Insecticide Resistance Surveillance of Malaria and Arbovirus Vectors in Papua New Guinea 2017-2022

doi:10.21203/rs.3.rs-1657177/v1

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Insecticide Resistance Surveillance of Malaria and Arbovirus Vectors in Papua New Guinea 2017-2022

https://doi.org/10.21203/rs.3.rs-1657177/v1

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Background: Insecticide resistance monitoring is key for evidence-based control of Anopheles and Aedes disease vectors in particular, since the vast majority of insecticide-based public health adult vector control tools are reliant on pyrethroids. While widespread pyrethroid resistance in Anopheles species and Aedes aegypti has been described in many countries, data for Papua New Guinea are scarce. Available data indicate the local Anopheles populations remain pyrethroid-susceptible, making regular IR monitoring even more important. Knowledge on Aedes IR in PNG is very limited, however, high levels of Aedes aegypti resistance have been described. Here we present IR monitoring data from across PNG generated between 2017 and 2022.

Methods: Mosquito larvae were collected in larval habitat surveys and through ovitraps. Mosquitoes were reared to adults and subjected to insecticide treated filter papers in WHO insecticide susceptibility bioassays. Subsets of Aedes mosquitoes were subjected to sequencing of the voltage-sensitive sodium channel (Vssc) region to identify resistance mutations.

Results: Overall, nearly 20,000 adult female mosquitoes from nine PNG provinces were used in the tests. We show that in general, Anopheline mosquitoes in PNG remain susceptible to pyrethroids but with worrying signs of reduced mortality in some areas. In addition, some Anopheles populations were indicated to be resistant against DDT. We show that Ae. aegypti in PNG are pyrethroid, DDT and likely bendiocarb resistant with a range of Vssc resistance mutations identified. We demonstrate that Ae. albopictus is DDT resistant and is likely developing pyrethroid resistance based on finding a low frequency of Vssc mutations.

Conclusion: This study represents the largest overview of IR in PNG. While Ae. aegypti is highly pyrethroid resistant, the Anopheline and Ae. albopictus populations exhibit low levels of resistance in some areas. It is important to continue to monitor IR in PNG and develop proactive insecticide resistance management strategies in primary disease vectors.

Mosquitoes

Insecticide Resistance

Malaria

Arbovirus

Vector control

Public Health

Insecticide resistance (IR) surveillance in mosquitoes is important for monitoring continued efficacy of insecticide-based vector control interventions and for guiding the selection and application of the most appropriate combinations of products and active ingredients [1, 2]. This includes the choice of products for long-lasting insecticidal nets (LLINs), indoor residual spraying, and other vector control tools. Ideally, a combination of products with active ingredients is used, against which no resistance in the population has been detected [3].

Papua New Guinea (PNG) is endemic for malaria and lymphatic filariasis transmitted by Anophelines [4, 5] and a wide range of Aedes-transmitted arboviruses including dengue, and chikungunya viruses [6, 7]. The primary Anopheles mosquito vector species in PNG belong to the Anopheles (An.) punctulatus complex including An. farauti sensu stricto (s.s), An. koliensis and An. punctulatus s.s. Secondary species that have been demonstrated to transmit malaria include An. longirostris complex and An. bancroftii [8, 9]. The arbovirus vectors Aedes (Ae.) aegypti are found in larger, more densely populated centres whereas Ae. albopictus dominates in rural settings and smaller urban centres [10]. Locally important secondary species include Ae. scutellaris [11, 12].

Over recent years we have reported IR status of malaria [13, 14] and arbovirus [15] vectors in several locations in PNG, determined using the World Health Organization (WHO) insecticide susceptibility bioassays. Our previous data were limited to five provinces for Anopheles spp. and two provinces for Aedes spp. We reported full susceptibility of all tested Anophelines against pyrethroids (deltamethrin), carbamates (bendiocarb) and organophosphates (malathion), and potential resistance to organochlorides (dichlorodiphenyltrichloroethane (DDT))[13, 14]. Furthermore, we reported pyrethroid resistance of Ae. aegypti and DDT resistance of Ae. albopictus found in Port Moresby and Madang. Genetic analysis indicated that all Ae. aegypti in PNG carried common target site resistance mutations conferring resistance to pyrethroids and DDT [15], but we were unable to identify genetic mutations conferring DDT resistance in Ae. albopictus and the Anophelines in this work. Further studies are required to better understand the mechanisms underlying the observed DDT resistance in these mosquito species.

