The transcriptional response in mosquitoes distinguishes between fungi and bacteria but not Gram types.

Mosquitoes are prolific vectors of human pathogens, therefore a clear and accurate understanding of the organization of their antimicrobial defenses is crucial for informing the development of transmission control strategies. The canonical infection response in insects, as described in the insect model Drosophila melanogaster, is pathogen type-dependent, with distinct stereotypical responses to Gram-negative bacteria and Gram-positive bacteria/fungi mediated by the activation of the Imd and Toll pathways, respectively. To determine whether this pathogen-specific discrimination is shared by mosquitoes, we used RNAseq to capture the genome-wide transcriptional response of Aedes aegypti and Anopheles gambiae (s.l.) to systemic infection with Gram-negative bacteria, Gram-positive bacteria, yeasts, and filamentous fungi, as well as challenge with heat-killed Gram-negative, Gram-positive, and fungal pathogens. From the resulting data, we found that Ae. aegypti and An. gambiae both mount a core response to all categories of infection, and this response is highly conserved between the two species with respect to both function and orthology. When we compared the transcriptomes of mosquitoes infected with different types of bacteria, we observed that the intensity of the transcriptional response was correlated with both the virulence and growth rate of the infecting pathogen. Exhaustive comparisons of the transcriptomes of Gram-negative-challenged versus Gram-positive-challenged mosquitoes yielded no difference in either species. In Ae. aegypti, however, we identified transcriptional signatures specific to bacterial infection and to fungal infection. The bacterial infection response was dominated by the expression of defensins and cecropins, while the fungal infection response included the disproportionate upregulation of an uncharacterized family of glycine-rich proteins. These signatures were also observed in Ae. aegypti challenged with heat-killed bacteria and fungi, indicating that this species can discriminate between molecular patterns that are specific to bacteria and to fungi.

GeCoNet-Tool: a software package for gene co-expression network construction and analysis.

BACKGROUND: Network analysis is a powerful tool for studying gene regulation and identifying biological processes associated with gene function. However, constructing gene co-expression networks can be a challenging task, particularly when dealing with a large number of missing values. RESULTS: We introduce GeCoNet-Tool, an integrated gene co-expression network construction and analysis tool. The tool comprises two main parts: network construction and network analysis. In the network construction part, GeCoNet-Tool offers users various options for processing gene co-expression data derived from diverse technologies. The output of the tool is an edge list with the option of weights associated with each link. In network analysis part, the user can produce a table that includes several network properties such as communities, cores, and centrality measures. With GeCoNet-Tool, users can explore and gain insights into the complex interactions between genes.

A global [Formula: see text] gene co-expression network constructed from hundreds of experimental conditions with missing values.

BACKGROUND: Gene co-expression networks (GCNs) can be used to determine gene regulation and attribute gene function to biological processes. Different high throughput technologies, including one and two-channel microarrays and RNA-sequencing, allow evaluating thousands of gene expression data simultaneously, but these methodologies provide results that cannot be directly compared. Thus, it is complex to analyze co-expression relations between genes, especially when there are missing values arising for experimental reasons. Networks are a helpful tool for studying gene co-expression, where nodes represent genes and edges represent co-expression of pairs of genes. RESULTS: In this paper, we establish a method for constructing a gene co-expression network for the Anopheles gambiae transcriptome from 257 unique studies obtained with different methodologies and experimental designs. We introduce the sliding threshold approach to select node pairs with high Pearson correlation coefficients. The resulting network, which we name AgGCN1.0, is robust to random removal of conditions and has similar characteristics to small-world and scale-free networks. Analysis of network sub-graphs revealed that the core is largely comprised of genes that encode components of the mitochondrial respiratory chain and the ribosome, while different communities are enriched for genes involved in distinct biological processes. CONCLUSION: Analysis of the network reveals that both the architecture of the core sub-network and the network communities are based on gene function, supporting the power of the proposed method for GCN construction. Application of network science methodology reveals that the overall network structure is driven to maximize the integration of essential cellular functions, possibly allowing the flexibility to add novel functions.

Serpins in arthropod biology.

