Life cycle and molecular characterization of the orange spiny whitefly Aleurocanthus spiniferus (Quaintance, 1903) (Hemiptera: Aleyrodidae) as a new quarantine pest of citrus fruits in The Republic of Croatia The orange spiny whitefly Aleurocanthus spiniferus (Quaintance, 1903) (Hemiptera: Aleyrodidae: Aleyrodinae) is a new entomological species in Croatia and currently has the status of a quarantine pest on the Union list, Part II, Annex B. A. spiniferus originates from Southeast Asia and belongs to the genus Aleurocanthus Quaintance & Baker, 1914, which includes about 90 species worldwide. Along with the species Aleurocanthus woglumi Ashby, 1915, it is one of the economically important pests. It was first detected in Europe in 2008 in southern Italy, and in subsequent years, it has spread to other Mediterranean countries. The adult stage of A. spiniferus is a small insect, measuring only 1.30-1.70 mm, characterized by dark-colored wings. The puparium of A. spiniferus is black and features white marginal wax filaments. It is a polyphagous pest, proven to attack more than 90 plant species from 38 plant families, with species from the genus Citrus spp., Pyrus spp., and Vitis spp., the main economically important host plants. It causes damage by feeding on plant sap on the undersides of leaves and by excreting abundant honeydew on which sooty mold fungi colonize, leading to reduced photosynthesis and evapotranspiration, defoliation, and stunted plant growth. In 2018, the first positive finding in a natural environment was confirmed in Vitaljina, southern Croatia. Along with the four whitefly species recorded on citrus in Croatia so far, Dialeurodes citri Ashmead, 1885, Parabemisia myricae Kuwana, 1927, Aleurothrixus floccosus Maskell, 1914 and Aleuroclava aucubae Kuwana, 1911, it is among the economically important citrus pests. Controlling A. spiniferus is challenging due to its polyphagy, high reproductive potential, overlapping generations, and all developmental stages in the orchard. As it is a newly introduced species, this quarantine pest's biological and ecological characteristics are insufficiently studied. In 2019, its spread towards northwest Croatia was recorded, posing a high phytosanitary risk, especially for the Neretva River valley area, the main citrus-growing region in Croatia. However, due to the limited presence of natural enemies in this area, there is a risk of establishing infestations on new crops and new plantations such as grapevines, pears, apples, etc. The aim of this study was to determine the life cycle of the pest under outdoor and controlled conditions in walk-in chambers at four different temperatures with the same relative humidity, to create a phenogram of the species' development, to determine potential host plants, and to perform analyses based on cytochrome c oxidase subunit I (COI) mitochondrial DNA (mtDNA) sequences to reconstruct possible invasion routes of A. spiniferus in Croatia. Field research on the life cycle of the pest A. spiniferus in natural conditions was conducted from January 1, 2020 to January 1, 2022, in the locality of Vitaljina, in a private mandarin orchard. The life cycle of A. spiniferus was determined based on monitoring and recording data on the occurrence of all developmental stages of the pest. Yellow sticky traps were used to signal the beginning of the flight of the adult stage, and every 7-10 days during the vegetation period, all present developmental stages were recorded on the underside of the leaves using a 60x hand magnifier. Subsequently, the time frames for the duration of each individual life stage were calculated. In addition to the life cycle, the population dynamics were determined by counting eggs, larvae, puparium and adults on the leaves. Mandarin leaves were also inspected throughout the year, every 7-10 days during the vegetation period and once a month during the dormant period. In 2020, 1620 leaves were analysed, and in 2021, 1512 leaves were from 18 trees. The sampled material was stored in plastic PVC bags and transported to the Institute for Mediterranean Cultures laboratory at the University of Dubrovnik. In the laboratory, the presence of each developmental stage, from eggs to puparia, was determined and recorded on the underside of the leaves using an Olympus SZ40 stereomicroscope. In contrast, the number of adult stages was determined by counting individuals on the underside of the leaves in the orchard. Each pre-imaginal stage of the pest was expressed as the number of eggs, larvae L1, L2-L3, and L4, as well as adults per 100 cm2 of leaf surface. Climate data from the nearest meteorological station (Dubrovnik airport) were recorded during the research to facilitate the determination of the occurrence of specific