Here we provide an updated data of IR monitoring in PNG that includes bioassay data from nine PNG provinces for Anopheles and Aedes mosquitoes conducted between 2017 and 2022. This update provides a comprehensive overview of the distribution and current IR status in PNG disease vector populations. Our studies focus on the low altitude areas of PNG where vector-borne disease transmission is intense [6, 16].

Study site and mosquito sampling

The study was conducted across nine provinces of PNG (Figure 1) with high burdens of vector-borne diseases transmission between 2017 and 2022. Specifically, the provinces included: National Capital District (NCD), Central Province, Milne Bay Province and Western Province in the southern region of mainland PNG; East New Britain and New Ireland in the New Guinea Islands region and East Sepik, Madang, Morobe, and West Sepik on the northern coast of mainland PNG. Most provinces were surveyed once, East Sepik Province was surveyed twice, and Madang Province was surveyed four times. Figure 1 shows a map of the surveyed provinces and the years the surveys were completed.

Provinces were surveyed for anopheline and aedine larvae simultaneously. Anopheline larval habitats included drainage channels, sand-barred streams, transient puddles, forest swamps, pig-wallows, wells, riverine puddles. The collection of larvae in each province was limited by access via the road network and collections were conducted mainly along the sides of roads accessible by vehicle. On average, each survey included 14 days of larval collections. Anopheles larvae were collected, preferably as 3^rd or 4^th instars, from their habitats using plastic scoops, placed into 500 mL PETT bottles, labelled and brought back to a field insectary or to the PNG Institute of Medical Research (PNGIMR) insectary in Madang. Larval habitat characteristics of each sampled larval source were recorded, including Global Positioning System (GPS) locations. Data were entered into the electronic data capture system Epicollect5 (https://five.epicollect.net/; V4.2.0 Centre for Genomic Pathogen Surveillance, 2022) using mobile electronic tablets. For reference, bioassays were also conducted with a fully susceptible An. farauti colony maintained at PNGIMR. The colony was originally from Rabaul, ENB and established at the PNGIMR in 2008. It has been routinely tested with susceptibility confirmed in various studies since 2009 [13, 14, 17, 18].

Aedes eggs and larvae were collected in ovitraps [19, 20] and through larval habitat surveillance. A total of around 20 ovitraps per survey were set up across consenting householders in an urban area (town or suburb) for five days. A single trap was placed in a protected location outside each house and the GPS location was logged electronically in EpiCollect5. Additionally, common aedine larval habitats were sampled and habitat characteristics recorded as described for anophelines.

Mosquito Rearing

Temporary insectaries were set up for each survey except in Madang province where the PNGIMR has a permanent insectary. Mosquito larvae (both Anopheles spp. and Aedes spp.) were reared in shallow photo developing trays (sizes 16.5×11 cm or 24.5×18.5 cm or depending on the number of larvae collected per site, with larval density ranging from 200 to 500 per respective tray size.). The larvae were fed with grounded Marine Master Tropical fish flakes (Marinepet Australia Pty Ltd). Pupae were collected daily into small plastic cups, and cups were placed into screened plastic containers (20 cm in diameter and 20 cm in height), or BugDorm-1 insect rearing cages (MegaView Science Co., Ltd, Taiwan, 1995-2022), with capacity to hold 300-500 adult mosquitoes at a time. Emerged adult mosquitoes were kept for 3-5 days in the cages before being used in WHO tube bioassays, with access to cotton balls soaked with 10% (w/v) sucrose solution placed on top of the cages and covered with damp cloth to keep ambient tropical temperature and humidity levels.

WHO insecticide susceptibility bioassays

Anopheles and Aedes mosquitoes were tested against eight insecticides. Anopheles mosquitoes were tested against WHO recommended discriminatory concentrations [2, 21]: pyrethroid type I insecticide, 0.75% permethrin; pyrethroid type II insecticides, 0.05% deltamethrin, 0.05% lambda-cyhalothrin and 0.05% alphacypermethrin; carbamate insecticide, 0.1% bendiocarb; organochloride insecticide, 4% DDT; and organophosphate insecticides, 5% malathion and, 0.25% pirimiphos-methyl. These concentrations were also used to test Aedes mosquitoes. We also included the discriminatory concentrations of 0.03% deltamethrin and 0.8% malathion against Aedes populations [21, 22] in Morobe and NCD. The number of insecticides tested varied across surveys (subject to availability of mosquito larvae), with insecticides such as deltamethrin and DDT prioritized because of the current insecticide usage (deltamethrin LLINs) and historical usage of DDT in the country, across the nine provinces.