Serpins are the largest known family of serine proteinase inhibitors and perform a variety of physiological functions in arthropods. Herein, we review the field of serpins in arthropod biology, providing an overview of current knowledge and topics of interest. Serpins regulate insect innate immunity via inhibition of serine proteinase cascades that initiate immune responses such as melanization and antimicrobial peptide production. In addition, several serpins with anti-pathogen activity are expressed as acute-phase serpins in insects upon infection. Parasitoid wasps can downregulate host serpin expression to modulate the host immune system. In addition, examples of serpin activity in development and reproduction in Drosophila have also been discovered. Serpins also function in host-pathogen interactions beyond immunity as constituents of venom in parasitoid wasps and saliva of blood-feeding ticks and mosquitoes. These serpins have distinct effects on immunosuppression and anticoagulation and are of interest for vaccine development. Lastly, the known structures of arthropod serpins are discussed, which represent the serpin inhibitory mechanism and provide a detailed overview of the process.

Mosquito Immunobiology: The Intersection of Vector Health and Vector Competence.

As holometabolous insects that occupy distinct aquatic and terrestrial environments in larval and adult stages and utilize hematophagy for nutrient acquisition, mosquitoes are subjected to a wide variety of symbiotic interactions. Indeed, mosquitoes play host to endosymbiotic, entomopathogenic, and mosquito-borne organisms, including protozoa, viruses, bacteria, fungi, fungal-like organisms, and metazoans, all of which trigger and shape innate infection-response capacity. Depending on the infection or interaction, the mosquito may employ, for example, cellular and humoral immune effectors for septic infections in the hemocoel, humoral infection responses in the midgut lumen, and RNA interference and programmed cell death for intracellular pathogens. These responses often function in concert, regardless of the infection type, and provide a robust front to combat infection. Mosquito-borne pathogens and entomopathogens overcome these immune responses, employing avoidance or suppression strategies. Burgeoning methodologies are capitalizing on this concerted deployment of immune responses to control mosquito-borne disease.

Structural and inhibitory effects of hinge loop mutagenesis in serpin-2 from the malaria vector Anopheles gambiae.

Serpin-2 (SRPN2) is a key negative regulator of the melanization response in the malaria vector Anopheles gambiae. SRPN2 irreversibly inhibits clip domain serine proteinase 9 (CLIPB9), which functions in a serine proteinase cascade culminating in the activation of prophenoloxidase and melanization. Silencing of SRPN2 in A. gambiae results in spontaneous melanization and decreased life span and is therefore a promising target for vector control. The previously determined structure of SRPN2 revealed a partial insertion of the hinge region of the reactive center loop (RCL) into ß sheet A. This partial hinge insertion participates in heparin-linked activation in other serpins, notably antithrombin III. SRPN2 does not contain a heparin binding site, and any possible mechanistic function of the hinge insertion was previously unknown. To investigate the function of the SRPN2 hinge insertion, we developed three SRPN2 variants in which the hinge regions are either constitutively expelled or inserted and analyzed their structure, thermostability, and inhibitory activity. We determined that constitutive hinge expulsion resulted in a 2.7-fold increase in the rate of CLIPB9Xa inhibition, which is significantly lower than previous observations of allosteric serpin activation. Furthermore, we determined that stable insertion of the hinge region did not appreciably decrease the accessibility of the RCL to CLIPB9. Together, these results indicate that the partial hinge insertion in SRPN2 does not participate in the allosteric activation observed in other serpins and instead represents a molecular trade-off between RCL accessibility and efficient formation of an inhibitory complex with the cognate proteinase.

Blood feeding induces hemocyte proliferation and activation in the African malaria mosquito, Anopheles gambiae Giles.

