Malaria is among the leading causes of childhood mortality in Guinea-Bissau, comprising 18% of mortality of children less than five years of age as of 2010 ( WHO, 2010). However, the human malaria incidence rate in Guinea Bissau varies considerably from year to year with a general decrease in recent years to about 3 children (<5 yrs of age) per thousand in some locations ( Ursing et al., 2014). Plasmodium falciparum predominates, causing 98% cases, followed by a few cases of Plasmodium malariae and Plasmodium ovale. Mixed infections of P. malariae, and to a lesser extent P. ovale, have been recorded but appear to be rare and highly variable in both Guinea-Bissau ( Snounou et al., 1993) and neighboring Senegal ( Fontenille et al., 1997a; Fontenille et al., 1997b).

Limited research has been conducted on the vectors and malaria parasite infection rates in Guinea-Bissau populations of Anopheles species in general and there is no data on comparative infection rates between A. gambiae and A. coluzzii and members of the A. gambiae complex. Variability is also high among the Anopheles spp. implicated as vectors in this region of West Africa in terms of both their temporal population dynamics as well as species composition among study sites ( Carnevale et al., 2010; Fontenille et al., 1997a; Jaenson et al., 1994; Snounou et al., 1993).

Here we present much needed data on P. falciparum infection of Anopheles spp. specimens collected from inside and around associated human habitations at eight sites in Guinea-Bissau ( Table 1).

Mosquitoes were collected by mouth aspiration from both the island and inland areas of Guinea-Bissau ( Figure 1) in 2009 between October and November, which corresponds with the time of year previously observed to have the highest infection rate in Anopheles species ( Jaenson et al., 1994). All mosquitoes were collected indoors with a single exception; samples in Ponta Anabaca were opportunistically collected outside while host-seeking at about 18:00hr. Each mosquito was dissected and the head and thorax were preserved in 100% ethanol for subsequent ELISA analysis. The remainder of each mosquito was preserved in 70% ethanol for genomic DNA extraction using the DNeasy extraction kit (Qiagen). Species determination of mosquitoes from the A. gambiae complex was made with the combination of species diagnostic assays ( Fanello et al., 2002; Favia et al., 2001; Santolamazza et al., 2008; Scott et al., 1993) and a divergence island SNP (DIS) genotyping assay ( Lee et al., 2014a) while other species were identified by morphology.

For the Scott PCR ( Scott et al., 1993) and the Fanello RFLP ( Fanello et al., 2002), we used four primers (UN [5'-GTG TGG CCC TTC CTC GAT GT-3'], GA [5'-CTG GTT TGG TCG GCA CGT TT-3'], ME [5'-TGA CCA ACC CAC TCC CTT GA-3'] and AR [5'-AAG TGT CCT TCT CCA TCC TA-3']). We excluded QD primer ( Scott et al., 1993) because our study site is well outside of the geographic range of this species (East Africa). A 25 µL PCR reaction containing 1X GeneAmp PCR Buffer (Applied Biosystems), 1mM MgCl ₂, 0.2mM of each dNTP, 0.12 µM of each primer and 0.05U AmpliTaq DNA polymerase (Applied Biosystems) was carried out for each individual. Scott PCR products were digested using Hha1 enzyme (New England Biosystems) following the protocol stated in ( Fanello et al., 2002). Thermocycler conditions were 95°C for 5 min followed by thirty-five cycles of 95°C for 45 s, 50°C for 30 s and 72°C for 45 s, with a final elongation at 72°C for 7 min, and a 4°C hold.

For the Favia PCR ( Favia et al., 2001), we used four primers (R5 [5'-GCC AAT CCG AGC TGA TAG CGC-3'], R3 [5'-CGA ATT CTA GGG AGC TCC AG-3'], Mopint [5'-GCC CCT TCC TCG ATG GCA T-3'] and B/S int [5'-ACC AAG ATG GTT CGT TGC-3']. A 25 µL PCR reaction containing 1X PCR Buffer (Applied Biosystems), 1.5mM MgCl ₂, 0.2mM of each dNTP, 0.2 µM of primer R5, 0.2 µM of primer R3, 0.16 µM of primer Mopint, 0.1 µM of primer B/S int and 0.02U DNA polymerase AmpliTaq (Applied Biosystems) was carried out for each individual. Thermocycler conditions were 95°C for 5 min followed by thirty-five cycles of 95°C for 30 s, 64°C for 30 s and 72°C for 30 s, with a final elongation at 72°C for 7 min, and a 4°C hold.