Bioassays were conducted using WHO standard procedures [2], ideally with 25 adult female anopheline mosquitoes per bioassay tube, and a full test having four replicates and two control tubes (n=150 adult female mosquitoes per test). However, if insufficient larvae were present to conduct a full test, tests were conducted with twenty to ninety mosquitoes (one to three replicates and 1 control per tests. Mosquitoes were exposed to insecticide-impregnated filter papers inside the replicate tubes for a total of 60 minutes, with knock down recorded at 5 minute intervals for 30 minutes, followed by 10 minute interval readings until 60 minutes. Mosquitoes were then transferred from each tube into separate holding tubes (controls included) and kept at an ambient tropical temperature and humidity for a 24-hour holding period, with access to 10% (w/v) sucrose solution. The primary endpoint was 24h mortality for all insecticides tested, however we also included a 48 hour holding period and mortality observation for DDT, as we observed much slower rates of knock down with this insecticide. Mosquitoes were graded as recommended by WHO [2]; alive mosquitoes were able to stand and flew in a coordinated manner whereas moribund mosquitoes were knocked down and or could not fly in a coordinated manner or fell down immediately after taking flight; dead mosquitoes showed no signs of life and could not stand. Mortality rates were calculated by dividing the sum of all dead and moribund mosquitoes by the total number of mosquitoes tested and multiplied by 100 [2].

Mortality from each holding tube was recorded after 24 hours, with all survivors separated for easy identification during later laboratory analysis. All mosquitoes that were alive after the 24hr holding period (test survivors and controls) were anesthetized at -20°C and morphologically identified using standard identification keys [23] along with the rest of the tested mosquitoes.

Molecular Analysis

Genomic DNA was extracted from a sample of adult mosquitoes (Ae. aegypti n=55, Ae. albopictus n=170) using the Roche High Pure PCR template kit (Roche Molecular Systems, Inc., Pleasanton, CA, USA) according to the instructions of the manufacturer, but with two elution steps (first elution in 60 µL elution buffer and second elution in 120 µL). The second elution was diluted 1:10 with water and used in the molecular analysis of resistance mutations.

Custom TaqMan^Ò SNP Genotyping Assays (Life Technologies, California, USA) developed for each of the three target site mutations in the voltage sensitive sodium channel (Vssc) of Aedes aegypti (codons 989, 1016, 1534) were run on the Roche LightCycler® 480 and analysed using the endpoint genotyping method [24]. The Vssc amino acid positions are labelled as S989P, V1016G and F1534C according to the sequence of the most abundant splice variant of the house fly, Musca domestica, Vssc (GenBank accession nos. AAB47604 and AAB47605 [25]).

The Vssc codon 1534 of Ae. albopictus was screened for mutations using PCR primers aegSCF7/aegSCR7 and sequenced with aegSCR8 [26]. 2 µL genomic DNA were amplified in a 25 µL PCR mix that included final concentrations of ThermoPol buffer Mg-free (1x) (New England Biolabs, Ipswich MA, USA), dNTPs (200 µM each), MgCl₂ (1.5 mM), 0.5 µM each of forward and reverse primers, 0.625 units of Immolase™ Taq polymerase (Bioline, London, UK), and PCR-grade H₂O. Thermocycling conditions followed those of Ahmad et al. [27]. PCR amplicons (740 bp) were sent to Macrogen Inc. (Seoul, South Korea) for Sanger sequencing on a 3730xl DNA analyser. Sequences were analysed, aligned, and trimmed (~180 bp) using the program Geneious Prime® 2020.0.4 (Biomatters Ltd.).

Data Analysis

Mosquito populations were classified as resistant or susceptible according to the WHO criteria [2] as follows; mortality rates of 98-100% is susceptible, 90-97% suggests possible resistance and less than 90% indicates confirmed resistance. Proportion of knocked down or dead mosquitoes was calculated and 24 h mortality was adjusted using ‘Abbott’s formula’ when the control mortality was between 5% to 20%. Analysis of proportions (95% CI) and one-sample z-tests using the online calculator Epitools (https://epitools.ausvet.com.au/; AusVet, 2022) were conducted to determine if the observed 24 h mortality was indicative of resistance or susceptibility. Analyses were conducted for each species (where possible), insecticide and province.

Species distribution of mosquitoes tested in the bioassays

A total of 11,210 anopheline mosquitoes and 8,294 aedine mosquitoes were used in the WHO tube bioassays across nine provinces in PNG between 2017 and 2022. The distribution of taxa identified based on morphology is shown in Figure 2. Bioassays were also conducted with a pyrethroid susceptible An. farauti colony at PNGIMR Madang (n = 1,762).