Malaria is a global public health problem, especially in sub-Saharan Africa, where the mosquito Anopheles gambiae Giles serves as the major vector for the protozoan Plasmodium falciparum Welch. One determinant of malaria vector competence is the mosquito's immune system. Hemocytes are a critical component as they produce soluble immune factors that either support or prevent malaria parasite development. However, despite their importance in vector competence, understanding of their basic biology is just developing. Applying novel technologies to the study of mosquito hemocytes, we investigated the effect of blood meal on hemocyte population dynamics, DNA replication and cell cycle progression. In contrast to prevailing published work, the data presented here demonstrate that hemocytes in adult mosquitoes continue to undergo low basal levels of replication. In addition, blood ingestion caused significant changes in hemocytes within 24 h. Hemocytes displayed an increase in cell number, size, granularity and Ras-MAPK signaling as well as altered cell surface moieties. As these changes are well-known markers of immune cell activation in mammals and Drosophila melanogaster Meigen, we further investigated whether a blood meal changes the expression of hemocyte-derived immune factors. Indeed, hemocytes 24 h post-blood meal displayed higher levels of critical components of the complement and melanization immune reactions in mosquitoes. Taken together, this study demonstrates that the normal physiological process of a blood meal activates the innate immune response in mosquitoes. This process is likely in part regulated by Ras-MAPK signaling, highlighting a novel mechanistic link between blood feeding and immunity.

The expanded immunoregulatory protease network in mosquitoes is governed by gene co-expression.

Serine protease cascades regulate key innate immune responses. In mosquitoes, these cascades involve clip-domain serine proteases and their non-catalytic homologs (CLIPs), forming a complex network whose make-up and structural organization is not fully understood. This study assessed the impact of 85 CLIPs on humoral immunity in Anopheles gambiae. By coupling RNAi with assays measuring antimicrobial activity and melanization, we identified 27 CLIPs as immunoregulators that together form two distinct subnetworks. CLIPs regulating antimicrobial activity were found to control infection resistance, as knockdowns reduced bacterial load and improved survival. Furthermore, our analysis of CLIP gene expression unveiled a novel immunoregulatory mechanism reliant on protease baseline co-expression rather than infection-induced upregulation. These findings underscore that despite its complexity mosquito immune regulation may be targeted for malaria interventions.

Insight into the structural hierarchy of the protease cascade that regulates the mosquito melanization response.

Serine protease cascades regulate important insect immune responses, including melanization and Toll pathway activation. In the context of melanization, central components of these cascades are clip domain serine proteases (CLIPs) including the catalytic, clip domain serine proteases (cSPs) and their non-catalytic homologs (cSPHs). Here, we define partially the structural hierarchy of An. gambiae cSPs of the CLIPB family, central players in melanization, and characterize their relative contributions to bacterial melanization and to mosquito susceptibility to bacterial infections. Using in vivo genetic analysis we show that the protease cascade branches downstream of the cSPs CLIPB4 and CLIPB17 into two branches one converging on CLIPB10 and the second on CLIPB8. We also show that the contribution of key cSPHs to melanization in vivo in response to diverse microbial challenges is more significant than any of the individual cSPs, possibly due to partial functional redundancy among the latter. Interestingly, we show that the key cSPH CLIPA8 which is essential for the efficient activation cleavage of CLIPBs in vivo is efficiently cleaved itself by several CLIPBs in vitro, suggesting that cSPs and cSPHs regulate signal amplification and propagation in melanization cascades by providing positive reinforcement upstream and downstream of each other.

A zone-of-inhibition assay to screen for humoral antimicrobial activity in mosquito hemolymph.

In insects, antibacterial immunity largely depends on the activation of downstream signaling and effector responses, leading to the synthesis and secretion of soluble effector molecules, such as antimicrobial peptides (AMPs). AMPs are acute infection response peptides secreted into the hemolymph upon bacterial stimulation. The transcription of innate immunity genes encoding for AMPs is highly dependent on several signaling cascade pathways, such as the Toll pathway. In the African malaria mosquito, Anopheles gambiae, AMPs hold a special interest as their upregulation have been shown to limit the growth of malaria parasites, bacteria, and fungi. Most of the current knowledge on the regulation of insect AMPs in microbial infection have been obtained from Drosophila. However, largely due to the lack of convenient assays, the regulation of antimicrobial activity in mosquito hemolymph is still not completely understood. In this study, we report a zone of inhibition assay to identify the contribution of AMPs and components of the Toll pathway to the antimicrobial activity of A. gambiae hemolymph. As a proof of principle, we demonstrate that Micrococcus luteus challenge induces antimicrobial activity in the adult female mosquito hemolymph, which is largely dependent on defensin 1. Moreover, by using RNAi to silence Cactus, REL1, and MyD88, we showed that Cactus kd induces antimicrobial activity in the mosquito hemolymph, whereas the antimicrobial activity in REL1 kd and MyD88 kd is reduced after challenge. Finally, while injection itself is not sufficient to induce antimicrobial activity, our results show that it primes the response to bacterial challenge. Our study provides information that increases our knowledge of the regulation of antimicrobial activity in response to microbial infections in mosquitoes. Furthermore, this assay represents an ex vivo medium throughput assay that can be used to determine the upstream regulatory elements of antimicrobial activity in A. gambiae hemolymph.

