Laboratory-based efficacy evaluation of Bacillus thuringiensis var. israelensis and temephos larvicides against larvae of Anopheles stephensi in ethiopia

Background Malaria, transmitted by the bite of infective female Anopheles mosquitoes, remains a global public health problem. The presence of an invasive Anopheles stephensi, capable of transmitting Plasmodium vivax and Plasmodium falciparum parasites was first reported in Ethiopia in 2016. The ecology of An. stephensi is different from that of Anopheles arabiensis, the primary Ethiopian malaria vector, and this suggests that alternative control strategies may be necessary. Larviciding may be an effective alternative strategy, but there is limited information on the susceptibility of Ethiopian An. stephensi to common larvicides. This study aimed to evaluate the efficacy of temephos and Bacillus thuringiensis var. israelensis (Bti) larvicides against larvae of invasive An. stephensi. Methods The diagnostic doses of two larvicides, temephos (0.25 ml/l) and Bti (0.05 mg/l) were tested in the laboratory against the immature stages (late third to early fourth stages larvae) of An. stephensi collected from the field and reared in a bio-secure insectary. Larvae were collected from two sites (Haro Adi and Awash Subuh Kilo). For each site, three hundred larvae were tested against each insecticide (as well as an untreated control), in batches of 25. The data from all replicates were pooled and descriptive statistics prepared. Results The mortality of larvae exposed to temephos was 100% for both sites. Mortality to Bti was 99.7% at Awash and 100% at Haro Adi site. Conclusions Larvae of An. stephensi are susceptible to temephos and Bti larvicides suggesting that larviciding with these insecticides through vector control programmes may be effective against An. stephensi in these localities. Supplementary Information The online version contains supplementary material available at 10.1186/s12936-023-04475-9.


Background
Malaria is a global public health problem that mainly affects tropical countries [1].It is transmitted by the bite of infective female Anopheles mosquito species.Globally, there are some 3530 species of mosquitoes under 43 genera in the family Culicidae, which are further divided into the subfamilies of Culicinae, Anophelinae and Toxorhynchitinae [2].Of these, malaria vectors belong to the genus Anopheles [2].
In Ethiopia, Anopheles arabiensis is the main malaria vector while Anopheles pharoensis, Anopheles funestus and Anopheles nili are secondary vectors [3].The recently reported invasive species, Anopheles stephensi in the country has exhibited the potential of transmitting Plasmodium falciparum and Plasmodium vivax [4,7].The species has also been reported from other countries in the Horn of Africa including Djibouti (2012), Sudan (2016), and Somalia (2019), raising concern about appropriate vector control strategies to target this invasive species [4,5].
Unlike other malaria vectors, An. stephensi is also considered an urban and peri-urban adapted malaria vector, which breeds in man-made habitats such as overhead tanks, ditches and canals [5][6][7].Anopheles stephensi feeds on both humans and animals, with a preference for the latter, and it exhibits more outdoor feeding [4].Anopheles stephensi in Ethiopia is resistant to most insecticides used in current malaria vector control tools, insecticide treated bed-nets (ITN) and indoor residual spraying (IRS) [7,9], so larviciding might be an effective control method [7].
Larval source management (LSM) is one of the oldest and primary strategies used throughout the world to control malaria targeting the immature stages of the mosquito vectors in their aquatic habitats; however it has been less commonly used in African countries following the introduction of indoor residual spraying (IRS) in the 1950s and long-lasting insecticide impregnated nets (LLINs) in the 1990s [10,11].Organophosphates larvicides such as temephos and pirimiphos-methyl interfere with the nervous system of the immature larval stages, whereas naturally occurring microbes such as Bacillus thuringiensis var.israelensis (Bti) and Bacillus sphaericus (Bs) kill larvae with their toxins when ingested [10,11].
In Ethiopia, concerted efforts have been made in the fight against malaria since the 1950s.The intervention strategies have included early diagnosis and prompt treatment of cases, IRS, prevention and control of epidemics, and recently, scale-up of LLINs and LSM through larviciding and environmental management at small scale, where breeding sites are few, findable and manageable [3,11].However, resistance to insecticides used in vector control by An. stephensi has been reported from within the country [9] and other places [5].Therefore, in order to tailor the local strategies to vector (s) susceptibility [7,12], it is crucial to investigate the efficacy of selected malaria vector control interventions towards the control of An. stephensi, especially in areas where research has not yet covered in a holistic manner.

