrobust. the total number of de genes for each comparison is shown in parentheses in each figure. gene ontology analysis of de genes was performed using david bioinformatics resources, and pie charts were generated using graphpad prism. up, upregulated de genes; down, downregulated de genes; sm, saline meal; pm, protein meal expression profiles of de genes related to immunity in response to chikv infection and their putative network interactions according to a heatmap based on fpkm values, >50% and >80% of over immunity related de genes were upregulated in midguts of pm fed mosquitoes at and dpf/pi, respectively, but not in those of sm fed mosquitoes. surprisingly, the majority of these immunity related genes were significantly upregulated when chikv was absent, suggesting that ingestion of non-viral protein can induce an immune response in the mosquito midgut. however, in midguts of sm fed mosquitoes at day only nine de genes related to immunity were significantly upregulated in response to chikv. these included genes encoding five anti-microbial peptides, three fibrinogen-related proteins, and a peptidoglycan recognition protein. at day post-infection with chikv, seven immunity related genes were significantly upregulated encoding three amps, a thioester-containing protein, a c-type lysozyme, a gram-negative binding protein, and a frep. in midguts of pm fed mosquitoes, only one de gene related to innate immunity, which encoded a gnbp was responsive to the presence of chikv at dpi and none at dpi.table immunity related genes significantly upregulated in the presence of chikv logfc logarithmic fold-change, cpm counts per million, fdr false discovery rate, pgrp peptidoglycan recognition protein, frep fibrinogen related protein, gnbp gram-negative binding protein, sm saline meal, pm protein meal, dpi days post-infection with chikv, dpf days post-feeding without chikv infection the maximum relevance minimum redundancy network analysis showed interactions of these immunity related genes in response to chikv infection. by implementing degree centrality statistics to predict key players within gene expression networks, we predicted a member of the frep family as a key player in establishing cross-talks among the immunity gene networks in response to chikv infection of sm and pm fed mosquitoes. freps are known to play an important role in anopheles as well as in aedes mosquitoes by triggering infection related immune responses. frep was predicted as the most central of all immunity genes in chikv infected midguts of sm and pm fed mosquitoes. the variation of the centrality score for frep between midgut rna-seq libraries of sm and pm fed mosquitoes indicates that the diet taken up by the midgut affected the gene's role in differentially modulating the cross-talk of immunity related genes.fig. maximum relevance minimum redundancy expression network of immunity related genes responding to chikv infection in midguts of sm and pm fed mosquitoes. the arrows indicate that frep was predicted as the most central immunity gene in chikv infected midguts of sm and pm fed mosquitoes when compared to non-infected midguts demonstrating its importance in the innate immune response to chikv infection of the mosquito midgut. the centrality score indicated is based on a degree centrality measure, showing how central the gene is to the given network. differences in the centrality scores underline their role in differentially modulating the cross-talk of immunity genes depending on sm or pm ingestion. genes used for the network analysis were: frep, frep, gnbp, pgrp, frep, ml26b, tep, cecg, tpx, att, lysc, defc, defd, frep, gam, cecn, and ceca expression of rna interference and apoptotic pathway related genes none of the rnai or apoptotic pathway related genes showed any significant de values in any of the libraries irrespective of the presence or absence of chikv. based on fpkm values, uniform expression patterns were observed in all rna-seq libraries for rnai pathway-related genes: with the exceptions of piwi, tsn, and ago, all rnai related genes were upregulated in chikv infected and non-infected midguts of pm fed mosquitoes at dpf/dpi. curiously, day earlier, most of these genes were downregulated, whereas in the sm rna-seq libraries their expression levels did not change. when analyzing apoptosis-related genes, the overall expression pattern occurred to be reversed. only casps and an inhibitor of apoptosis-like protein encoding gene showed upregulation in any of the pm rna-seq libraries whereas all other apoptosis-related genes were upregulated in the sm rna-seq libraries. casps was described as a putative sensor or enhancer for casps as the former is lacking its own catalytic activity. however, casps was not upregulated in the pm rna-seq libraries. of all caspases, casps exhibited the strongest overall gene expression levels followed by casps thus, in presence of sm there was a tendency for an apoptotic response whereas pm ingestion had the tendency to trigger rnai and other immune responses. de genes in midguts of sm and pm fed mosquitoes in comparison to midguts of sugarfed mosquitoes sm feeding induced in midguts de genes at day and de genes at day when compared to midguts of sugarfed mosquitoes. the median logfc value for upregulated genes in