developmental stages of the pest in the study of its biology in the southern region of Croatia. Identification of other beneficial entomofauna on the collected leaves was carried out to determine the potential presence of natural enemies of A. spiniferus. Determination of entomofauna was conducted at the Plant Protection Center, CAAF, based on morphological characteristics using relevant keys available in the literature, using an Olympus BX51 binocular light microscope equipped with an Olympus DP25 digital microscopic camera and analySIS doc morphometry software. The pest's life cycle was investigated in controlled conditions according to the method of Argov et al. (1999) in walk-in climate chambers. Four mandarin seedlings were placed in each individual entomological cage in the climate chambers with fluorescent light, set to a photoperiod of L16:D8, and a relative humidity of 80 ± 10%, while the temperature varied in four treatments: 20 °C, 25 °C, 30 °C, and 35 °C. Subsequently, an artificial infestation with 300 individuals of adult developmental stages of the pest was conducted for 24 hours. The leaf examination was carried out using a hand magnifier with 60x magnification, and the biological development of the species in climate chambers was conducted every three days. All leaves on which pre-imaginal stages were present were marked. Average values of the development length for each developmental stage at different temperatures and under natural conditions. Sexual index determination and oviposition of A. spiniferus were conducted in walk-in chambers. Molecular analysis of A. spiniferus was performed using the PCR method on ten samples. DNA isolation was carried out using the Dneasy Blood & Tissue isolation kit (Qiagen® GmbH, Hilden, Germany), and the isolated DNA was stored at -20°C. The mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) region was amplified using polymerase chain reaction (PCR) according to the method of Uesugi et al. (2016). Visualization of PCR products was performed by agarose gel electrophoresis. A 200 bp DNA ladder containing 13 fragments of known lengths was used as a molecular weight standard for DNA determination. After electrophoresis, the gel was examined and photographed using the UVIdoc HD2 system. The amplified mtCOI fragments of 670 bp were purified using the GenElute® PCR Clean-up Kit and sent to Macrogen Europe® service for sequencing. The obtained sequences were compared with sequences from the GenBank database using the BLAST program. In both years of the study, in natural conditions, adult developmental stages were recorded in April. In the climate conditions of southern coastal Croatia, A. spiniferus has three generations per year and overwinters in the fourth larval stage. The total development of A. Spiniferus from egg to adult, in the first year of the study, lasted from 41-105 days, while in the second year it lasted from 49-93 days. Overlapping generations were recorded. In monitoring the population dynamics, maximums of individual developmental stages were recorded in both years of the study. In contrast, for the larvae of the fourth developmental stage, it was not possible due to overlapping generations, and some larvae remained on the leaves and overwintered in that stage. In determining other entomofauna to find natural enemies of A. spiniferus, the Coccinelidae species Clitostethus arcuatus (Rossi, 1974) was confirmed. The determination of the sexual index at different temperatures revealed that females predominated in the overall population (0-7-0.8), while the highest number of deposited eggs was observed at 25 °C (506 eggs/24 h). None of the six tested host plants were found to be suitable hosts. This scientific paper represents a significant contribution to entomological science as it provides a deeper understanding of the biological development of the quarantine pest A. spiniferus. The research was conducted under natural conditions, which allowed for the establishment of a scientific basis for the control and prevention of the spread of this pest, particularly in the area of intensive citrus production in the Neretva Valley, which is at high risk of infestation by this pest. Furthermore, this study presents the ideal conditions that favor the development of this pest, which can serve as a basis for future research on this species. Moreover, this paper also contributes to the genetic identification of the species, which is essential in the context of controlling the introduction of plant material from other countries to prevent the possibility of spreading this pest to new areas and host plants. These findings are crucial for preserving citrus orchard productivity in the Neretva Valley and the other regions at risk of infestation by this pest.