Anopheles stephensi larval and pupal collection sites
Larvae and pupae of An. stephensi were collected from Awash Subah Kilo Town (also spelled as Awash Sebat Kilo in other publications) and Haro Adi around Metehara from January 2021 to June 2021.Awash Subah Kilo Town is located in Administrative Zone 3 of the Afar Regional State, just above a gorge of the Awash River, after which it is named.The town lies on the Addis Ababa-Djibouti Railway line at about 217 km from Addis Ababa.This town is the largest settlement in Awash Fentale district, lying at a longitude of 08°59′N 40°10′E at an elevation of 986 m.Metehara is also a town in central Ethiopia; located in the East Shewa Zone of the Oromia Regional State, on a longitude of 08°54′N 39°55′E, at an elevation of 947 m above sea level.Haro Adi village, from where the larvae and pupae of An. stephensi were collected, is a village to the south of Metehara Town along Lake Beseka located about two kilometers away from Metehara Town (Table 1 and Fig. 1).
A total of 45 breeding sites/habitats, in and around the towns of Awash Subah Kilo and Metehara and Haro Adi village areas were visited for larval and pupal surveys.Of these, 31 breeding habitats were from Awash Subah Kilo Town, 7 from Metehara Town, and 7 from Haro Adi village.The survey of An. stephensi larvae and pupae was carried out in three sites, namely; Awash Subah Kilo Town, Metehara Town and Haro Adi around Metehara Town (Fig. 1).The survey for An.stephensi larvae and pupae was conducted in metal tanks near houses under construction, in jerry cans where water is reserved for daily household consumption, on cemented water banks for daily household consumption, on water reservoirs with domestic plastic (near Metehara health centre), overhead water tanks, and in cemented burrows of water reserved for production of cement blocks.These sites were selected based on the previous reports of the presence of An. stephensi [4,6].The sampling of breeding sites was conducted based on the WHO guidelines for laboratory and field testing of mosquito larvicides [13].All natural and man-made breeding sites around the study areas were assessed for the presence or absence of An. stephensi larvae.

Anopheles stephensi larval and pupal collection
Larvae and pupae were collected using a World Health Organization (WHO) standard dipper and transferred into a plastic jar of five-litre capacity with a handle and a cover with plenty of holes to allow air circulation.The jar was used for handling and transporting the larvae and pupae.The scooped larvae and pupae were filtered using clean cheesecloth prepared for this purpose and transferred to a plastic jar.Then, approximately 1-1.5 L of water along with plant debris from the natural breeding sites was added to the jar for larvae to feed on until they reached the insectary facility.

Rearing Anopheles stephensi mosquitoes
All larval instars and pupae collected from the field were transported to and subsequently reared to adults in a biocontained insectary facility at the Aklilu Lemma Institute of Pathobiology (ALIPB).The insectary has two secured doors, with a double door at the entrance and each separate unit of the insectary has its own door and sealed glass windows, which prevent mosquitoes from escaping.During mosquito rearing laboratory conditions, such as maintaining the temperature at 27 ± 2 °C and 75 ± 10% relative humidity, were met and monitored.Upon arrival in the insectary, larvae were transferred into a white enamel plastic tray.Once larvae were removed from their natural water source in the plastic container using plastic of 1 ml micropipettes, a diet of baker's yeast was added to the larval tray.After 5 min the tray was swirled to distribute the powder and prevent suffocation from undiluted/accumulated powder [14].Larvae were provided with food twice per day, and trays were checked to see if food remained unconsumed.If food remained unconsumed, no additional food was added.Sorting pupae from larvae was undertaken on a daily basis.Pupae were picked with plastic pipettes and transferred into a beaker with fresh deionized water and then transferred to adult holding cages.Adults in the cage were provided with sugar solution using soaked/wetted cotton ball placed on the top of the meshed cage.The cotton was maintained wet so that mosquitoes could feed on the sugar.The cotton balls were changed every 5 or 6 days, in order to avoid the growth of mold spores and/or fungus on the pad exposed to sugar [14].Concurrent with sugar feeding, 3-7 days old female mosquitoes were fed on rabbit blood meals twice per week (ethical approval was obtained from Addis Ababa University-Aklilu Lemma Institute of Pathobiology (AAU-ALIPB) Ethical Review Board)).Water filled petri-dish and/or wet filter paper supported with cotton and placed on a petri dish were provided for mosquitoes to lay eggs on.Breeding of wild-collected An. stephensi colonies continued until the end of the study.The tests were done on F 0 , F 1 and F 2 generations of the field-collected larvae and pupae.