For the SINEX PCR ( Santolamazza et al., 2008), we used S200 X6.1 forward [5'-TCG CCT TAG ACC TTG CGT TA-3'] and reverse [5'-CGC TTC AAG AAT TCG AGA TAC-3'] primers. A 25 µL PCR reaction containing 1X PCR Buffer (Applied Biosystems), 2mM MgCl ₂, 0.4mM of each dNTP, 0.2 µM of each primer and 0.1U DNA polymerase AmpliTaq (Applied Biosystems) was carried out for each individual. Thermocycler conditions were 95°C for 5 min followed by thirty-five cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s, with a final elongation at 72°C for 10 min, and a 4°C hold.

The resulting PCR products were analyzed on a Qiaxcel capillary electrophoresis instrument (Qiagen) using a DNA Screening Cartridge (Qiagen).

For DIS genotyping, we used Sequenom iPLEX Gold Genotyping Reagent Set (Catalog number: Sequenom 10158) and ran on the MassArray (Sequenom) mass spectrometer at the UC Davis Veterinary Genetics Laboratory. A mosquito was considered a hybrid if at least 5 out of 7 DIS on the X chromosome were in a heterozygous state.

P. falciparum infection was determined by enzyme linked immunosorbent assay (ELISA) of circumsporozoite protein (CSP) ( Burkot et al., 1984; Wirtz et al., 1987) from the head and thorax of mosquito specimens in an attempt to capture the parts of the mosquito that would indicate that they were infective. All chemicals except for substrate solutions (Item 5 on page 5 of the supplemental ELSA protocol document) were ordered from Sigma-Aldrich. Monoclonal antibodies (capture and conjugate) were obtained from Kirkegaard & Perry Laboratories. P. falciparum sporozoite protein for positive controls was ordered from the Centers for Disease Control and Prevention (CDC). We followed the Sporozoite ELISA directions provided by the CDC (Sep, 2009 version) with a few modifications (see supplemental document for the modified ELISA protocol). Samples were considered positive if absorbance values were three or more standard deviations from the negative control samples (99% CI) on each ELISA plate ( Beier et al., 1987; De Arruda et al., 2004).

The results of the ELISA were analyzed for both CSP concentration, adjusted for plate-to-plate variation, with an analysis of variance and for a binary outcome using a χ ² test implemented in SPSS 16.0 ( SPSS, 2007). The data were analyzed for differences between species and among collection sites, using a G-test implemented in Deducer library under R software ( http://www.r-project.org/). A Confidence interval (CI) was calculated using adjusted Wald confidence intervals using an online calculator ( www.measuringu.com/wald.htm). Mosquito species. P. falciparum infection state and CSP concentration for each individual are provided in Dataset 1.

Four species, A. coluzzii, A. gambiae, A. melas, A. pharoensis were collected during sampling. A number of A. coluzzii x A. gambiae hybrids were also collected. All mosquitoes were collected indoors with a single exception; samples in Ponta Anabaca were opportunistically collected outside of a human habitation while apparently host-seeking immediately after sunset at about 18:00 hr, which is earlier than reported observations for members of the A. gambiae complex in The Gambia (West Africa) ( Lindsay et al., 1989; Snow et al., 1988). All species were collected at multiple sites except A. pharoensis, which was only collected at the more inland site of Bambadinca. A. pharoensis is not generally considered a significant vector in West Africa but the distribution observed in this study matches the previously observed pattern in Senegal ( Carrara et al., 1990). Anopheles arabiensis was absent from collections.

No significant differences were observed for CSP concentration or in the analysis of positive samples with χ ². This is probably due to the variation in the distribution of vector species and P. falciparum in the environment at the time of sampling. Table 1 presents CSP rate data and the total number of each individual species collected at each site.