Overall insecticide resistance profile of PNG vector species

Table 1 shows the overall insecticide susceptibility profiles of An. farauti colony mosquitoes, An. punctulatus s.l, Ae. albopictus, Ae. aegypti, and secondary species (combined) as determined in the present study.

An. punctulatus s.l populations (Table 1) showed susceptibility to all tested insecticides at discriminatory concentrations except for DDT, for which resistance was indicated, with 95.8% (95% CI: 94.8-96.7%) 24 h mortality. An. spp. (mainly An. bancroftii and An. longirostris) numbers were too low to draw any conclusions for most insecticides except for deltamethrin, where susceptibility was indicated.

Ae. aegypti populations present in urban centres across PNG exhibit pyrethroid and DDT resistance as indicated by an average mortality of 31.8% (95% CI: 26.9-37.0%) against deltamethrin, 32.0% (19.5-46.7%) for lambda-cyhalothrin and 18.9% (95% CI: 12.5-26.8%%) for DDT (Table 1). Bendiocarb resistance was observed with 94.0% mortality (95% CI: 88.0-97.0%). Resistance to other pyrethroid insecticides, alphacypermethrin and permethrin was inconclusively indicated due to low sample numbers tested against the insecticides. The species remained susceptible to malathion with 99.5% (95% CI: 97.0-100.0%) and 100% mortality (95% CI: 91.0-100.0%) against pirimiphos-methyl.

Ae aegypti tested against deltamethrin and malathion in Morobe and NCD at the discriminatory concentrations recommended for Aedes mosquitoes by WHO in 2016 [22] at 0.03% and 0.8% respectively, showed deltamethrin and malathion resistance. However as shown above, these vector populations had high mortality rates against the anopheline recommended discriminatory concentration. This malathion concentration is approximately six times higher than the interim recommended Aedes discriminatory concentration. According to the WHO intensity bioassay criteria [2], the species when combined across PNG shows a low level of resistance intensity.

Ae. albopictus showed resistance to DDT (78.5%, 95% CI: 75.4-81.4%) and possible resistance to malathion (97.2%, 95% CI: 95.5-98.4%). Ae. albopictus showed susceptibility to deltamethrin (98.7%, 95% CI: 98-100%), lambdacyhalothrin (100%), permethrin (100%) and bendiocarb (99%, 95% CI: 98-100%). Similar to Ae. aegypti populations in Morobe and NCD, Ae. albopictus was resistant to malathion (43%, 95% CI: 36-50%) and possible resistance to deltamethrin (97%, 95% CI: 90-99%) at the 2016 WHO recommended discriminatory concentrations for Aedes mosquitoes [22].

Other Aedes species (which are mainly Ae. scutellaris from the northern PNG) were present in numbers too low to draw definitive conclusions. However, results suggest phenotypic resistance against DDT with 24 h mortality of 89.5% (95% CI: 66.9-98.7%).

Spatial and species-specific results of Anopheles punctulatus s.l morphospecies

Species-specific mortality for the three primary Anopheles morphospecies An. farauti, An. punctulatus s.s. and An. koliensis in each province is shown in Figure 3. When data were stratified by morphospecies and province, sample size usually became quite small, limiting the confidence of inferences made from the analyses.

Data indicate susceptibility against all tested insecticides in all morphospecies and provinces, including deltamethrin. Results from the An. koliensis population in East Sepik province indicate higher than average levels of DDT and lambda-cyhalothrin resistance, with mortality of 33.3% (95% CI: 7.49-70.1%) and 83.3% (95% CI: 51.6 - 97.7%) respectively. Results from the An. punctulatus population also indicate higher than average levels of DDT resistance in Milne Bay province, with a mortality of 88.2% (95% CI: 72.6-96.7%) and lambda-cyhalothrin resistance in West Sepik province, mortality of 87.5% (95% CI: 47.4-99.7%).

DDT results also indicated possible resistance in the An. farauti population in Madang, Milne Bay and Western provinces with the mortality rates ranging between 94%-97%, and in the An. punctulatus population of East New Britain, East Sepik and Madang provinces, with mortality rates ranging between 92%-97% (Figure 3).

Spatial and species-specific results for Aedes aegypti and Ae. albopictus

Species-specific mortality for the two primary Aedes species Ae. aegypti and Ae. albopictus, in each province is shown in Figure 4.