response to sm feeding was for day and for day whereas that for downregulated genes was for day and for day. however, pm ingestion induced a considerably higher number of de genes, genes at day and genes at day when compared to midguts of sugarfed mosquitoes. the median logfc value for upregulated genes in response to pm feeding was for day and for day whereas that for downregulated genes was for day and for day.fig. differential expressed genes induced by sm and pm at and days post-feeding and functional annotation of de genes at dpf. a venn diagram representing shared de genes between sugarfed controls and sm/pm samples at and dpf. b histogram presentation of functional categories of upregulated and downregulated de genes shared between sm and pm samples at dpf. gene ontology was analyzed using biomart at vectorbase. the x-axis denotes the number of genes in a functional category similar to the functional categories revealed for de genes in response to chikv infection, de genes in response to sm or pm feeding belonged to the six largest categories unknown > catalytic activity > cellular process > binding > metabolic > single organism process. profound changes in transcript abundance between sm and pm fed mosquitoes and between and dpf occurred for the go categories transporter activity, response and stimulus, regulation of biological processes, localization, signaling, and single organism process. at day sm and pm rna-seq libraries shared upregulated de genes and downregulated genes. one day later, the number of commonly upregulated de genes decreased by about half to genes and that of commonly downregulated genes to just the sm libraries of day and had nine upregulated and downregulated de genes in common. pm had by far the strongest effect on de gene upregulation in the midgut. of those upregulated de genes, were commonly expressed at and dpf and both libraries shared downregulated de genes. the profound increase in the number of upregulated genes in the pm libraries when compared to sm libraries suggests that most of these genes were involved in protein digestion. common de genes among the sm and pm rna-seq libraries obtained from midguts of non-infected mosquitoes at dpf belonged to the categories catalytic activity, hydrolase activity, serine-type endopeptidase activity, metalloproteinase activity, protein binding, and oxidoreductase activity, three of which may contain proteinase genes involved in bl degradation and remodeling. comparing expression levels of genes encoding trypsins, metalloproteinases, and serine-type endopeptidases in view of their possible involvement in bl degradation and remodeling, we focused on three groups of proteinase genes: trypsins, metalloproteinases, and serine-type endopeptidases whose relative expression levels are presented in heatmaps based on fpkm values. sm or pm ingestion caused increased expression of >50% of the trypsin, metalloproteinase and serine-type endopeptidase encoding genes, including genes for two mmps, aemmp and aemmp, two adams/adamts, and two predicted serine collagenases. as a general trend, most of the trypsin, metalloproteinase, and serine-type endopeptidase genes showed upregulation in the pm rna-seq libraries in comparison to the sugarfed library. ten, seven, and four de genes encoding trypsins, serine-type endopeptidases, and metalloproteinases, respectively, were significantly upregulated in midguts of sm or pm fed mosquitoes at dpf. in addition, two serine collagenase-like genes and one glutamate carboxypeptidase encoding gene were identified. most of these genes were highly expressed in midguts and their expression levels were increased by logfc >  in sm and logfc >  in pm.fig. median-normalized expression levels of three groups of proteinase genes. data show a comparison between sugarfed controls and sm/pm samples at and days post-feeding and are presented as heatmaps for each transcript at each time point, with yellow and blue indicating high and low levels of expression, respectively logfc logarithmic fold-change, cpm counts per million, fdr false discovery rate, sm saline meal, pm protein meal, dpf days post-feeding without chikv infection mmps, adams/adamts, and collagenases are known to be involved in extracellular matrix remodeling and tissue repairing. it is possible that trypsins, other metalloproteinases, and serine-type endopeptidases are also involved in midgut bl degradation and/or remodeling either directly by taking part in proteinase activation cascades or indirectly by participating in relevant signaling processes. assessing the catalytic activities of aesp and aelt in vitro being a major component of the midgut bl, we wanted to investigate whether collagen iv abundance was changed in mosquito midguts following sm ingestion. when we conducted a time series study of collagen iv degradation in midguts of sm fed mosquitoes, it became evident that midgut collagen iv was less abundant at and h post-feeding in comparison to other time points. concurrent with midgut collagen iv degradation, generic mmp activity was significantly increased in mosquito midguts at day post-sm in comparison to the sugarfed control. no significant difference was observed between midguts of sm and sugarfed mosquitoes at dpf while bm and pm acquisition resulted in a significantly