There is a wide array of anopheline mosquitoes capable of transmitting malaria in Africa, and each species has varied behaviours, distribution, and seasonality that influences their roles in malaria transmission [25]. For vector control to be most effective, precise data on target mosquito species are essential for selecting the best intervention and measuring impact [26]. During the 7-year surveillance period (2016–2022) eight anopheline species were collected from the mangrove /forest ecological zone of Mpat-Enim LGA in Akwa Ibom State, Nigeria, including members of An. coustani, An. funestus group, An. gambiae s.l., An. maculipalpis, An. marshallii group An. moucheti An. nili.and An. obscurus The identification of An. marshallii group is new within the vector species composition of mosquitoes identified in Akwa Ibom, starting in 2021.

According to Gilles and Coetzee [11], the An. marshallii group consists of 16 species in the Marshallii-Hancocki section (series Myzomyia). The species are Anopheles austenii, Anopheles berghei, Anopheles brohieri, Anopheles gibbinsi, Anopheles hancocki, An. hargreavesi, Anopheles harperi, Anopheles hughi, Anopheles kosiensis, Anopheles letabensis, An. marshallii s.s., Anopheles mortiauxi, Anopheles mousinhoi, Anopheles njobiensis, Anopheles seydeli and Anopheles upemba [12]. As with other species groups/complexes, these species have different biting/feeding behaviours, larval breeding habitats, host preferences, vectorial capacity, and other characteristics. However, the absence of accurate molecular identification methods hinders studies on this species group [12]. In agreement Harbach, [27] referred to An. marshallii as a complex comprising 16 known species inclusive of four identical morphological species: An. kosiensis, An. letabensis, An. marshallii s.s. and An. hughi. However, since some of these 16 species can be separated or distinguished by pupa and egg morphology, they are better referred to as a group and not a complex.

The challenge in the morphological identification of the An. marshallii group was due to the damage done by the suction of the CDC LT collection method on mosquito samples including loss of vital parts such as legs, wings and the scales on the thorax. The PSC samples also did not fare better as similar damage was done on the vital parts on account of the tremors caused by exposure to pyrethroids. This study was able to confirm some of these samples by morphological identification via scale morphology, but most required molecular analysis via genetic fingerprinting of ITS2 and CO1.

The samples that were identified as An. hargreavesi using the key characteristic of scale morphology were subject to further molecular identification confirmation analysis. Using phylogenetic analysis, the closest relationship between the morphologically identified An. hargreavesi was to an An. marshallii isolate (ITS2: PP915781-PP915785; COI: PP919268-PP919271), compared to other members of the An. marshallii group. Those samples that were too damaged for morphological identification or that otherwise were unamplified and aligned by phylogenetic analysis with the same An. marshallii isolate may be An. hargreavsi, but they could be other members of the An. marshallii group. Unfortunately, this is a limitation because An, hargreavsi sequences are not in the nucleotide database. As such the identification remains tentative and these mosquitoes were recorded as An. marshallii group.

In response to these data findings, a refresher training was organized for the Akwa Ibom site technicians to strengthen their capacity on morphological identification skills, focusing on characteristic features of the An. marshallii group, resulting in the reduction of unamplified samples. Apart from taxonomy skills demonstrated, the usefulness of molecular tools in confirming the identity of damaged, unamplified and/or cryptic species, enhanced vector surveillance outcomes [28, 29]. There is a need to further develop specific PCR assays and/or submission of nucleotide sequences of species members to provide more accurate identification and understanding of this group.

Several secondary vectors have been recognized across Africa, including An. coustani, An. moucheti, An. pharoensis, An. pretoriensis, An. rivulorum, An. rufipes, An. squamosus, as well as the An. marshallii group [30,31,32]. Some of these have been found across various entomological monitoring sites in Nigeria and have been considered of negligible importance because of their strong zoophilic tendencies [3]. Secondary vectors have been recognized for their importance in sustaining malaria transmission during periods when the population of the primary vectors reduces, most often due to lack of rainfall in the dry season [33, 34]. Understanding the role of these secondary vectors relative to the primary vectors will help design sustainable vector control strategies [35].

Ecological variables such as quality of larval habitats, vegetation, topography, climate change and flow speed of rivers are known to have a strong influence on mosquito abundance and distribution [36]. In this study, the co-existence of three major vector species, An. gambiae s.l., An. coluzzii and An. marshallii group (alongside An. moucheti and An. nili) were observed in coconut plantations within the mangrove ecozone in Akwa Ibom. Similar reports from southern Cameroon have shown the co-existence of An. marshallii alongside An. moucheti and An. nili in one sentinel site (Nyabessang) out of the five sites surveyed. The ecology of Nyabessang is dense, moist forest [32], similar to the mangrove forest in Akwa Ibom. These findings provide additional information on the possible and peculiar ecology that can be exploited by the An. marshallii group, An. moucheti and An. nili. This flora should be considered when describing An. marshallii group habitat and when planning vector surveillance and control.

The data on indoor resting density indicated the co-existence of both An. gambiae s.l. and An. marshallii group mosquitoes with either species having potential to dominate the other indoors during specific periods of the year. It is apparent that both species have strong indoor resting tendencies, which are influenced seasonally. This means that vector control interventions targeting indoor resting may protect household members year-round.

Differences in HBR between Anopheles species have previously been observed to vary seasonally and in different eco-geographical locations [32]. The indoor and outdoor biting rate of the An. marshallii group in Akwa Ibom peaked when rains waned. This biting activity suggests the An. marshallii group could perpetuate malaria transmission during the dry season, followed by transmission largely by An. gambiae s.l. at the onset of the rains. Similar variation in the biting patterns at different periods of the rainy season have been reported in Mendong in Cameroon. A sharp increase in the biting rates of An. funestus was recorded at the beginning of the rainy periods compared to An. gambiae s.l., which was present year-round but peaked during the rainy season [37].

In Akwa Ibom, the An. marshallii group were found biting both indoors and outdoors in the early evenings. Residual malaria transmission due to early evening and outdoor biting vectors could pose a challenge to core malaria control interventions that focus on indoor vector behaviours. Supplemental vector control tools targeting early evening and outdoor biting behaviours may need to be considered.

The sporozoite rate reported here should be carefully interpreted as limited samples were positive for P. falciparum. This evaluation has confirmed the An. marshallii group to be infected with P. falciparum and to demonstrate human blood feeding preferences (determined from PSC collections) similar to members of the An. gambiae s.l. This is consonant with the findings of Fondjo et al. [32], WHO reported an infection rate of 0.11% in An. marshallii mosquitoes in Cameroon.