Study area
Meme River Basin is situated in the rain forest of the South West Region of Cameroon approximately 60 km inland from the Atlantic Ocean. The topography is very diverse; the main feature is a mountain range (Rumpi Hills, characterized by a volcanic ridge culminating at 1764 m with a northeast orientation [14]. The volcanic ridge is broken by several valleys and constitutes a watershed that is the source for several rivers, the Manyu, Meme, Mungo and Ndian. These rivers go down steep slopes generating fast currents and appropriate flow rates that favour the establishment of permanent Simulium breeding sites conducive to high intensity of onchocerciasis transmission. The climate is characterized by 8 months of rainfall and a short dry season from December to March. The annual rainfall varies between 2500 and 4000 mm with annual temperatures ranging from 25 to 32 °C.
River Meme and its tributaries originate from the Rumpi Hills and flow down precipitous slopes some kilometres from the selected communities. The main tributaries of the Meme River are the Mbile River, which flows pass Marumba I and II, and the Uve River in Bakumba and Big Massaka. These tributaries create important Simulium breeding sites as they enter the Meme River.
Identification of breeding sites
Potential Simulium breeding sites in the Meme River were selected using a topographical map of 1:200,000 scale [28] followed by an entomological survey to check for the presence of rapids, trailing vegetation and larvae to confirm that it is a breeding site. The Melange River in Ndian Division was set as the control area, which will be used to monitor the stability of entomological indicators after ground larviciding. The larvae and adults were identified using standard identification keys [15, 16]. All breeding sites were geo-referenced using a GPS. The co-ordinates were used to generate a topographical map of the breeding sites (Fig. 1a, b).
Study sites
The study was conducted from March to July 2017. River Meme and its tributaries were surveyed, 20 breeding sites from 12 communities (Betenge, Small Massaka Lokando Big Butu, Small Butu, Big Massaka, Kumukumu, Bombele, Bobanda Kwakwa Nake and Marumba 1) were visited, but due to absence of larvae in some sites at the time of the study, only 8 sites from six communities (Betenge, Small Massaka, Big Massaka, Bombele, Kwakwa and Marumba 1) were chosen for fly and larvae collection. Due to accessibility, two sites near one community (Berenge) were surveyed in the Melange River Basin (control area). Flies were collected and dissected from one of the sites that had larvae to monitor the stability of entomological indicators in an untreated area after ground larviciding. Some sites in the Meme River Basin did not have enough larvae, so larvae for cytotaxonomy were collected only from three sites (Big Massaka, Bombele and Kwakwa). In the study arm, the breeding sites chosen were at:
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Marumba I (River Mbile, a tributary of Meme). Here, S. damnosum larvae were collected and a fly dissection point was set up at the river bank.
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Bombele, where the Meme River runs over several rock features developing rapids, which constitute breeding sites along its way to join the Atlantic Ocean.
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Kwakwa (along River Mbile, a tributary of Meme). Here, many larvae were found and a fly collection point was set up at the bank of the river.
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Big Massaka (River Uve, tributary of Meme). A survey of this tributary revealed many breeding sites which contained S. damnosum larvae. A catching point was therefore located in this community.
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Betenge, where the Meme runs. A fly collection and dissection point were located in this community
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Small Massaka (River Ka, tributary of Meme). A fly collection and dissection point were located in this community
Susceptibility of S. damnosum larvae to temephos (Abate 500EC)
Larvae of S. damnosum were collected from several breeding sites, namely Marumba 1, Big Massaka, Bombele and KwaKwa in the Meme River Basin located in the South West Region of Cameroon. These larvae were exposed using a bowl containing local bottled water (Supermont) to ten concentrations of temephos (0.0005 mg/l to 0.5 mg/l) (Additional file 1: Text S1) for a period of 3 h [17]. After this, mortality was determined by observation of larvae motility response to stimulation [18].
Dosage-mortality regression line
Adjusted mortality for each concentration was calculated utilising the control mortality frequency that ran in parallel with the test. If the control mortality was between 5 and 20%, then Abbot’s formula [19] was used to correct the mortality and ensure that other factors were not contributing to the mortality of the larvae. No adjustment was done if control mortality was < 5% and results were discarded for any testing with control mortality > 20%.
Abbot’s formula
$${\text{Corrected test mortality }}\;{ = }\;\frac{{\left( {{\text{\% test mortality }} - {\text{ \% control mortality}}} \right){ }}}{{{ }\left( {{100} - {\text{\% control mortality}}} \right)}} \, \times {100}$$
Temephos dosage-dependent mortality curve was constructed using prism 7.0 (Graphpad, San Diego, CA, USA).