higher mmp activity. these results support the conclusion that bm, sm, and pm feeding can stimulate or support bl remodeling by affecting the abundance of collagen iv via an increased mmp activity. the results also suggest that genes encoding proteinases involved in collagen iv degradation/remodeling were expressed in midguts of sm fed mosquitoes. the potential involvement of aemmps in midgut bl remodeling has been investigated in a recent study.fig. effects of sm feeding on midgut collagen iv abundance and generic mmp activity. a western blot showing abundance of midgut collagen iv at different time points post-sm feeding using polyclonal antibodies generated against human collagen iv. β-actin was detected as a loading control. molecular masses in kda are indicated. b generic mmp activity of midgut lysates prepared from mosquitoes that had received a bm, pm, or sm at and dpf. mean values with standard deviations from three independent experiments are shown. different letters indicate significant differences based on one-way analysis of variance followed by tukey's multiple comparisons test. bm, blood meal; sm, saline meal; pm, protein meal here, we focus on the potential involvement of two putative collagenase genes, aelt and aesp in midgut bl degradation/remodeling. as revealed by qrt-pcr, both genes were significantly upregulated in midguts at dpf/pi in conjunction with bm or pm ingestion; however, only aelt was significantly upregulated in sm fed midguts at dpf/pi. as shown by western blot analysis, expression of aelt and aesp in drosophila s cells resulted in recombinant proteins with sizes of kda and ~ kda for aelt and aesp respectively, when using an anti-his-tag specific monoclonal antibody for detection. following their activation via incubation with tpck-treated bovine trypsin, both proteins produced band signals that were reduced in size by ~ kda indicating that pro-domain cleavage had occurred. in vitro, activated recombinant aelt showed strong generic mmp activity using fs- as substrate whereas raesp did not. further, addition of the recombinant aedes aegypti tissue inhibitor of metalloproteinases did not inhibit raelt activity indicating that the catalytic domain of the proteinase is structurally different from that of mmps or adams both of which can be inhibited by timps.fig. drosophila s cell expression of two putative serine collagenase genes and catalytic activities of the recombinant proteins. a western blot detection of recombinant aelt and aesp proteins using an his tag-specific monoclonal antibody. blank, s cells were transfected with non-insert containing plasmid vector; raelt and raesp s cells were transfected with plasmid vectors expressing aelt or aesp molecular masses in kda are indicated. b sds-page showing purified raelt and raesp with or without additional trypsin activation. molecular masses in kda are indicated. kinetics of raelt and raesp in vitro activities and their inhibition by raetimp. twenty ng of purified raelt or raesp were preincubated with/without tpck-treated trypsin for min at rt, then incubated with ng of raetimp, or reaction buffer at rt for h, followed by addition of fs- substrate. fluorescence intensity was measured every min. raelt-pro and raesp1-pro, pro-protein of raelt and raesp1; raelt and raesp activated raelt and raesp1. discussion bloodmeal uptake by the mosquito midgut leads to a global upregulation of endogenous genes, many of which encode proteins specifically involved in bloodmeal digestion. thus, offering mosquitoes minimal meals lacking any substantial nutritional value seemed to be a promising strategy to limit gene expression to those candidate genes responding to structural changes in the midgut due to tissue expansion rather than to genes involved in digestion. ingestion of pm, which consisted of bsa and this way was of nutritional value, caused upregulation of several thousand genes making this meal type physiologically look similar to a bloodmeal. however, in contrast to blood, bsa is a highly purified protein and free of proteinases, enzymes and growth factors, which all are highly abundant in blood sources. thus, the midgut transcriptome of pm fed mosquitoes should predominantly reflect any effects associated with feeding, protein digestion, and changes in tissue structure. replacing protein with a substance without nutritional value such as salt in sm, should strongly reduce the expression of genes involved in digestion. in agreement with this hypothesis, we only recorded de genes that were upregulated by sm at dpf, compared to genes which were upregulated in presence of pm at dpf or genes upregulated at h post-bm. the presence of chikv in sm induced expression of de genes in total at and dpi, which was similar to the number of genes observed to be upregulated in the chikv containing pm libraries. to compare with another study, denv induced expression of and de genes in midguts of ae. aegypti at and dpi via bm, respectively. most of the de genes responding to chikv challenge along with sm feeding had been previously reported to be responsive to arboviruses such as denv wnv and yfv. interestingly, ingestion of chikv along with sm or pm significantly reduced the midgut infection intensity of the virus but increased its carcass infection intensity in