DNA Barcode Reference Library for the African Citrus Triozid, Trioza erytreae (Hemiptera: Triozidae): Vector of African Citrus Greening.

Citrus (Citrus spp.) production continues to decline in East Africa, particularly in Kenya and Tanzania, the two major producers in the region. This decline is attributed to pests and diseases including infestation by the African citrus triozid, Trioza erytreae (Del Guercio) (Hemiptera: Triozidae). Besides direct feeding damage by adults and immature stages, T. erytreae is the main vector of 'Candidatus Liberibacter africanus', the causative agent of Greening disease in Africa, closely related to Huanglongbing. This study aimed to generate a novel barcode reference library for T. erytreae in order to use DNA barcoding as a rapid tool for accurate identification of the pest to aid phytosanitary measures. Triozid samples were collected from citrus orchards in Kenya, Tanzania, and South Africa and from alternative host plants. Sequences generated from populations in the study showed very low variability within acceptable ranges of species. All samples analyzed were linked to T. erytreae of GenBank accession number KU517195. Phylogeny of samples in this study and other Trioza reference species was inferred using the Maximum Likelihood method. The phylogenetic tree was paraphyletic with two distinct branches. The first branch had two clusters: 1) cluster of all populations analyzed with GenBank accession of T. erytreae and 2) cluster of all the other GenBank accession of Trioza species analyzed except T. incrustata Percy, 2016 (KT588307.1), T. eugeniae Froggatt (KY294637.1), and T. grallata Percy, 2016 (KT588308.1) that occupied the second branch as outgroups forming sister clade relationships. These results were further substantiated with genetic distance values and principal component analyses.

Cold tolerance and disinfestation of Bactrocera invadens (Diptera: Tephritidae) in 'Hass' avocado.

Bactrocera invadens (Diptera: Tephritidae) has spread rapidly across Africa and currently poses a phytosanitary threat to the fruit industry of South Africa. In reaction a cold mitigating treatment to provide phytosanitary security to importing countries was developed in Nairobi, Kenya. Using laboratory reared fruit flies, the rate of development in 'Hass' avocado (Persea americana Miller) was determined at 28 degrees C. Fruit ripeness or softness was found to be a factor improving larval fruit fly survival. Using this information the egg and larval developmental stages were subjected to 2 degrees C cold treatment and it was found that the third instars were the most cold tolerant life stage and that it was expected that between 16 and 17 d treatment would provide phytosanitary security. There were no survivors in the treatment of an estimated 153,001 individuals in four replicates at an average fruit pulp temperature of 2 degrees C satisfying the Probit 9 level of efficiency at a confidence of >95%. These data provide evidence that a continuous cold treatment of 1.5 degrees C or lower for 18 d would provide phytosanitary security in that any consignment entering an importing country poses no risk of accidental importation of B. invadens.

Cold susceptibility and disinfestation of Bactrocera invadens (Diptera: Tephritidae) in oranges.

To develop a cold disinfestation treatment for the fruit fly Bactrocera invadens Drew, Tsuruta & White (Diptera: Tephritidae) that is rapidly spreading across Africa, research was conducted in Nairobi, Kenya, using flies from a laboratory culture and 'Valencia' orange (Citrus sinensis L. Osbeck) as the host. The developmental rate of B. invadens in Valencia oranges was determined at 28 degrees C, and the third instar was found to be the least susceptible of the egg and larval life stages to cold treatment at 1.1 degrees C in oranges. When 22,449 B. invadens third instars were exposed in oranges to a cold treatment with an approximate midpoint of 1.1 +/- 0.5 degrees C, the results suggested that a period of 16 d would be worthwhile verifying on a larger scale in oranges. Results from the first replicate of 16,617 larvae showed no survivors, but the second replicate of 23,536 larvae had three survivors. Because a longer cold treatment based on a mean temperature of 1.1 degrees C would create logistical difficulties for some export markets, further replicates were conducted at an approximate midpoint of 0.5 degrees C and at mean hourly maximum of 0.9 +/- 0.5 degrees C, for 16 d. After three replicates, in which 65,752 B. invadens third instars in total were treated with no survivors, the Japanese requirement of 99.99% mortality at the 95% confidence level was surpassed. The following treatment protocol for B. invadens larvae in oranges can therefore be recommended: fruit pulp to be maintained at temperatures of 0.9 degrees C or lower for 16 consecutive days.

Field infestation, life history and demographic parameters of the fruit fly Bactrocera invadens (Diptera: Tephritidae) in Africa.

Field infestation rates of an invasive fruit fly species, Bactrocera invadens Drew Tsuruta & White on mango was determined at different localities in Kenya. At most of the locations and especially at low elevations, B. invadens frequently shared the same fruit with the indigenous fruit fly species Ceratitis cosyra (Walker) but often occurred at higher numbers than C. cosyra. The level of infestation varied with location ranging from 3.0 to 97.2 flies per kg of fruit. There was a significant inverse relationship between numbers of flies per kg of fruit and elevation at which fruit was collected, suggesting that B. invadens is a predominantly lowland pest. On an artificial diet, development of B. invadens immatures lasted 25 days; egg incubation required 1.2 days, larval development 11.1 days and puparia-adult development 12.4 days. About 55% of eggs developed to the adult stage. Life expectancy at pupal eclosion was 75.1 days in females and 86.4 days in males. Average net fecundity and net fertility were 794.6 and 608.1 eggs, respectively, while average daily oviposition was 18.2 eggs. Daily population increase was 11% and mean generation time was 31 days. Results are discussed in relation to the biology and ecology of the insect and in the development of mass rearing and control measures for B. invadens.