Aedes aegypti were found in four population centres, namely Port Moresby (NCD), Lae (Morobe), Madang (Madang Province), and in Kokopo and Rabaul (East New Britain Province). Resistance was observed against deltamethrin, lambdacyhalothrin, DDT, malathion and bendiocarb (Figure 4). Deltamethrin tested across all four Ae. aegypti populations showed mortality percentages ranging from 17% to 63%. Lambdacyhothrin resistance was observed in Madang with mortality rates of 32.4% (95% CI: 18.5-49.8%) and 25% mortality (95% CI: 7.3-52.4%) in Morobe. DDT resistance with mortality ranging from 0% to 71% was detected in across all four provinces. Malathion susceptibility was observed in Morobe, NCD and Madang, but possible resistance was detected in East New Britain with mortality of 93.8% (95% CI: 69.8%-99.8%). Possible resistance to bendiocarb with mortality ranging from 89-95% was observed in Morobe, NCD, ENB and Madang.

Ae. albopictus was found in all nine provinces surveyed. Populations across the nine provinces showed mainly susceptibility or possible resistance against the panel of insecticides tested except for DDT (Table 1). DDT resistance was observed with rates between 60% and 79% in West Sepik, Milne Bay, Western, Morobe, New Ireland and Madang. Possible resistance was observed in East New Britain with 96.3% (95% CI: 81.0%-99.9%) mortality and in NCD with 97.9% (95% CI: 88.7%-100%) mortality. Possible deltamethrin resistance was observed in Morobe (95.3%, 95%CI: 90.5-98.1%)) and in NCD (94.6% (95% CI: 85.1-98.9)). All Ae. albopictus in the seven provinces outside of Morobe and NCD were deltamethrin susceptible with mortality rates ranging from 98 to 100%. The species was fully susceptible to the other pyrethroid insecticide, lambdacyhalothrin, in Madang, WSP and Western (100% mortality). Western population was also fully susceptible to permethrin. Possible malathion resistance was observed in Morobe with mortality rates of 97.8% (95% CI:92.2%-61%), NCD with 97.2% (95% CI:85.5-99.9%). 96.3% (95% CI: 89.6-99.2%) in Madang and 92.8% (95% CI: 86.3-96.8%) in Western province. Ae. albopictus in New Ireland and WSP were malathion susceptible with 24 h mortalities between 99% and 100%. Bendiocarb susceptibility was indicated with mortality percentages ranging from 99 to 100% in all provinces except in Morobe province where possible resistance was indicated with 92.1% (95% CI: 86.5-95.8) mortality. In pirimiphos-methyl susceptibility in Ae. albopictus was observed in Western Province (100% (95%CI: 95.6-100.0%) and possible resistance in Morobe with 97.9 (95% CI: 88.7-100%) mortality.

Molecular Analyses in Aedes aegypti and Ae. albopictus.

Mutations in the Vssc gene in Ae. aegypti were found to be common at codons 1016, 1534 and 989 in the PNG sample. Six composite Vssc genotypes arising from four haplotypes were identified in the n=55 samples of Ae. aegypti from five PNG provinces (Table 2).

Three of the genotypes are known to confer pyrethroid resistance. No wild-type genotype or haplotype was identified in the sample.

The most frequent genotype (frequency = 0.27) in the sample was a homozygous mutation at codon 1016 and 989 with the wildtype homozygote at codon 1534. Another common genotype found was the triple heterozygote at V1016G, F1534C and S989P (genotype frequency = 0.26). A new genotype (1016G in the homozygous state and not linked to 989P - now known as genotype M) for PNG was found at a frequency of 11% in the sample. The distribution of Vssc genotypes across the sampled provinces of PNG revealed resistance mutations to be widespread.

Genotyping by sequencing the region including codon 1534 of the Vssc in Aedes albopictus showed that most mosquitoes in the sample were wildtype for the resistance mutation. However, one homozygote and four heterozygotes for this mutation were found in a sample of 166 individuals. The mosquitoes carrying this mutation were collected in Milne Bay and Morobe Provinces (Table 3).

The present study provides the largest currently available dataset on IR status of malaria and arbovirus vectors in PNG. Using standard WHO tube bioassays, we characterized phenotypic resistance of Anopheles and Aedes species against important insecticides used in public health programs in nine provinces experiencing high vector-borne disease burden.

The three primary malaria vector species, An. farauti, An. punctulatus s.s. and An. koliensis, have been previously profiled using the same methods as susceptible to deltamethrin, lambda-cyhalothrin and DDT in Madang, East Sepik, Manus, Milne Bay and East New Britain provinces [13, 14]. Susceptibility of An. punctulatus s.l to the AIs used in malaria control in PNG, in particular deltamethrin, which has been the single AI used in LLINs in PNG for over 15 years, indicates sustained bioefficacy against these vector species. This is interesting as PNG has achieved over 80% LLIN coverage nationwide with over 50% usage rates since 2014 [28]. Additionally, vector behaviours leading to reduced contact with LLINs such as outdoor, early evening biting and opportunistic feeding behaviour of these vectors could play a role in maintaining susceptibility to deltamethrin [29].