Anopheles stephensi species identification
Mosquito species identification was undertaken morphologically under a dissecting microscope.Before commencing any efficacy test of the selected larvicides against An.stephensi, 30 adult female mosquitoes were randomly aspirated from cages.Then these mosquitoes were transferred into a glass tube and exposed to chloroform by a cotton ball wetted at the tip.Each of these mosquitoes was laid under a Olympus SZ stereomicroscope at 40X for morphological identification using the updated key to the females of Afro-tropical Anopheles mosquitoes, which includes An. stephensi [15].All were confirmed to be An.stephensi.Fewer Culex and Aedes larvae were collected compared to An. stephensi from the same habitats.Though there were a few Culex and Aedes species larvae collected with An. stephensi, all emerged adults aspirated from the cage were An.stephensi.Typical features of the morphology of An. stephensi are (i) the appearance with 3 pale bands in the palpus and the two apical pale bands are very broad with speckling on palpus segment 3 and (ii) in the 2nd main dark area on the vein 1of its wing, there are 2 pale interruptions [15].

Bacillus
thuringiensis var.israelensis (Bti); FourStar ® Briquets of a solid form; produced by DBA FourStar Microbials LLC.1501 East Woodfield Road, #200W (https:// www.centr almos queto contr ol.com/ allprodu cts/ fours tar/ fours tar-briqu et-180) in January 2019 and with expiry date of December 2023, were acquired from ICIPE/ILRI.The powder form of this bacterial larvicide was weighed on digital weighing scale and prepared in increasing doses of 0.05 g, 0.1 g and 0.2 g, in such a way that it was to be applied in a container of 2000cm 2 with one litre water volume until the dose mortality response was reached.Based on this design, first, the lowest prepared concentration of Bti (0.05 g/l), was added to deionized water and kept for 48 h by covering the container to prevent insects from landing or laying egg in it [16].In order to ensure no insects entered into the larvicidetreated water, the tray remained closed.The subsequent tests were conducted following the same procedure.
In preparation to expose the larvae to larvicides, late third to early fourth instar larvae were sorted into disposable cups containing water using pipettes.Larvae were filtered first through cheesecloth over a separate container for this purpose.The filtered larvae were immediately transferred into plastic containers with an area of 2000cm 2 and containing one litre of deionized water treated with Bti of 0.05 g, as per the application recommended for spot spray [17].Batches of 25 larvae were exposed per testing container.Simultaneously, an equal number of larvae (negative controls) were tested using untreated deionized water with same number of larvae per container.The tests were conducted in four replicates.The experiment was repeated three times on different days and repeated for larvae collected from each site.Only, the lowest prepared concentration of Bti (0.05 g) was tested as a result of vector's larvae susceptibility response to the larvicide.
Temephos, an emulsifiable liquid concentrate containing 500 g of active ingredient per liter, brand name BASF-Abate ® 500E, developed in Malaysia in 2018 (https:// www.mkhar dware.com.my/ pages/ pages_ id/ 13613/) was acquired from the Ethiopian public health institute (EPHI), and tested against An.stephensi larvae.Following the same procedure used for Bti testing, temephos of 0.25 ml/l, 0.5 ml/l and 1 ml/l was prepared in increasing concentrations, until the dose response was saturated.Temephos (0.25 ml) was added to a container of 2000cm 2 with one litre of deionized water volume using 1000 ml capacity micropipette.Four replicates were set up for each concentration each was run three times on different days.An equal number of negative controls were set up simultaneously with deionized water.The late third and early fourth stage larvae, collected from the field and from reared adults (F 0 , F 1 and F 2 ), were used for the larvicidal test.Larvae were first collected from the tray using pipettes into disposable plastic cups containing water.Then 25 larvae were filtered and immediately transferred into the container of 1000 ml deionized water treated with 0.25 ml of temephos.
While conducting the efficacy tests of both larvicides, larval mortality was recorded after 24 h [15,17,18].Same to Bti, only the lower prepared concentration of temephos (0.25 ml) was tested, as a result of larvae susceptibility response.Larvae that sank down to the bottom of water, in the case of temephos, and appeared floating on the water with swollen and blackened bodies, in the case of Bti, were considered dead.The WHO guidelines for laboratory and field testing of larvicides, states that the test should be rejected if the control mortality is > 20% or pupation is > 10% [13].