The Potential of a New Beauveria bassiana Isolate for Mosquito Larval Control.

The African malaria mosquito, Anopheles gambiae Giles (Diptera: Culicidae), and the Asian tiger mosquito, Aedes albopictus Skuse (Diptera: Culicidae) are of public health concern due to their ability to transmit disease-causing parasites and pathogens. Current mosquito control strategies to prevent vector-borne diseases rely mainly on the use of chemicals. However, insecticide resistance in mosquito populations necessitates alternative control measures, including biologicals such as entomopathogenic fungi. Here we report the impact of a new Beauveria bassiana (Balsamo) Vuillemin (Hyprocreales: Cordycipitaeceae) isolate, isolated from field-collected Ae. albopictus larvae on mosquito survival and development. Larval infection bioassays using three B. bassiana conidial concentrations were performed on the second and third larval instars of An. gambiae and Ae. albopictus mosquitoes. Larvae were monitored daily for survival and development to pupae and adults. Our results show that B. bassiana MHK was more effective in killing An. gambiae than Ae. albopictus larvae. We further observed delays in development to pupae and adults in both mosquito species exposed the varying concentrations of B. bassiana as compared to the water control. In addition, larval exposure to B. bassiana reduced adult male and female survival in both mosquito species, further contributing to mosquito population control. Thus, this study identifies a new B. bassiana isolate as a possible biological control agent of two mosquito species of public health concern, increasing the arsenal for integrated mosquito control.

Insight into the structural hierarchy of the protease cascade that regulates the mosquito melanization response.

Serine protease cascades regulate important insect immune responses, including melanization and Toll pathway activation. In the context of melanization, central components of these cascades are clip domain serine proteases (CLIPs) including the catalytic, clip domain serine proteases (cSPs) and their non-catalytic homologs (cSPHs). Here, we define partially the structural hierarchy of An. gambiae cSPs of the CLIPB family, central players in melanization, and characterize their relative contributions to bacterial melanization and to mosquito susceptibility to bacterial infections. Using in vivo genetic analysis we show that the protease cascade branches downstream of the cSPs CLIPB4 and CLIPB17 into two branches one converging on CLIPB10 and the second on CLIPB8. We also show that the contribution of key cSPHs to melanization in vivo in response to diverse microbial challenges is more significant than any of the individual cSPs, possibly due to partial functional redundancy among the latter. Interestingly, we show that the key cSPH CLIPA8 which is essential for the efficient activation cleavage of CLIPBs in vivo is efficiently cleaved itself by several CLIPBs in vitro, suggesting that cSPs and cSPHs regulate signal amplification and propagation in melanization cascades by providing positive reinforcement upstream and downstream of each other.

The transcriptional response in mosquitoes distinguishes between fungi and bacteria but not Gram types.