We show here that the An. punctulatus in ESP and ENB are showing signs of emerging phenotypic resistance to DDT and lambda-cyhalothrin. If cross resistance to deltamethrin and or alphacypermethrin arises, this may compromise the effectiveness of the mainly pyrethroid-based malaria control strategies in PNG in the future.

Our data also indicate DDT resistance in An. farauti and An. koliensis, however this could not be conclusively shown due to the low numbers of the species tested in ESP. An. koliensis was reportedly resistant to DDT in neighboring Irian Jaya, Indonesia [30]. It is imperative that future IR surveillance confirm the resistance status of these important malaria vector species.

Whether the observed signs of DDT resistance in Anopheline populations in some areas are a remnant of historic DDT usage in PNG [31] , agricultural usage of DDT and or pyrethroids [32] or emerging cross-resistance with pyrethroids [33] is yet to be elucidated. Our data showing resistance against lambda-cyhalothrin as well as DDT in some provinces suggests that is a potential cross-resistance of DDT and lambdacyhalothrin. Furthermore, it is unclear whether the cross-resistance profile (DDT-lambdacyhalothrin) observed in the PNG vector species may be driven by either target site mutations or through a metabolic detoxification pathway such as that in the South American malaria vector, An. darlingi [34].

There is an urgent need to confirm the underlying resistance mechanisms using molecular analysis tools such as Vssc genotyping and population genomic tools such as Whole Genome Sequencing and investigation of gene copy number variation [35]. Furthermore, biochemical and biological assays are essential to confirm these underlying mechanisms [36]. This includes a role for expanding bioassay tools to include use of synergists to identify components of metabolic resistance. Widespread emergence of pyrethroid resistance in PNG would compromise current public health vector control strategies.

Ae. aegypti and Ae. albopictus are the two common arboviral vectors in PNG. Previous profiling of these species in Madang and Port Moresby (NCD) reported pyrethroid resistance in Ae. aegypti in Port Moresby (NCD) and Madang. DDT resistance was detected in Ae. albopictus in Madang. While both species in the two areas were susceptible to malathion, Ae. aegypti indicated resistance to DDT and bendiocarb (not confirmed due to low sample size) and Ae. albopictus was susceptible to lambdacyhalothrin [15].

The emergence of resistance to deltamethrin, lambdacyhalothrin, DDT and malathion in Ae. aegypti and Ae. albopictus, confirmed in several populations, highlights a critical need to understand the landscape of chemical use for pest control (private sector, public health, agricultural or industrial) in PNG. DDT was last used almost 50 years ago in a national malaria control program [37], however it has been reported to be used illegally within the country for either public health or agriculture [32]. Products containing a number of AIs including malathion and lambdacyhalothrin are frequently used in PNG [32] suggesting that selective pressure from these AIs could be contributing to the high resistance in Ae. aegypti and the emergence in Ae. albopictus.

The capital cities of Port Moresby (NCD) and Lae (Morobe) are the most urbanized and densely populated areas in PNG. Both Aedes species thrive in these environments at high densities similar to elsewhere in Asia [38], Latin America [39], and Africa [10], posing a high risk of arboviral transmission, in particular dengue fever, chikungunya and Zika within these cities. Widespread resistance in Ae. aegypti and Ae. albopictus to multiple insecticides in these provinces (Morobe and NCD) highlights a critical need for a harmonized vector control plan that considers these resistance profiles. Furthermore, building capacity for routine mosquito surveillance in PNG and strengthening partnerships across PNG research institutions, government departments and health laboratories is critical to reduce these important mosquito vector populations and thus reduce disease transmission [40].

The pyrethroid resistance within the Ae. aegypti populations in PNG is conferred, at least in part, by three or more different kdr genotypes. The most frequent genotype, a homozygous mutation at codon 1016 and 989 with the wildtype homozygote at codon 1534, is a common genotype also found in Bali and other locations throughout Southeast Asia and the Pacific, and confers a high level of resistance to Type I and II pyrethroids [24, 41]. The heterozygote at V1016G, F1534C and S989P is found commonly in Ae. aegypti in other countries in the region and confers a low to moderate level of resistance to Type I and II pyrethroids [42]. The homozygote 1534C occurring alone is rare in the PNG sample, but confers a low level resistance to Type I pyrethroids [42]. A new genotype for PNG (1016G in the homozygous state and not linked to 989P - now known as genotype M) has not been found in recent studies of Ae. aegypti in the Indo-Pacific region [24], but does exist in Taiwan and its association with resistance has not been ascertained [43]. The two remaining genotypes identified in this study contain the haplotype that makes up genotype M and their association with pyrethroid resistance is yet to be tested. The role of metabolic resistance in the PNG mosquito populations has not been investigated. For both Aedes species studied in PNG, it will be important to determine whether the Vssc resistance mutations have arisen due to local selection or have arrived with invasive mosquitoes as has been shown in other parts of the Indo-Pacific region [24]. Whatever their origin, the continued presence of the Vssc mutations implies that they are being maintained due to selection with pyrethroid insecticides in PNG as there are known fitness costs associated at least with the 1016G/989P mutations [44, 45].