Identification of cytospecies of S. damnosum in the Meme River Basin
Cytotaxonomy was used to identify the different cytospecies in the Meme River Basin. Cytotaxonomy requires the extraction and analysis of polythene chromosomes (giant chromosomes caused by cells that have undergone repeated rounds of replication) present in the silk gland of black fly larvae. Larvae for cytotaxonomy were collected from three breeding sites (Big Massaka, Bombele and Kwakwa) because the other breeding site did not have enough larvae for cytotaxonomy. The sixth and seventh stage larvae were collected and put in Carnoy’s fixative (3 parts ethanol: 1part acetic acid) that was freshly prepared at the riverside. A minimum of 30 larvae were put in 24 ml of Carnoy. The Carnoy’s solution was changed after 3 h and 24 h. The silk glands were dissected followed by hydrolysation to enable all the silk in the glands to be removed. Thereafter, chromosomes were stained for 5 min with Feulgen stain, which stains the DNA pink. Continual addition of orcien to the silk gland occurred to prevent dehydration. The gonads of the larvae were inspected to identify the sex. If male, the gonads are round, but if female, the gonads are elongated in shape. Squashing was done to separate the chromosomes while a light microscope was used to visualize and identify them. Photographs were taken at × 400. Identification of each larval chromosome was done using information from pictorial guide and identification key by Post et al. [20].
Collection and identification of Simulium flies
Simulium flies were collected from seven different sites: Bombele and Betenge (River Meme), Big Massaka (River Uve), Kwakwa and Marumba I (River Mbile), Small Massaka (River Ka) and Berenge (River Melange). Residents of endemic communities for onchocerciasis were trained to carry out collections; Simulium flies landing on exposed legs for a blood meal were captured using suction tubes or mouth aspirators (locally adapted) before they bit [21].
Adult flies were either dissected to determine the parity and infectivity rate or stored in 80% alcohol for future use. Onchocerca volvulus transmission indicators (parous rate, infection rate, infectivity rate and monthly transmission potential) were computed after fly collection and dissection as described by Walsh et al. [22].
Dissection of Simulium flies
Captured flies were killed using chloroform, counted and dissected in physiological saline under a dissecting microscope (Humanscope, HUMAN Diagnostics Worldwide, USA). Flies caught were dissected on an hourly basis to determine parity and infection status. This consisted of holding the fly with a needle in the thorax, piercing the abdomen with a dissecting needle at the posterior end and then pulling out the different internal organs to examine the fat bodies, state of the Malpighian tubules and ovaries to distinguish parous from nulliparous flies (nulliparous ovary is generally clear with follicles that are bigger and have voluminous fatty substance while parous flies generally have yellowish, spotted ovaries because of follicular relicts. The follicles are much smaller with little or no fatty substance). The head, thorax and abdomen of parous flies were further dissected separately and examined for O. volvulus developing larvae (L1, L2 and L3). Any larvae found were counted and recorded on a dissection sheet [23] and the entomological indices computed [22]
Survey of some non-target aquatic fauna
Survey of some non-target aquatic fauna was conducted to obtain a picture mainly of the common fish species and also other common vertebrate as well as invertebrate non-target fauna to serve as indicators to guide measurement of any detrimental effect of temephos during ground larviciding. For this to be done, fishermen and -women were contacted to determine the species of aquatic vertebrates common in the Meme River and its tributaries. Thereafter, they were requested to carry out fishing exercises to determine the vertebrate aquatic fauna found at the different study sites. Locally made fishing nets were placed in water overnight to trap any available vertebrate aquatic fauna as modified from the protocol adopted by OCP in 1991 [24]. During the collection of Simulium larvae, invertebrate aquatic fauna present on the same substrate as the Simulium larvae were observed. Any aquatic fauna observed were grouped as either vertebrate or invertebrate. The vertebrate aquatic fauna were identified with reference to Temegne and Momo [25] while the invertebrate aquatic fauna were identified using a key by Danladi et al. [26].
Data analysis
Fly collection data were entered in a template created in Epi info version 3.5.3 (https://www.cdc.gov/epiinfo/index.html), exported to Microsoft Excel and then later to SPSS version 24 for analysis. The different entomological indicators generated were biting densities, daily biting rate, monthly biting rate, parous rates infection rates, infective rates and monthly transmission potential. The chi-square test for trend was used to establish whether there was any association among parous rate, infection rate and infective rate.
Entomological indices were calculated using the following formulae:
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Parous rates were computed as the number of parous flies divided by the total number of flies dissected and multiplied by 100.
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Infection rates were computed as number of flies carrying any developmental form of O. volvulus larvae divided by the total number of parous flies dissected and multiplied by 100.
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Infective rates were computed as the total number of flies carrying infective larvae in the head divided by the total number of parous flies dissected and multiplied by 100.
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Biting density (number of flies collected at different hours plotted against the hours collected).
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Monthly transmission potential: (number of days in month × number of infective larvae)/number of fly collection days × (number of flies collected/number of flies dissected).