The percentage of P. falciparum positive samples from members of the A. gambiae species complex observed in this study (overall 10.2%; CI:7.45-13.6%) were similar to earlier studies in other regions in Guinea-Bissau (12.0% ( Snounou et al., 1993) and 9.6–12.4% ( Jaenson et al., 1994)). The overall CSP positive rate for A. gambiae was 12.6% (CI 8.88-17.6%) and 11.1% (CI:1.86-34.1%) for A. melas. Previously published CSP positive rates for A. gambiae s.s. (= A. gambiae and A. coluzzii) range between 2.24% in Guinea ( Carnevale et al., 2010) to 9.6% in Guinea-Bissau ( Jaenson et al., 1994). Earlier studies when individual species within the A. gambiae complex were not identified, infection rate of A. gambiae s.l. ranged from as high of 17.73% in the eastern regions of The Gambia ( Thomson et al., 1994) to 12% in Guinea-Bissau ( Jaenson et al., 1994; Snounou et al., 1993). The CSP positive rate was significantly higher in A. gambiae (12.6%) than A. coluzzii (4.3%) (Wilcoxon rank sum test P-value=0.0384). This is consistent with the earlier study in Senegal ( Ndiath et al., 2011) but differs from a recent survey conducted in Mali ( Fryxell et al., 2012). The study site in Senegal located in the village of Dielmo (13°43'N, 16°24'W) ( Ndiath et al., 2012) was geographically closer (200km) than Mali sites (>800km) to our collection sites in Guinea-Bissau. The Senegal study site at Dielmo and nine of our study sites were proximal (<50km) to the Atlantic Ocean, while Mali is a land-locked country at least 500km away from the Atlantic Ocean. Therefore, the discrepancy among studies may be due to climatic and environmental pressure on the different genetic backgrounds of A. gambiae observed in this area of West Africa ( Lee et al., 2013). More robust sampling over a larger number of collection sites would help in confirming this trend.

In this study, a few A. pharoensis (N=6) were collected, half of which were CSP positive. Other studies in this region of West Africa have found that A. funestus and A. arabiensis may also be important vector species at different times in nearby Senegal ( Fontenille et al., 1997a; Fontenille et al., 1997b). A. arabiensis was not collected in our study while a small number (N<10) of A. funestus were observed but not collected.

Recent studies on the prevalence of malaria parasites in humans have suggested that infection rates in Guinea-Bissau may be in decline due to widespread use of effective treatment and insecticide treated bed nets (ITNs and long lasting insecticide treated bed nets, LLINs) by the most high-risk groups ( Rodrigues et al., 2008; Ursing et al., 2014). The malaria parasite life cycle is complicated and may not directly relate to the prevalence of human cases but it is possible that the lack of data during periods of political unrest has concealed a more stochastic pattern than was previously observed in Guinea-Bissau ( Ursing et al., 2014).

Outdoor mosquito collection was not the focus of this survey and was only made at Ponta Anabaca Hotel grounds when we fortuitously noted mosquitoes biting. Consequently no general comments about the degree of exophily of A. gambiae in Guinea-Bissau can be made. However, evidence of exophily by the major malaria vector A. gambiae in this study and by others in West Africa ( Reddy et al., 2011; Tchouassi et al., 2012) raises the concern of the long term effectiveness of Indoor Residual Spraying (IRS) and Long lasting Insecticide-treated Nets (LLINs) in reducing outdoor transmission of malaria especially before bedtime and by people sleeping outdoors. The relatively high infection rate of 11.1% of A. melas in Guinea-Bissau together with its tendencies to be both endophilic and exophilic and have a high human blood index ( Sharp et al., 2007; Tuno et al., 2010) make the species a significant vector, which may also be hard to control by reliance on ITNs and LLINs.

The high CSP rate of 33.3% in the 6 indoor collected A. pharoensis might implicate a significant role in malaria transmission in drier inland Guinea Bissau, however this should be viewed with caution due the small sample size. This contrasts the recent finding in Mozambique where none of the 4390 A. pharoensis samples were positive for CSP-ELISA ( Charlwood et al., 2013). Very low infection rates and absence of malarial parasites, traditionally found in West and Central African populations of A. pharoensis has always led to the conclusion that this mosquito plays little role in malaria transmission despite its anthropophilic habits and that it can be easily experimentally infected ( De Meillon, 1947; Ndiath et al., 2012; Tchouassi et al., 2012). In drier Sahel regions of Africa where the major vectors of malaria are absent or very rare and irrigated rice and other crop lands are increasing, A. pharoensis is considered more important at maintaining low levels of malaria ( Kerah-Hinzoumbé et al., 2009; Kibret et al., 2010). More rigorous sampling effort and infection confirmation using multiple approaches (ELSA + SNP genotyping assays ( Lee et al., 2014b) will be required to confirm the definitive role of this species in malaria transmission in this region.

The data referenced by this article are under copyright with the following copyright statement: Copyright: � 2014 Sanford MR et al.

Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

figshare: ELISA results identifying Plasmodium falciparum infection status in Anopheles spp. collected in Guinea-Bissau. doi: 10.6084/m9.figshare.1200058 ( Sanford et al., 2014).