PNG has so far avoided pyrethroid resistance in Anopheline vectors. It is important to aim to maintain pyrethroid susceptibility in PNG vectors by promoting sustainable and responsible use of insecticides in both public health and agriculture sectors in PNG. The WHO-recommended IR management guidelines for malaria vectors suggest strategies such as rotation of insecticides and combination of interventions such as LLINs and IRS to delay resistance development [46]. In addition, increased capacity-building and better resourcing of IR monitoring efforts in PNG are critical. A national strategic plan for vector surveillance and control, as well as strong leadership by the National Department of Health are required. Facilitation and coordination of subnational partnerships between public health and private sector (e.g., agricultural) stakeholders is critical for effective IR management in PNG [40].

We show that An. punctulatus, an important malaria vector species is resistant to DDT, with a possible cross resistance to the pyrethroid insecticide, lambdacyhalothrin. Further monitoring of the IR status of other malaria vector species needs to be maintained. Vector control of arbovirus vector species such as Ae. aegypti in PNG needs to be streamlined and regulated under one regulatory body especially at provincial level. The existing toolbox for controlling these vectors is compromised due to pyrethroid, organochloride and organophosphate resistance. Monitoring of all the important vector species at both phenotypic and molecular levels is crucial to identify and develop effective strategies in a timely manner to mitigate the spread of resistance across PNG.

IR: Insecticide resistance;

LLINs: long-lasting insecticidal nets;

PNG: Papua New Guinea;

WHO: World Health Organization;

DDT: dichlorodiphenyltrichloroethane;

Vssc: voltage sensitive sodium channel

Acknowledgements

We would like to thank the PNG communities where mosquito samples were collected for their support. We acknowledge the support from Provincial Health Authorities in all surveyed provinces. We thank the PNG National Malaria Control Program (NMCP), Rotarians Against Malaria PNG, and the PNGIMR Project Management Unit for the Global Fund Grant for management support, especially Tim Freeman, Munir Ahmed, Michelle Auryan, Sharon Jamea and Serah Kurumop. We thank the PNG National Agriculture and Quarantine Inspection Authority, especially David Tenakanai and Orlando Mercado for enabling operational support in several provinces. We acknowledge the Lihir Malaria Elimination Project team and the vector control unit International ISOS (Newcrest Lihir) for operational support, especially Pere Millat, Barbara Baro and Tony Tandrapah.

We thank current and former staff of the Entomology section at the PNGIMR Vector Borne Diseases Unit in Madang and the PNGIMR Maprik branch for contributions to logistical organization, sample collection and WHO tube bioassays, especially Michael Kurumop, Lincoln Timinao, Rebecca Vinit, Elias Omera and Peter Kaman, Benson Kiniboro and Kenny Rupa. We thank Rachael Farquhar (Burnet Institute) and Annie Dori (PNG NDoH) for STRIVE PNG partnership coordination and logistics support. We thank Tanya Russell (JCU) for support and technical guidance, and with drafting of standard operating procedures.

We thank the STRIVE PNG Vector Working Group and the Technical Working Group of the NMCP, especially Tom Burkot, Nigel Beebe, Trevor Kelebi, Enoch Waipeli, Lucy Dally, Melinda Susapu and John Deli for helpful discussions and guidance.

Funding

This study was funded, in part, by the Global Fund to Fight Aids Tuberculosis and Malaria, STRIVE PNG - Department of Foreign Affairs and Trade, Australian Government [Grant No. 74430], Indo-Pacific Centre for Health Security and the Lihir Malaria Elimination Project. SK is supported by an NHMRC Career Development Fellowship (GNT1141441). MK was supported by a Wellcome Trust International Masters Fellowship. AH and NE were supported by National Health and Medical Research Council (Program Grants 1037003, 1132412). LJR is supported by NHMRC Career Development Fellowship (GNT1161627).