Data analysis
The data were recorded using the WHO larvicide efficacy evaluation result recording form [13].The data from all replicates were pooled and entered into an excel spreadsheet for analysis using STATA version 14.0.
If the control mortality was between 5 and 20%, the mortalities of treated groups were corrected according to Abbott's formula.Tests with control mortality greater than 20% or pupation greater than 10% were discarded.
The mortality of the test sample was calculated by summing the number of dead larvae across all exposure replicates expressed as a percentage of the total number of exposed larvae.

Ethical considerations
This study involved no human subjects and it was implemented after obtaining ethical clearance (Ref.

Collection of Anopheles stephensi larvae and pupae
Larvae and pupae were found only in two of the surveyed sites (Awash Subah Kilo Town and Haro Adi village around Metehara), and only in water reserved for the production of cement blocks in small manual factories for construction purposes.Larvae and pupae of Anopheles species were found in cement concrete water reservoirs and cisterns, while Culex/Aedes larvae were observed in plastic tankers, overhead metal tankers, barrels, jerry cans, domestic water reservoir plastics, burrow and some cisterns.Of the 45 total surveyed habitats, 5 out of 31 (16.2%) in Awash Subah Kilo Town, 3 out of 7 (42.9%) in Haro Adi, and none of the 7 in Metehara Town were found to be positive for An.stephensi.The map of An. stephensi larvae and pupae collection sites is visualized in Fig. 1.

Additional reading regarding positive and negative breeding habitats
For the detailed descriptions of breeding habitats and timeliness on visit of habitats for larval and pupae collections in the study sites refer to Table 1 and Supplement 1. Habitats represented as 17-26 in Additional file 1 were visited during March 28-30/2021 and all were found negative for An.stephensi larvae, but Culex/Aedes species were found.Habitat 1 in Awash Subah Kilo Town and habitats 1 and 2 in Haro Adi village of Table 1 were found positive for larvae of An. stephensi, and all surveyed habitats in Metehara Town and habitats represented as 1-16 in Awash Subah Kilo Town were negative for larvae of An. stephensi during 2-3 February 2021.During 1-3 March of 2021, in Awash Subah Kilo Town habitat 1 in Table 1 and all surveyed habitats in Metehara Town and in Haro Adi village habitats 1-4 in Additional file 1 were negative for larvae of An. stephensi, whereas habitats 4 and 5 in Awash Subah Kilo Town and habitat 3 in Haro Adi village, as presented in Table 1 were positive for larvae of An. stephensi.During 8-10 of April 2021, habitats represented as 1-3 both in Awash Subah Kilo Town and in Haro Adi village, Table 1, were found positive for larvae of An. stephensi.On June 3, 2021, three sites (all cisterns) were found positive for An.stephensi in Awash Subah Kilo Town, and three sites (all cisterns) were found positive in Haro Adi village.

Efficacy of Bacillus thuringiensis var. israelensis against Anopheles stephensi larvae
Out of the total of 600 exposed larvae, only one survived this bacterial larvicide after 24 h.All other larvae exposed in each replicate died within 24 h, and all larvae appeared floating on the water with swollen and blackened bodies.All 600 larvae under negative control conditions survived during the course of the experiment (24 h).The two higher doses (0.1 g and 0.2 g Bti) were not tested because larvae had already responded to the lowest dose of Bti (0.05 g).

Efficacy of temephos against Anopheles stephensi larvae
All exposed larvae from both sites were susceptible to temephos and sank down to the bottom of the water within a short period of time (starting at 2 h post exposure).All of the 600 larvae exposed to 0.25 ml/l concentration of temephos were found dead within 24 h (Table 3).The two higher doses (0.5 ml and 1 ml of temephos) were not tested because larvae had already responded to the lowest doses of temephos (0.25 ml).
Statistical analysis was not done rather only descriptive statistics were used because nearly all control larvae survived and nearly all treated larvae died.From the total of 600 larvae in the control group, only 1 (0.2%) died and the test was accepted, without correction.