Mosquitoes are prolific vectors of human pathogens; a clear and accurate understanding of the organization of their antimicrobial defenses is crucial for informing the development of transmission control strategies. The canonical infection response in insects, as described in the insect model Drosophila melanogaster , is pathogen type-dependent, with distinct stereotypical responses to Gram-negative bacteria and Gram-positive bacteria/fungi mediated by the activation of the Imd and Toll pathways, respectively. To determine whether this pathogen-specific discrimination is shared by mosquitoes, we used RNAseq to capture the genome-wide transcriptional response of Aedes aegypti and Anopheles gambiae ( s.l. ) to systemic infection with Gram-negative bacteria, Gram-positive bacteria, yeasts, and filamentous fungi, as well as challenge with heat-killed Gram-negative, Gram-positive, and fungal pathogens. From the resulting data, we found that Ae. aegypti and An. gambiae both mount a core response to all categories of infection, and this response is highly conserved between the two species with respect to both function and orthology. When we compared the transcriptomes of mosquitoes infected with different types of bacteria, we observed that the intensity of the transcriptional response was correlated with both the virulence and growth rate of the infecting pathogen. Exhaustive comparisons of the transcriptomes of Gram-negative-challenged versus Gram-positive-challenged mosquitoes yielded no difference in either species. In Ae. aegypti , however, we identified transcriptional signatures specific to bacterial infection and to fungal infection. The bacterial infection response was dominated by the expression of defensins and cecropins, while the fungal infection response included the disproportionate upregulation of an uncharacterized family of glycine-rich proteins. These signatures were also observed in Ae. aegypti challenged with heat-killed bacteria and fungi, indicating that this species can discriminate between molecular patterns that are specific to bacteria and to fungi.

CLIPB4 is a central node in the protease network that regulates humoral immunity in Anopheles gambiae mosquitoes.

Insect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist of clip domain serine proteases (cSPs) and/or their non-catalytic homologs (cSPHs), which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquito Anopheles gambiae as a central node in this protease network. CLIPB4 is tightly co-expressed with SRPN2 and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network in An. gambiae, by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.

CLIPB4 Is a Central Node in the Protease Network that Regulates Humoral Immunity in Anopheles gambiae Mosquitoes.

Insect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist of clip-domain serine proteases (cSPs) and/or their non-catalytic homologs, which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquito Anopheles gambiae as a central node in this protease network. CLIPB4 is tightly co-expressed with SRPN2 and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network in An. gambiae, by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.

Characterization of a regulatory unit that controls melanization and affects longevity of mosquitoes.

Melanization is an innate immune response in arthropods that encapsulates and kills invading pathogens. One of its rate-limiting steps is the activation of prophenoloxidase (PPO), which is controlled by an extracellular proteinase cascade and serpin inhibitors. The molecular composition of this system is largely unknown in mosquitoes with the exception of serpin-2 (SRPN2), which was previously identified as a key negative regulator of melanization. Using reverse genetic and biochemical techniques, we identified the Anopheles gambiae clip-serine proteinase CLIPB9 as a PPO-activating proteinase, which is inhibited by SRPN2. Double knockdown of SRPN2 and CLIPB9 reversed the pleiotrophic phenotype induced by SRPN2 silencing. This study identifies the first inhibitory serpin-serine proteinase pair in mosquitoes and defines a regulatory unit of melanization. Additionally, the interaction of CLIPB9 and SRPN2 affects the life span of adult female mosquitoes and therefore constitutes a well-defined potential molecular target for novel late-life acting insecticides.

Discovery of Plasmodium modulators by genome-wide analysis of circulating hemocytes in Anopheles gambiae.

Insect hemocytes mediate important cellular immune responses including phagocytosis and encapsulation and also secrete immune factors such as opsonins, melanization factors, and antimicrobial peptides. However, the molecular composition of these important immune cells has not been elucidated in depth, because of their scarcity in the circulating hemolymph, their adhesion to multiple tissues and the lack of primary culture methods to produce sufficient material for a genome-wide analysis. In this study, we report a genome-wide molecular characterization of circulating hemocytes collected from the hemolymph of adult female Anopheles gambiae mosquitoes--the major mosquito vector of human malaria in subSaharan Africa. Their molecular profile identified 1,485 transcripts with enriched expression in these cells, and many of these genes belong to innate immune gene families. This hemocyte-specific transcriptome is compared to those of Drosophila melanogaster and two other mosquitoes, Aedes aegypti and Armigeres subalbatus. We report the identification of two genes as ubiquitous hemocyte markers and several others as hemocyte subpopulation markers. We assess, via an RNAi screen, the roles in development of Plasmodium berghei of 63 genes expressed in hemocytes and provide a molecular comparison of the transcriptome of these cells during malaria infection.

Feature learning and network structure from noisy node activity data.