comparison to bm acquired chikv. these observations support the conclusion that blood may contain additional components that are advantageous for viral cell entry. our data show that chikv induced expression of numerous genes related to immunity including those encoding amp, frep and pdgr. we identified additional genes that were significantly upregulated in the presence of chikv and had previously been reported to be involved in arbovirus infection of mosquitoes. these genes encoded three synaptic vesicle proteins, unc93a, cysteine-rich venom protein, and two c-type lectins. in mammals, unc93a and sv proteins are involved in intracellular transport and their transcripts were enriched more than 40-fold during sindbis virus or denv infection of ae. aegypti. unc93a and sv seem to play a role in virus assembly and/or budding from the infected cell as revealed by transient gene silencing of sv or unc93a in mosquitoes infected with sinv. crvp proteins are known to contain a trypsin inhibitor-like domain, and therefore could be functioning as serine protease inhibitors. one of the ae. aegypti crvp genes, crvp, has recently been reported to be significantly upregulated in mosquitoes infected with the flaviviruses yfv or wnv. further, crvp expression proved to be essential during infection of ae. aegypti mosquitoes or aag cells with denv. ctls are a family of proteins possessing carbohydrate-binding activity. several mosquito ctls have been reported to be interacting with arboviruses as ligands to facilitate mosquito infection. ctls play a role in maintaining gut microbiome homeostasis by manipulating the bactericidal capacity of amps. in ae. aegypti, the galactose specific c-type lectin- increased the attachment efficiency of wnv to the cell membrane via binding to a membrane-bound protein. furthermore, mosgctl- enhanced denv infection via interacting with the e protein of the virus. besides those genes whose possible functions during arbovirus infections have been analyzed by others, we also observed numerous genes to be significantly upregulated in presence of chikv, whose functions do not appear to be directly related to virus infection. these genes, for example, include a cytochrome p gene, two lethalessential for life efl) genes, a juvenile hormone-inducible protein gene, a heat shock protein gene, and a microtubule binding protein gene. in a recent study in which we compared the vector competence of two ae. aegypti strains for chikv, we suggested that the virus exits the midgut tissue by crossing the bl surrounding the organ instead of using tracheal cells or the cardia as a conduit for dissemination. in agreement with this finding, we observed here that the abundance of collagen iv, a major component of the bl, was diminished between 18– h post-sm, which coincided with an increased generic mmp activity. in eukaryotes, bl degradation/remodeling involves the activities of numerous metalloproteinases and inhibitors and an aim of this study was to identify potential candidate genes involved in mosquito bl degradation/remodeling. in insects, several mmps such as drosophila mmp and mmp have been shown to cleave collagen in vitro. here, we also observed transcriptional upregulation of two mmps, aemmp1and aemmp and two adams/adamts in response to sm and/or pm ingestion. in addition, two putative serine collagenase genes were significantly upregulated in the midgut rna-seq libraries of sm and pm fed mosquitoes and their expression patterns were further confirmed by qrt-pcr. in insects, a serine collagenase was for the first time identified in the heel fly hypoderma lineatum with the purified enzyme being able to degrade native collagen but unable to degrade synthetic peptide substrates specific for trypsin or chymotrypsin. despite its designation as a trypsin, bacterially expressed recombinant aelt did not affect cleavage of the trypsin specific substrate dl-bapna, whereas transient silencing of aelt had no effect on the midgut infection efficiency of denv suggesting that aelt may actually lack any trypsin activity. based on structural similarities, aelt does not resemble a canonical trypsin but a serine collagenase instead. we expressed the full-length aelt in drosophila s cells and the resulting raelt showed a high level of generic mmp activity. therefore, our results strongly support the conclusion that aelt, indeed, is a serine collagenase. in contrast, recombinant aesp lacked any mmp activity, which is not surprising because in aesp his and ser in the archetypal his57-asp102-ser catalytic triad motif are changed to gln and val.  substituting bm for pm or sm did not affect the persistent infection of ae. aegypti with chikv, although virus titers in midguts were significantly lower when the virus was acquired along with sm instead of a bloodmeal. on the other hand, pm and sm did not negatively affect the dissemination rates of the virus, indicating that the protein content of the ingested meal was not a major determinant of chikv midgut escape from the midgut. instead, stretching of the midgut tissue due to meal ingestion may be the critical mechanism or trigger underlying viral midgut escape. gene expression patterns in pm fed midguts largely resembled those previously observed for bloodfed midguts. the presence of chikv in any of the ingested meals had relatively minor effects on the overall gene expression profiles in midguts, as only genes were being differentially expressed, of which were related to immunity. in comparison to pm feeding resulting in the upregulation of genes at day and sm feeding substantially reduced the gene expression profile in the mosquito midgut with only genes being significantly upregulated, many of which belonged to the functional category catalytic activity. these genes included seven serine-type endopeptidases, ten trypsins, two serine collagenases, four m zinc metalloproteases, and a glutamate carboxypeptidase, which could be involved in the midgut escape of chikv when traversing the midgut bl. the recombinant serine collagenase, aelt, showed strong generic mmp activity in vitro. 