Discussion
In this study, larvicide bioassays revealed that larvae of An. stephensi from two study localities in Ethiopia (Awash Subah Kilo and Haro Adi around Metehara Towns) were susceptible to both Bti powder and temephos liquid formulation at the lowest prepared doses.The findings also suggested that there is no difference in susceptibility status to the tested larvicides between larvae collected from the two sites.
The specimens were not stored for further molecular confirmation because of financial limitations and the inability to preserve the specimens for a longer time.However, rearing of the colony in the insectary has continued.F 0 , F 1 and F 2 larvae were also used to avoid shortage of test mosquitoes and also for confirmation of their species.
The bacterial larvicide, Bti, was efficient in killing 99.7% of exposed An. stephensi larvae, at the concentration of 0.05gm/l water.This finding is inline with studies conducted in Iran, though tested with different concentration units of 512 and 4096 ppm for Bio-flash ® granules and powder formulation after 24 h post-treatment, that An. stephensi larvae were seen 100% susceptible [20].The finding of this study, aligns with the laboratory test findings on 0.046 mg/L and 0.149 mg/L, and 0.05, 0.1, 0.2, 0.5, and 1 g/m 2 dosages of Bti against An.stephensi in Pakistan [21] and in India [22], respectively, that have shown high efficacy against An.stephensi larvae within 24 h of post treatment.
A field based study conducted in western highlands in Kenya [23], has also revealed that larvae of Anopheles gambae complex and An.funestus mosquitoes decreased from 7.56 larvae per dip during pre-intervention period to 3.09 post application of Bti FourStar ® and 20 week follow up.A Bti susceptibility study conducted against Aedes aegypti in Brazil [19], using laboratory colony and Bti powder IPS82 as a reference to field populations, has shown that all Ae.aegypti populations were susceptible to Bti.The review of 39 studies conducted on Bti and/  or BS across sub-Saharan African countries in laboratory and semi-field conditions against Anopheles gambiae complex and An.funestus have also revealed that these larvicides were effective in reducting larval density, vector density, vector biting and malaria transmission in most of the tested areas [18].The findings of these studies, though with different methodological approach and on different mosquitoes species, strengthen the result of this study.Future work could consider testing the lower concentrations of Bti against larvae.The residual efficacy of the larvicides was not included in this study.
In this study, temephos showed 100% efficacy in killing all exposed An. stephensi larvae.This finding is similar to findings from other countries and other studies within Ethiopia [7].Laboratory based studies in India and southern Iran revealed that larval bioassays on An. stephensi collected from the field were susceptible to a temephos larvicide diagnostic dose of (0.25 mg/l) [24,25].The only larval habitats found to be positive for An.stephensi were permanent water containers filled with water for the purpose of cement block production during January through March 2021 larval collections.Unlike findings by others [6][7][8], barrels, domestic plastic water reservoirs, overhead water containers, and Jerry Cans were negative (for unexplained reason) for larvae and pupae of An. stephensi in Awash Subah Kilo, Metehara and Haro Adi, around Metehara Town.

Conclusion
The present study revealed that An. stephensi larvae from two locations, Haro Adi around Metehara Town and Awash Subah Kilo Town, are susceptible to Bti and temephos larvicides.Both Bti and temephos were found to be 99.7% and 100% effective in killing An. stephensi larvae, respectively.The preference of this vector for breeding in artificial habitats suggests possible control through the application of larvicides to these fixed habitats.Further laboratory and field-based studies are necessary to determine efficacy of larvicides against An.stephensi and other malaria vectors at different localities and presumably under field settings.Exhaustive assessment of breeding sites and identifying the cohabitants of this vector can also help in identifying effective tool(s) to control these vectors in an integrated approach.

Table 1
Description of larvae and pupae positive habitats visited from February 2021 to June 2021 See Additional file 1 for complete list of sites including habitats negative for An. *

Stephensi Site Geographic location Region Climate zone Anopheles species Awash Subah Kilo Habitat type Latitude Longitude Altitude Afar Semi-arid An. stephensi
Fig. 1 Map of Anopheles stephensi larvae and pupae collection habitats

Table 2
Anopheles stephensi larval mortality after 24 h of exposure to Bti, collected from Awash Subah Kilo and Haro Adi around Metehara Towns, January-March, 2021Generally, Bti caused mortality of 100% and 99.7% in larvae from Awash Subah Kilo and Haro Adi around Metehara Town, respectively

Table 3
Anopheles stephensi larval mortality after 24 h of exposure to temephos, collected from Awash Subah Kilo and Haro Adi around Metehara Towns, January-March, 2021