In the studies of network structures, much attention has been devoted to developing approaches to reconstruct networks and predict missing links when edge-related information is given. However, such approaches are not applicable when we are only given noisy node activity data with missing values. This work presents an unsupervised learning framework to learn node vectors and construct networks from such node activity data. First, we design a scheme to generate random node sequences from node context sets, which are generated from node activity data. Then, a three-layer neural network is adopted training the node sequences to obtain node vectors, which allow us to construct networks and capture nodes with synergistic roles. Furthermore, we present an entropy-based approach to select the most meaningful neighbors for each node in the resulting network. Finally, the effectiveness of the method is validated through both synthetic and real data.

Patterns of fungal community assembly across two Culex mosquito species.

In the aquatic environment, mosquito larvae encounter bacteria and fungi that assemble into bacterial and fungal communities. The composition and impact of mosquito-associated bacterial community has been reported across larvae of various mosquito species. However, knowledge on the composition of mosquito-associated fungal communities and the drivers of their assembly remain largely unclear, particularly across mosquito species. In this study, we used high throughput sequencing of the fungal Internal transcribed spacer 2 (ITS2) metabarcode marker to identify fungal operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) associated with field-collected Culex restuans and Culex pipiens larvae and their breeding water. Our analyses identified diverse fungal communities across larval breeding sites collected on a fine geographic scale. Our data show that the larval breeding site is the major determinant of fungal community assembly in these mosquito species. We also identified distinct fungal communities in guts and carcasses within each species. However, these tissue-specific patterns were less evident in Cx. restuans than in Cx. pipiens larvae. The broad ecological patterns of fungal community assembly in mosquito larvae did not vary between OTU and ASV analyses. Together, this study provides the first insight into the fungal community composition and diversity in field collected Cx. restuans and Cx. pipiens larvae using OTUs and ASVs. While these findings largely recapitulate our previous analyses in Aedes albopictus larvae, we report minor differences in tissue-specific fungal community assembly in Cx. restuans larvae. Our results suggest that while the fungal community assembly in mosquito larvae may be generalized across mosquito species, variation in larval feeding behavior may impact fungal community assembly in the guts of mosquito larvae.

Evaluating RNAlater® as a preservative for using near-infrared spectroscopy to predict Anopheles gambiae age and species.

BACKGROUND: Mosquito age and species identification is a crucial determinant of the efficacy of vector control programmes. Near-infrared spectroscopy (NIRS) has previously been applied successfully to rapidly, non-destructively, and simultaneously determine the age and species of freshly anesthetized African malaria vectors from the Anopheles gambiae s.l. species complex: An. gambiae s. s. and Anopheles arabiensis. However, this has only been achieved on freshly-collected specimens and future applications will require samples to be preserved between field collections and scanning by NIRS. In this study, a sample preservation method (RNAlater(®)) was evaluated for mosquito age and species identification by NIRS against scans of fresh samples. METHODS: Two strains of An. gambiae s.s. (CDC and G3) and two strains of An. arabiensis (Dongola, KGB) were reared in the laboratory while the third strain of An. arabiensis (Ifakara) was reared in a semi-field system. All mosquitoes were scanned when fresh and rescanned after preservation in RNAlater(®) for several weeks. Age and species identification was determined using a cross-validation. RESULTS: The mean accuracy obtained for predicting the age of young (<7 days) or old (≥ 7 days) of all fresh (n = 633) and all preserved (n = 691) mosquito samples using the cross-validation technique was 83% and 90%, respectively. For species identification, accuracies were 82% for fresh against 80% for RNAlater(®) preserved. For both analyses, preserving mosquitoes in RNAlater(®) was associated with a highly significant reduction in the likelihood of a misclassification of mosquitoes as young or old using NIRS. Important to note is that the costs for preserving mosquito specimens with RNAlater(®) ranges from 3-13 cents per insect depending on the size of the tube used and the number of specimens pooled in one tube. CONCLUSION: RNAlater(®) can be used to preserve mosquitoes for subsequent scanning and analysis by NIRS to determine their age and species with minimal costs and with accuracy similar to that achieved from fresh insects. Cold storage availability allows samples to be stored longer than a week after field collection. Further study to develop robust calibrations applicable to other strains from diverse ecological settings is recommended.