Availability of data and materials

Data used in this present study is presented within the article.

Author Contribution

The study was conceived by SK, LM, ML, LR, AH. Sample collection and experiments was conducted by MK, SL, NEH, SD, JG, NV, MS, AD, LK, SG, CP, SK; The data was analyzed by MK, SL, NEH, SK and the first draft of the manuscript written by MK, SL, SK and reviewed by MK, SL, NEH, AH, LM, LR, ML, SK.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors have declared they have no competing interests.

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Table 1: Summary of phenotypic resistance bioassay results of the primary malaria and arbovirus vector species in PNG. Results are shown as 24 h mortality in percent with 95% confidence intervals given (95% CI) along with the total number of mosquitoes exposed [N]. Susceptibility status based on mortality rates are classified according to the WHO criteria as follows; S (susceptible) denotes 98-100%, PR (possible resistant) denotes 90-97% and R (resistant) denotes less than 90% mortality. Cells designated NA are indicative of no data available as no bioassays were conducted, or data were omitted due to sample size being too low. ^c Insecticide susceptible An. farauti s.s colony maintained at PNGIMR. *Inconclusive because of low number of mosquitoes tested.

Insecticide

An. farauti s.s^c

An. punctulatus s. l

An. spp.

Ae. albopictus

Ae. aegypti

Ae. spp.

% [N]

(95% CI)

Status

% [N]

(95% CI)

Status

% [N]

(95% CI)

Status

% [N]

(95% CI)

Status

% [N]

(95% CI)

Status

% [N]

(95% CI)

Status

0.05% Deltamethrin

100 [487]

99.5 [2241]

100 [45]

98.7 [1059]

31.8 [346]

100 [48]

(99.2-100)

(99.2-99.8)

(92.1-100)

(97.8-99.3)

(26.9-37.0)

(92.6-100)

0.05% Lambdacyhalothrin

100 [186]

99.2 [757]

99.7 [387]

32 [50]

100 [14]

(98.0-100)

(98.5-99.8)

(98.6-100)

(19.5-46.7)

(76.8-100)

0.05% Alphacypermethrin

100 [101]

100 [172]

100 [62]

64.3 [42]

(96.4-100)

(97.9-100)

(94.2-100)

(48.0-78.5)

0.75%

Permethrin

100 [99]

100 [106]

100 [88]

10.7 [28]

(96.3-100)

(96.6-100)

(95.6-100)

(2.3-28.2)

4 %

DDT

100 [94]

95.9 [1812]

78.5 [762]

18.9 [127]

89.5 [19]

(96.2-100)

(94.9-96.8)

(75.4-81.4)

(12.5-26.8)

(66.9-98.7)

Malathion

100 [97]

100 [1051]

97.2 [567]

99.5 [203]

100 [51]

(96.3-100)

(99.7-100)

(95.5-98.4)

(97.0-100.0)

(93.0-100)

0.25%

Pirimiphos-methyl

100 [114]

100 [287]

99.2 [129]

100 [39]

(96.8-100)

(98.7-100)

(95.8-100)

(91.0-100)

0.1%

Bendiocarb

100 [96]

99.9 [1177]

98.5 [922]

94 [133]

100 [45]

(96.2-100)

(99.5-100)

(97.5-99.2)

(88.0-97.0)

(92.1-100)

Table 2: Six composite genotypes identified in Aedes aegypti in PNG.

Codon
1016	1534	989	n	frequency
GG	TT	CC	15	0.273
TT	GG	TT	2	0.036
TG	TG	TC	14	0.255
GG	TT	TC	9	0.164
TG	TG	TT	9	0.164
GG	TT	TT	6	0.109

Table 3: Sequencing results for the region including codon 1534 of the Vssc in Aedes albopictus. Most samples were wildtype but low frequencies of resistance mutations were identified in Milne Bay, Morobe and Western Provinces.

Province	1534		n	Freq. in Province
East New Britain	TT	WILDTYPE	26	1.00
East Sepik	TT	WILDTYPE	44	1.00
Milne Bay	TT	WILDTYPE	9	0.82
Milne Bay	TG	F1534C	1	0.09
Milne Bay	GG	1534C	1	0.09
Morobe	TT	WILDTYPE	20	0.91
Morobe	TG	F1534C	2	0.09
New Ireland	TT	WILDTYPE	12	1.00
West Sepik	TT	WILDTYPE	10	1.00
Western	TT	WILDTYPE	41	0.98
Western	TG	F1534C	1	0.02

No competing interests reported.

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Insecticide Resistance Surveillance of Malaria and Arbovirus Vectors in Papua New Guinea 2017-2022

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