to understand the transcriptomic basis of midgut escape and to reveal genes involved in the process, we conducted a comparative transcriptomic analysis of midgut samples from mosquitoes which had received a saline meal or a protein meal containing chikv. aedes aegyptichikungunya virusrna-seqprotein mealsaline mealimmunity- related genemidgut escape barriercollagen iv degradationbasal laminaserine collagenasehttp://dx.doi.org/ /100000060national institute of allergy and infectious diseasesr01ai091972franz alexander w. e. http://dx.doi.org/ /100007165university of missouristart-up fundsfranz alexander w. e. issue-copyright-statement© the author 2017 chikv which was orally acquired by a mosquito along with a sm or pm productively infected the midgut epithelium and disseminated to secondary tissues. by substituting a bloodmeal for sm, we identified midgut-expressed genes not involved in blood or protein digestion. the presence of chikv in any of the ingested meals had relatively minor effects on the overall gene expression profiles in midguts. the mosquito aedes aegypti is the primary vector for medically important arthropod-borne viruses, including chikungunya virus. by comparison, pm feeding induced de genes at day and genes at day twenty-three de genes encoding trypsins, metalloproteinases, and serine-type endopeptidases were significantly upregulated in midguts of mosquitoes at day following sm or pm ingestion. these included genes coding for trypsins, metalloproteinases, and serine-type endopeptidases, which could be involved in facilitating midgut escape for arboviruses in ae. in vitro, recombinant aelt showed strong matrix metalloproteinase activity whereas recombinant aesp did not. following oral acquisition, an arbovirus has to persistently infect several organs in the mosquito before becoming transmissible to another vertebrate host.
 the mosquito aedes aegypti is the primary vector for medically important arthropod-borne viruses, including chikungunya virus. following oral acquisition, an arbovirus has to persistently infect several organs in the mosquito before becoming transmissible to another vertebrate host. a major obstacle an arbovirus has to overcome during its infection cycle inside the mosquito is the midgut escape barrier, representing the exit mechanism arboviruses utilize when disseminating from the midgut. to understand the transcriptomic basis of midgut escape and to reveal genes involved in the process, we conducted a comparative transcriptomic analysis of midgut samples from mosquitoes which had received a saline meal or a protein meal containing chikv.  chikv which was orally acquired by a mosquito along with a sm or pm productively infected the midgut epithelium and disseminated to secondary tissues. a total of rna-seq libraries from midguts of mosquitoes that had received pm or sm containing chikv at and days post-feeding were generated and sequenced. fewer than genes responded differentially to the presence of chikv in midguts of mosquitoes that had acquired the virus along with sm or pm. sm feeding induced differential expression of genes at day and genes at day when compared to midguts of sugarfed mosquitoes. by comparison, pm feeding induced de genes at day and genes at day twenty-three de genes encoding trypsins, metalloproteinases, and serine-type endopeptidases were significantly upregulated in midguts of mosquitoes at day following sm or pm ingestion. two of these genes were ae. aegypti late trypsin and serine collagenase precursor. in vitro, recombinant aelt showed strong matrix metalloproteinase activity whereas recombinant aesp did not.  by substituting a bloodmeal for sm, we identified midgut-expressed genes not involved in blood or protein digestion. these included genes coding for trypsins, metalloproteinases, and serine-type endopeptidases, which could be involved in facilitating midgut escape for arboviruses in ae. aegypti. the presence of chikv in any of the ingested meals had relatively minor effects on the overall gene expression profiles in midguts. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords aedes aegyptichikungunya virusrna-seqprotein mealsaline mealimmunity- related genemidgut escape barriercollagen iv degradationbasal laminaserine collagenasehttp://dx.doi.org/ /100000060national institute of allergy and infectious diseasesr01ai091972franz alexander w. e. http://dx.doi.org/ /100007165university of missouristart-up fundsfranz alexander w. e. issue-copyright-statement© the author 2017 